Chronic Exposure to Artificial Light at Night Dampens Rhythms in Locomotor Activity, Metabolism, and Sleep in Female Mice

Abstract

Nearly every organism on Earth displays circadian organization of physiology and behavior, which is entrained to 24 h by daily exposure to bright light early during the day. However, the widespread presence of nocturnal electric lighting has blurred the boundary between day and night, leading to temporal desynchrony among individuals. Indeed, less than 6 weeks exposure to low levels of artificial light at night (ALAN) alters metabolism, energy, and sleep homeostasis in male mice. We hypothesized that ALAN would similarly affect female mice following prolonged exposure. After ≥20 weeks of exposure to dim ALAN, female mice had dampened locomotor rhythms, altered heat generation, respiratory exchange ratio (RER), and sleep. ALAN exposure decreased daily caloric intake, without altering body composition. The period and phase of the rhythm of sleep were significantly increased by ALAN, elevating the time spent sleeping during the active phase, without altering total sleep bout length. Taken together, these data indicate that ALAN impairs the rhythms in core physiological processes in female mice, but the mechanisms mediating these alterations may differ from those in males.

1. Introduction

In the absence of environmental light cues, circadian rhythms in physiology and behavior persist, with a period close to, but not precisely, 24 h. Among mammals, temporal synchronization of individuals with the environmental light-dark cycles is mediated by the suprachiasmatic nuclei (SCN), a set of paired nuclei located in the anterior hypothalamus that serves as the ‘central clock’. The SCN synchronize physiological processes to the environmental light dark cycle by daily exposure to the most potent zeitgeber [1, 2], bright light. As the central clock, the SCN communicates with peripheral clocks, synchronizing them via humoral and neural signals. These synchronized internal rhythms allow for optimal timing of physiological processes, such as daily fluctuations in body temperature, immune function, metabolism, and sleep.

Life on Earth evolved under conditions of dark nights and brightly illuminated days, which has allowed precise synchrony of internal circadian rhythms to the 24 h solar days. However, modern technology and electrical lighting have altered this natural light-dark demarcation by extending the illuminated ‘day’ well into the nighttime. As a result of the widespread adoption of electric lighting, the nocturnal light-scape of modern times has dramatically changed. Indeed, a recent study recorded a minimum 49% increase between 1992 – 2017, in satellite-observable light emissions [3]. As a result, over 80% of the World’s population is exposed to nocturnal light pollution [4], with skyglow from light emanating from buildings, streetlights, advertisement billboards, and other display lighting. Mounting evidence has demonstrated the noxious effects of exposure to artificial light at night (ALAN) on physiology and behavior. Indeed, exposure to ALAN has been associated with psychiatric [5–8], and metabolic [9–13] disorders, as well as various cancers [14–18]. Preclinical studies have recapitulated many of these human observations in other mammals [19–22] and have begun to provide mechanistic insight into how exposure to ALAN may affect health [23–28].

Although night shift workers are at elevated rates of ALAN exposure, which, in turn, leads to disrupted circadian rhythms and increased risk for developing diseases and other disorders [16, 29–32], the general population is also now increasingly affected by ALAN. Sources of light at night infiltrating homes range from uncontrollable streetlights and other outdoor lighting, to controllable exposure from the voluntary use of indoor lights and LED light from ubiquitous personal electronic devices. To study the effects of these lifestyle factors in controlled laboratory settings, circadian desynchronization models have been implemented in preclinical studies. For instance, variations in light regimes such as the duration of exposure, the intensity of the illumination source, and the spectral composition (i.e., wavelength) are commonly used [22, 33–35]. One frequently used preclinical circadian desynchrony paradigm mimics urban environmental ALAN exposure by exposing animals to dim artificial light at night between 2 – 10 lux of intensity [20, 26, 28, 36], with reported significant effects of dim ALAN exposure on diverse outcomes in paradigms ranging from 1 night [37] to, 24 weeks [38], to 12 months [39]. However, the majority of these previous studies did not include females.

The physiological differences of health and behavioral outcomes as a result of biological sex have become increasingly evident [40–44] as research is beginning to include sex as a biological variable (SABV). For instance, the prevalence and progression of cardiovascular risk factors and diseases, such as hypertension and diabetes vary by sex [41], and the prognosis of some cardiovascular diseases, such as coronary artery disease, is worse for males than females, but only for women under the age of 60 (prior to menopause) [45, 46]. Beyond that age, prognosis drops to that of males aged over 80 [45]. Further, biological sex differences in circadian function, regulation, and response to circadian rhythm desynchrony have been observed in rest-activity states, metabolic regulation neuroendocrine responses, and others [47–50]. Thus, in all studies seeking to appropriately represent pathophysiology and health outcomes, SABV must be considered [43, 51–53]. Recently, studies in male rodents have reported that various durations of exposure to low levels of ALAN shifts the timing of food intake to the inactive phase [20, 26, 54, 55], elevates body mass gain [20, 21, 56], suppresses clock gene expression [20, 57, 58], alters glucose processing [21, 22, 28, 59], and insulin resistance [21, 60, 61], and influences sleep architecture [36, 62]. Although males and females do not generally differ in photic processing from the retina through the SCN, some sex differences have been identified in circadian rhythms downstream of the SCN that strongly suggest that male data cannot be assumed to apply to females [63], and very few studies have investigated the effects of ALAN in females. Thus, the goal of the present study was to begin to fill these gaps in our knowledge by testing the hypothesis that prolonged exposure to ALAN alters physiology and metabolic parameters in female mice.

2. Methods

2.1. Animals and Lighting Conditions.

Twenty (7 weeks of age) female Swiss Webster (CFW) mice were obtained from Charles River Labs (Wilmington, MA, USA) (Figure 1). Mice were individually housed in polypropylene cages (30 cm × 18 cm × 14cm) at an ambient temperature of 22 ± 2°C and provided with Teklad 2018 chow (Madison, WI, USA) and filtered tap water ad libitum. Upon arrival, all mice were maintained under 14 h light (125 lux) and 10 h dark (0 lux) light/dark (LD) conditions for one week of acclimation to the vivarium. A 14:10 LD cycle was maintained as these mice are outbred and could interpret shorter (e.g. 12:12) LD cycles as either a short or long photoperiod [64], leading to increased variability in outcome measures. Mice were then randomly assigned to remain in dark nights (LD) or exposed to dim light at night (ALAN), 14 h light (125 lux) and 10 h dim light (5 lux), for the duration of the study. ALAN was supplied by standard LUMA5 LED light strips (Hitlights Inc.; 1.5 W/ft, 5000 K “cool white”, 1200 lumens), hung at mid-cage height as previously reported [65], daytime light for all animals was provided by the general room overhead lighting. Cage light levels in both light and ALAN phases were measured and confirmed inside each cage with a Mavolux 5032C illuminance meter (Nürnberg, Germany) and an Ophir Starbright irradiance meter (Jerusalem, Israel). Meters were placed in the center of the cage with the light sensor up and cage lid in place, to verify 125 daytime lux, and either 0 lux (dark nights) or ~5 lux (ALAN) of light exposure inside of each cage. To avoid behavioral escape from ALAN, mice were housed singly and given one puck of nestlet material, they did not have access to huts that could potentially block ALAN exposure. We have previously shown that 24 weeks exposure to dim ALAN alters outcomes in female mice [38], so we staged our experiment to explore the outcomes centered around this time point. All experimental procedures were approved by West Virginia University Institutional Animal Care and Use Committee, and animals were maintained in accordance with the guidelines established by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals.

Figure 1: Experimental timeline.

2.2. Metabolic monitoring.

Following 20 weeks of experimental light exposure, mice (n = 8 per light condition, run sequentially) were placed in the Comprehensive Laboratory Animals Monitoring System (CLAMS 8 chamber system; Oxymax, Columbus Instruments, Columbus, OH, USA) to collect whole body metabolic measures for 4 days (Figure 1B). The first experimental day in the CLAMS served as an acclimation period, data from days 2 – 4 were then collected for experimental measures. For the ALAN cohort nighttime light exposure was provided from light sources as described in section 2.1. Mice were weighed immediately before housing in the CLAMS chambers and body mass was used as a covariate for metabolic analyses. Volume O₂ (ml/kg/h), volume CO₂ (ml/kg/h), respiratory exchange ratio (RER; (VCO₂/VO₂)), heat production (kcal/h) (VO₂ ×(3.815 + (1.232 × RER)) and locomotor activity (x and y axis infrared beam breaks) were binned every 20 min for data output each animal. For some analyses data were binned by light and dark cycle, or into 2 h bins for rhythm analyses. Mice had access to food and water ad libitum, and food consumption data were also recorded via the CLAMS system. After the 4-day metabolic assessment period, mice were returned to their home cages.

2.3. Sleep assessment.

After 25 weeks in lighting conditions, by group, mice that had been assessed in the CLAMS were transferred and individually housed in PiezoSleep mouse behavioral tracking system cages (23.5 cm × 15.25 cm × 15.25 cm; Signal Solutions LLC, Lexington, KY, USA) to track sleep (Figure 1C). This system uses piezoelectric sensors detect minute changes in pressure produced by movement, including subtle respiratory movements, to assess sleep and wake states [66, 67]. After 24 hr acclimation to the system, individual sleep data from days 2–4 were collected and analyzed using the default scoring in SleepStats v2.18 (SignalSolutions LLC, Lexington, KY, USA). Sleep parameters assessed include: (1) average total sleep percent over 24 h, (2) average sleep percent per assigned phase (day and night); (3) average sleep bout length over 24 h, and (4) average sleep bout length per phase.

2.4. Body composition.

At the conclusion of sleep assessment, after 26 weeks of ALAN exposure, all mice (n = 10 per condition; Figure 1D) were weighed and then had body composition assessed by EchoMRI TM-100 (Echo Medical Systems, Houston, TX). Parameters including fat mass, lean mass, free water mass, and total water mass were normalized to percent of body mass for each animal [68, 69]. At the conclusion of body composition analysis, mice were euthanized and final tissues masses were assessed.

2.5. Statistical analyses.

Outliers were identified and removed using the Grubbs' test, with an alpha value set to 0.05; at most one data point per group was defined as an outlier. All non-circadian longitudinal data were analyzed using a repeated-measures two-way analysis of variance (RM ANOVA), unless otherwise noted, and a mixed effects model was used if any values were missing. Time was categorized as the within subject factor and light treatment as the between subject factor. For significant ANOVAs, post-hoc comparisons were conducted using a Fisher’s LSD test. Comparisons between lighting conditions with respect to weight gain, body composition, tissue masses, percentage of daytime food intake, and area under the curve were conducted using a Studenťs t-test. All statistical analyses were performed using GraphPad Prism 9.5.0. In all cases, differences between group means were considered statistically significant at p ≤ 0.05.

2.6. Nycthemeral (diel) analyses.

To determine the effect of ALAN on the daily parameters of locomotion, RER, heat production, and sleep, data were analyzed using BioDare2 analysis software (Edinburgh, UK) [70, 71]. MFourFit analysis method was used to calculate the period, phase, and amplitude for each group, as previously described [72].

3. Results

3.1. ALAN alters food consumption without affecting weight gain.

Female mice exposed to dark nights or ALAN did not differ in body mass at the beginning of the study and we did not observe effects of light condition on body mass across the experiment (F _{(1, 14}) = 0.1424; p = 0.7115). Absolute and percent body mass increased over time for all mice (Figure 2 A,B; F_{(1.257, 15.33)} = 88.75; F_{(1.640, 19.13)} = 106.2; p < 0.0001 for both analyses, respectively). Although we did not track food consumption by phase for the duration of this experiment, during metabolic testing in the 20^th experimental week food consumption was monitored by the CLAMS system (Figure 2C,D). Female mice housed in ALAN for 20 weeks shift the timing of food intake to the inactive phase (Figure 2C; (F_{(1.677, 24.15)} = 8.550; p = 0.0024). Post hoc comparisons revealed a significant increase in light phase eating in ALAN-exposed mice during the third (p = 0.0019) and fourth (p = 0.0054) days, with concurrent decreases in food intake during the third (p = 0.0019) and fourth (p = 0.0054) nights of the study. Females consumed less chow overall and thus fewer total daily calories than their LD counterparts (Figure 2D; t₁₄ = 4.706, p = 0.0003).

Figure 2: Long-term exposure to ALAN shifts the timing of food intake, but does not increase body mass gain in female Swiss Webster mice.

(A) No significant differences by light conditions were recorded in weekly raw body mass throughout the study, nor in the (B) percent change in body mass between baseline and current experimental week, except at week 26 (p = 0.0143). (C) Female mice in ALAN shift the time of consumption of the majority of their calories to their inactive phase (daytime; interaction of phase and light, p < 0.001), and (D) consume fewer total daily calories than their LD counterparts (p = 0.003). All data are presented as mean ± SEM; n = 8/group. & p<0.05 for time, # p<0.05 for light; $ p<0.05 for phase. Food consumption data were collected during week 20 of the experimental timeline.

3.2. Body composition is not altered by chronic exposure to ALAN in female mice.

At the time of EchoMRI testing during week 26, body mass (Figure 3A; t₁₈ = 1.059; p = 0.3035), lean mass (Figure 3B; t₁₈ = 0.4111; p = 0.6858), fat mass (Figure 3C; t₁₈ = 0.7131; p = 0.4849) and hydration ratio (Figure 3D; t₁₈ = 1.462; p = 0.1610) did not differ between lighting conditions. At the conclusion of the experiment (week 30) heart mass (Figure 3E, t₁₄ = 0.4414, p = 0.6667), spleen mass (Figure 3F, t₁₄ = 1.300, p = 0.2192), and adrenal mass (Figure 3G, t₁₄ = 1.390, p = 0.1883) did not differ due to ALAN exposure.

Figure 3: ALAN does not alter body composition or organ masses in female Swiss Webster mice.

In females body mass (A), lean mass (B), fat mass (C), hydration ratio (D), heart mass (E), spleen mass (F), and adrenal mass (G) were not affected by 26 weeks of ALAN exposure (p > 0.05). Lean and fat masses are reported as percent body mass. Data are presented as mean ± SEM; n = 10/group.

3.3. Long-term exposure to ALAN dampens the amplitude of locomotor rhythms without altering total daily activity.

The majority of activity in mice held in dark nights occurred during the dark phase, whereas for ALAN exposed counterparts, less than half (44%) of their activity occurred during the dark phase (Figure 4A; interaction of light condition and phase, F_{(5, 72)} = 24.75; p < 0.0001). Total 24 hour locomotor activity was not affected light treatment (Figure 4B; t₁₄ = 0.6913; p = 0.5007), and area under the curve (AUC) analysis further supports the lack of differences in total daily activity (Figure 4C, t₁₄ = 2.030; p = 0.0618). Dark night-exposed (LD) mice exhibited characteristic nocturnal activity with peak shortly after the onset of the dark phase (~ZT14–16) with a minor activity bout around the onset of the light phase (~ZT0–2), whereas ALAN exposure blunted these peaks in overall daily activity rhythms spreading them out across the 24 hour cycle (Figure 4D). The period (Figure 4E; t₁₂ = 2.091; p = 0.0584), and amplitude of the rhythm (Figure 4G; t₁₄ = 1.789; p = 0.0952) did not differ between LD and ALAN mice. However, ALAN exposure reduced the phase and advanced the peak of activity compared to mice in dark nights (Figure 4F; phase = 12.51; t₁₂ = 3.444; p = 0.0049).

Figure 4: Long-term exposure to ALAN blunts diel rhythms in locomotor activity without altering total daily activity.

(A) Long-term (20 weeks) exposure to dim ALAN shifts the majority of 24 hour locomotor activity from the night (dark) phase to the daytime (**p < 0.005; ***p < 0.001). (B) Total daily average of activity did not differ between light conditions (p > 0.05). (C) Light exposure did not affect area under the curve analyses of data shown in D; total beam breaks (p > 0.05). (D) Locomotor activity (beam break count in 2 h bins) across 3 experimental days. (E) Period, (F) phase, and (G) amplitude of activity after 20 weeks of exposure to ALAN. ALAN exposure reduced the phase, resulting in an advance of the activity peak compared to dark nights (F; phase = 12.51; p = 0.0049). Data are mean ± 1 SEM, n = 8/group. & p<0.05 for time, @ p<0.05 for interaction of time-of-day and lighting condition. Mean of group inside bar in E, F. Locomotor activity data were collected during week 20 of ALAN exposure.

3.4. ALAN dampens diel rhythms in heat generation, but promotes sustained levels across 24 h, in female mice.

Continuous calorimetry analysis using the CLAMS system revealed an overall increase in heat produced and a desynchrony of the 24 h rhythm of heat production in ALAN-housed mice compared to those in dark nights (Figure 5A, B, C) (time-of-day; F_{(6.814, 93.31)} = 2.713; p = 0.0140). Time-of-day and nighttime light conditions also interacted to alter rhythms of heat production (Figure 5A; F_{(6.814, 93.31)} = 3.357; p = 0.0034). AUC was significantly different between light treatments (Figure 5B, t₁₄ = 2.996, p = 0.0096), with increased heat generation during the 2 h period prior to lights off (ZT12) throughout the three days of testing (p < 0.05 on day 1, p < 0.005 on day 2, and p < 0.0001 on day 3, Figure 5A). Indeed, heat production was elevated at ZT12, at the onset of the active phase (Figure 5A, C; p = 0.0013), with a significant effect of time-of-day potentiating these observations in the 2 h bins before and after light cycle transition periods (Figure 5C; F_{(1.633, 21.77)} = 5.413; p = 0.0167). Although this increase in heat production did not alter the period compared to that of LD-housed mice (Figure 5D; t₁₀ = 1.921, p = 0.0837), the phase of heat production was significantly reduced (Figure 5E; t₁₀ = 3.009, p = 0.0131), resulting in an advance in the acrophase of heat output. Although ALAN increased overall heat production and altered the phase of the rhythm, changes in the amplitude of the rhythm did not reach statistical significance (Figure 5F, t₁₀ = 1.157, p = 0.2744).

Figure 5: ALAN exposure alters diel rhythms and increases total 24 h heat generation in female mice.

(A) Bihourly-binned plot of heat production over 72 h, depicting intragroup variability in heat generation (thin lines), along with averages for each light treatment group (thick lines). (B) ALAN-exposed females increased heat production across the day (AUC; p = 0.0096), with the highest differential in heat production occurring during the 2 h prior to the onset of the dark phase (C; ZT12, p < 0.001). The period (D) and amplitude (F) of heat generation rhythms were not altered by ALAN, but the acrophase of heat generation was advanced by ALAN exposure (E; phase = 9.43; p = 0.0131). Data are presented as mean ± SEM; n = 8/group). & p<0.05 for time of day, @ p<0.05 for interaction between light and time-of-day. Mean of group inside bar in D, E. Heat data were collected during week 20 of the experimental timeline.

3.5. Long term exposure to ALAN blunts daily rhythms in RER and increases oxygen consumption.

Time-of-day and ALAN interacted to affect RER (F_{(36, 490)} = 4.292; p = <0.0001), and ALAN decreased RER variability across the 24 h day (Figure 6A, F _{(1, 14)} = 45.01; p < 0.0001). ALAN reduced overall RER across the three-day measurement period (AUC Figure 6B; t₁₄ = 19.41; p <0.0001; daily intervals Figure 6C; F_{(1.958, 26.77)} = 3.407; p = 0.0489) suggesting a shift towards fat as their primary metabolic substrate. There were no differences in the period (Figure 6D; t₁₁ = 0.1759, p = 0.8636) or phase of the RER waveform (Figure 6E; t₁₁ = 0.8611, p = 0.4075) due to ALAN, however the amplitude of the rhythm was dampened by ALAN exposure (Figure 6F; t₁₁ = 2.685, p = 0.0212). Consistent with the decrease in RER, ALAN exposed mice had elevated oxygen consumption (Figure 6H; t₁₄ = 9.201; p <0.0001), and this was time-of-day-specific (Figure 6G, F_{(5.893, 80.37)} = 2.620; p = 0.0233). Time-of-day and light exposure interacted to affect oxygen consumption (F_{(36, 491)} = 2.860; p <0.0001), with the highest consumption in the ALAN mice occurring during the light phase.

Figure 6: Long term exposure to ALAN blunts daily rhythms in RER and increases oxygen consumption.

(A) Bihourly-binned plot of RER over three days in the CLAMS system, depicting intragroup variability in RER (thin lines), along with averages for each light treatment group (thick lines). (B) ALAN decreased total RER across 3 days, with significant effects during all of the 2 h periods before and after phase transitions (C). Period (D) and phase (E) of the RER rhythm were not altered by ALAN. However, the amplitude of the RER rhythm was significantly blunted (F). (G) Bihourly-binned plot of oxygen consumption across three days, depicting intragroup variability (thin lines), along with averages for each light treatment group (thick lines). ALAN exposed mice had elevated oxygen consumption (H), mainly during the inactive phase (G). All data are presented as mean ± SEM, n = 8/group. # p<0.05 for light, & p<0.05 for time-of-day, and @ p<0.05 for interaction between light and time-of-day. Mean of group inside bar in D, E. RER data were collected during week 20 of the experimental timeline.

3.6. ALAN dampens sleep rhythms in females, delays sleep acrophase, and increases sleep bout length at the onset of the active phase.

Across the 3 days of sleep monitoring, both light exposure (F_{(1, 14)} = 9.496; p = 0.0081) and time-of-day (F_{(6.929, 95.27)} = 5.144; p < 0.0001) independently affected sleep, and both factors interacted to further alter sleep patterns (F_{(36, 495)} = 4.669; p < 0.0001) (Figure 7A). Specifically, housing with dim ALAN reduced the time sleeping during the light (inactive) phase (~ZT6–8; p < 0.05), and increased the percentage of time spent sleeping early in the active (dark) phase (ZT14–16, p < 0.05)(Figure 7A). Percent time total spent in sleep across the monitoring period was not affected by ALAN (Figure 7B; t₁₄ = 1.716; p = 0.1081). However, when analyzing sleep by phase on each day of monitoring we observed a significant increase in sleep in the ALAN group during the third experimental night (Figure 7C; p = 0.0345). The diurnal wake ratio (daytime:nighttime sleep) was altered by light treatment (F_{(1, 14)} = 13.14; p < 0.0028), with significant increases in nighttime (active phase) sleep in ALAN-exposed females during days 2 (p = 0.0052) and 3 (p = 0.0338)(Figure 7C). Moreover, ALAN exposure altered sleep rhythms; the period of sleep was increased (t₁₄ = 3.440; p = 0.0040, Figure 7E, Table 1), the phase of peak amplitude of sleep was delayed (t₁₄ = 3.516; p = 0.0034, Figure 7F, Table 1), without affecting the amplitude of the sleep rhythm (t₁₄ = 1.107; p = 0.2870, Figure 7G, Table 1).

Figure 7: ALAN alters sleep rhythms in female mice, delays sleep acrophase, and increases early the active phase sleep bout length.

(A) Bihourly binned plot of percent sleep across 3 experimental days (72 h) of observation. ALAN dampened diel rhythms in sleep by increasing daytime and decreasing nighttime percent sleep (A), without altering total percent of time spent in sleep (B). ALAN increased total sleep during the active phase on the third night of the study (C), and significantly reduced the diurnal wake ratio on Days 2 (p < 0.005) and 3 (p < 0.05) of the observation period (D). The period of the sleep rhythm was increased (E) and phase of the percent sleep distribution was delayed by ALAN exposure (F), while the amplitude remained unaltered (G). ALAN exposure alters the bout length pattern (H), but did not alter the bout length distribution by phase of activity (I). All data are presented as mean ± SEM, n = 8/group. # p<0.05 for light; & p<0.05 for time-of-day; $ p<0.05 for phase; @ p<0.05 for interaction of time-of-day and light treatment. Mean of group inside bar in E, F. Sleep data were collected during week 25 of the experimental timeline.

Table 1.

Circadian parameters.

	Group	Period (±SEM)	Phase (±SEM)	Amplitude (±SEM)	N rhythmic	N arrhythmic
Heat	LD	23.11 ± 0.33	18.01 ± 0.43*	0.052 ± 0.004	5	3
	ALAN	24.17 ± 0.40	9.43 ± 2.36*	0.043 ± 0.006	7	1
RER	LD	24.08 ± 0.32	15.82 ± 0.72	0.054 ± 0.004*	7	1
	ALAN	24.20 ± 0.62	13.64 ± 2.63	0.038 ± 0.005*	2	6
Sleep	LD	23.94 ± 0.19*	4.62 ± 0.40*	17.27 ± 1.88	4	4
	ALAN	25.05 ± 0.26*	13.46 ± 2.49*	14.18 ± 2.06	4	4

Similar to the changes observed in sleep percent distribution (Figure 7A), time-of-day independently altered sleep bout length (F_{(7.794, 104.1)} = 3.035; p = 0.0045), and interacted with light to alter sleep bout length (F_{(36, 481)} = 2.695; p < 0.0001) (Figure 7H). Specifically, across the observation period ALAN increased sleep bout length early in the active (dark) phase, and had variable effects on bout length during the inactive (light) phase (Figure 7H). When analyzed across the observation period by phase and day (Figure 7I), instead of in 2 h bins, sleep bout length varied by phase (F_{(2.755, 36.50)} = 3.442; p = 0.0297), but was not affected by ALAN exposure ( F_{(1, 14)} = 1.15; p = 0.302).

4. Discussion

4.1. These data provide evidence that long term exposure to ALAN in female mice alters food intake and sleep patterns, and dampens the daily rhythms in metabolism and rest-activity states, without affecting body composition. However, the alterations in daily heat rhythms observed may suggest sex differences in metabolic responses to ALAN exposure buffer against body mass gain in female mice. Male mice housed in LD conditions typically consume ~30% of their calories during the daytime and ~70% during the nighttime [26], whereas exposure to 5 lux of ALAN shifts the timing of the majority of food intake to the light phase [20, 55, 73], in common with restricting food availability to the light phase [54], resulting in elevated body mass [20, 26, 54, 55]. In the present study, we observed no changes in body mass (Figure 2A, B) or body composition in females with exposure to ALAN (Figure 3), despite a similar shift of the timing of food consumption to the inactive phase (Figure 2). Moreover, unlike its effects reported in males during 8 weeks of exposure [73], 5 lux of ALAN for >20 weeks decreased total daily caloric intake in females (Figure 2). To our knowledge, this is the first study to report elevated inactive phase food consumption with decreased total food intake and no differential effects on body mass, in female mice. A recent study in adult female Wistar rats exposed to 5 weeks of ALAN at 5–7 lux, similarly reported no effects on body mass, although they did not report increased daytime (inactive phase) feeding [74]. Together, these results may suggest that the effects of ALAN on body mass, caloric intake, and feeding patterns in adult females may differ from adult male rodents. However, factors in addition to biological sex might also influence the responses to ALAN, such as developmental age at the onset of ALAN exposure. For instance, adolescent male, but not age-matched female mice, exposed to 5 lux of ALAN for 6 weeks starting at 5 weeks of age increased body mass and daytime food intake [55], suggesting a role of age in modulating the effects of ALAN. Alternatively, restricting total caloric intake, even when calories are consumed during the inactive phase, may limit, in females, the typically-observed body mass gain in ALAN-housed males. This hypothesis is in line with data reporting the benefits of time-restricted feeding. Specifically, restricting food availability to the inactive phase, with [75] or without [76] a reduction in daily caloric availability (relative to controls), or administering a high fat diet restricted to a limited time window within the inactive phase [77], limits body mass gain in males. Furthermore, caloric restriction, irrespective of the time-of-day of food availability, reduced whole body fat mass relative to ad libitum-fed counterparts [75]. The absence of increased fat mass in females exposed to ALAN, and the decreased daily RER, may indicate that the voluntary, temporally-constrained, caloric reduction by ALAN females in our study, may mediate the observed results. Future studies specifically examining the effects of ALAN on time- and calorie-restricted feeding in female mice, should explore this possibility.

As previously stated in section 1, ALAN increases body mass without altering total 24 h locomotor activity in males after 1 week of exposure and persisting for at least 8 weeks [26, 78], whereas it did not in females (current study). Thus, we next sought to determine if ALAN altered locomotor patterns in females contributed to the observed results. Although we observed a decrease in the amplitude and an advance in phase of activity rhythms in ALAN-exposed females (Figure 4C), total daily locomotor activity was unaltered by ALAN. This suggests that the lack of body mass difference induced by ALAN exposure was not mediated by altered locomotor activity, but perhaps blunted activity rhythms combined with sustained activity (Figure 4D) led to an increase in post-exercise energy expenditure and improved metabolic processing of fuels [79].

We next assessed metabolism to determine whether ALAN-induced alterations offset the predicted weight gain in female mice. In two well-established models of disrupted circadian rhythms, sleep restriction [80] and ALAN exposure [74], reduced food consumption is associated with changes in metabolism irrespective of body mass alterations. In the present study, chronic ALAN increased basal oxygen utilization (Figure 6G, H), dampened the rhythm in energy expenditure (heat; Figure 5A), and overall heat generation was increased relative to dark-night conspecifics (Figure 5B). Furthermore, ALAN decreased RER (Figure 6). RER values within the 0.8 – 0.9 range (as observed in ALAN females), indicate that between 32 – 68% of the energetic source was coming from (stored) lipids, whereas LD mice presented with values between 0.95 – 1.1, which indicate that <15% of their energy was derived from lipids, while >85% of it came from dietary carbohydrates. As a consequence, LD mice likely were not oxidizing as much fat as ALAN mice. Taken together, it is likely that increased fat oxidation in ALAN and decreased fat oxidation in LD contributed to offset the predicted ALAN-induced weight gain seen in males under similar conditions. It may also be the case that, in contrast to ALAN, dark night housed females elevated de novo lipid synthesis following their "typical" feeding time. Although typical RER values range between 0.7 and 1.0, we observed a rise in values above 1.0 during the active (primary feeding) phase in LD mice. Indeed, a diet high in carbohydrates may increase post-meal lipid synthesis and drive RER values above 1.0 [81]; this phenomenon has been observed in rodents [82, 83]. Hence, elevation in the synthesis and storage of dietary lipids in LD females, may also have offset the predicted effects on mass accumulation as observed in ALAN males. Nonetheless, the molecular and physiological mechanisms underlying this effect remain to be determined.

Sleep is one of the most important physiological processes; allowing for rest and recovery, and supporting growth and development [84], in addition to allowing the brain to clear itself from toxins and metabolic waste products [85]. Furthermore, its physiological importance is evidenced by its persistence across virtually all organisms [86] and events that disrupt the daily rhythms in sleep, may have pathological downstream effects. Sleep patterns for female LD mice were consistent with previous reports analyzing sleep in various strains of mice, and in which female Swiss Webster mice were reported to sleep ~60% of the light phase, and ~20% of the dark phase [87]. Although ALAN-housed females slept for a similar total amount of time across the day as their LD counterparts, the distribution of time slept during light and dark phases was altered, so that ALAN mice slept similar amounts of time in each phase (Figure 7). ALAN altered sleep patterns (Table 1) but did not affect total sleep bout length in female mice, which is consistent with a previous study conducted in male Swiss Webster mice [88]. That study further examined sleep architecture using EEG and EMG and established that ALAN (at 5 lux) did not affect slow-wave sleep or rapid eye movement (REM) sleep, and the time or quality of sleep, were similarly unaffected. Rats exposed to ALAN dampen the amplitude of REM and non-REM sleep, but in common with our study, do not decrease total 24 h sleep time [62]. Whether this represents a disruption of sleep, or a simple shift in sleep patterns is equivocal from our current data as ALAN did not alter the number of mice displaying rhythmic sleep patterns (Table 1). Differences among these studies, in addition to sex, could be due to species or strain differences [89], or the length and intensity of ALAN exposure prior to sleep testing, or variation in light intensities during the light cycle. That our sleep data in females differ from some studies in males is not unexpected. Various studies have reported differences in sleep patterns by sex (both humans and rodents) across the sleep stages (reviewed in [90, 91]), some of which are linked to ovarian hormones [92, 93]. In humans, sex differences are often documented in self-reported [94, 95], and objective sleep measures [96–98]. Moreover, these differences extend to health outcomes when sleep is impaired [99], underscoring the importance of considering sex as an important variable in these studies. Because sleep and circadian timing are interconnected, the timing of sleep onset relative to the circadian cycle, is also an important factor to consider. For instance, despite similar timing of sleep onset and total duration, women have a shorter circadian period [100] resulting in earlier timing in the rhythms of melatonin and temperature relative to men [101]. This apparent disconnect between the behavioral and circadian timing of sleep may account for the self-reported perception of less restorative sleep, typically reported by women [94, 95], but remains to be further explored. Furthermore, the increasing evidence associating sleep disturbances with night shift work (and consequently, ALAN) [102, 103], and the elevated association among biological sex, sleep disturbances, and negative outcomes [104–107], suggest more attention should be devoted to the study of the interplay among these factors. Thus, to increase rigor, future studies should consider time-of-day and sex as biological variables, and light intensities both day and night, in the examination of the effects of ALAN on metabolism and sleep [108].

4.2. Limitations of the study.

One of the limitations of this study is that it was only performed in females. Although effects of ALAN on altered rhythmicity, sleep, and physiological outcomes have been reported for exposure periods ranging from 1 night to 1 year, a direct comparison of males and females longitudinally and concurrently in the same experiment would help to resolve any issues associated with the differences in experimental design among ALAN studies. Additionally, although our sampling was longitudinal, it was not continuous. Future studies can expand on the differences we describe in this study and focus on more detailed sampling of single measures with concurrent biochemical analyses across time. This study also bridged developmental windows from late adolescence into adulthood, future studies could more specifically address the effects of ALAN in specific developmental stages. Lastly, it is possible that relocation from home cages to semi-novel assay cages (e.g. CLAMS and PiezoSleep) without habituation periods longer than 24 hours could affect some measures. However, we did not note any evidence of chronic or acute stress in this study.

Highlights.

• 20 weeks dim ALAN exposure dampened locomotor rhythms, altered heat generation, respiratory exchange ratio (RER), and sleep in female mice.

• ALAN exposure decreased daily caloric intake, without altering body composition

• ALAN increased period and phase of the rhythm of sleep, elevating the time spent sleeping during the active phase, without altering total sleep bout length.

Data Availability Statement

All data will be made available upon reasonable request.