How Long-Term Light Schedule Changes in Adolescence Affect Body Clocks, Brain Activity, and Behavior in Mice

Female and male mice used in this study were on a C57BL/6 J genetic background, except for GAD67- GFP mice that were on CD1 background. The Gad1-tm1.1Tama (GAD67-GFP knock-in) mouse line was provided by Y. Yanagawa (Gunma University Graduate School of Medicine, Japan). Mice were heterozygous for insertion of the gene encoding GFP under the control of the GAD67 gene promoter. They were used to label the inhibitory GABAergic neurons by enhancing the GFP signal with an anti-GFP antibody. Mice were bred and reared in an on-site animal facility at UofSC under controlled environmental conditions and maintained in standard group housing cages with ad libitum access to normal chow and water. Light intensity was approximately 600 lux at the top of the cage, and the ambient temperature was 23° ± 2°C. The time referred to as “lights on” is defined as ZT0. We minimized the number of animals used in each experiment (sample size based on power analysis) and the animals’ pain and distress. Mouse studies were conducted in accordance with regulations of the Institutional Animal Care and Use Committee at UofSC.

Adolescent mice (post-natal day 30) were transferred into light-tight circadian cabinets (Actimetrics, IL, United States) and subjected to one of the following lighting conditions for a period of 4 weeks: (1) standard 12:12 h light–dark cycles (12 L:12D) (light at ∼600 lux) (Control group); (2) 19 h of light and 5 h of darkness for 5 days and 12 L:12D for 2 days per week (LCD group) (Figure 1). LED light (~600 lux) is turned on during the night phase of the animals’ cycle, resulting in increased duration of light exposure for 5 days of a 7-day cycle. After 4 weeks of either LCD or control conditions, mice were placed in 12 L:12D for 5 days and tested for memory and avoidance behaviors and then sacrificed. Three cohorts of mice were used: one for assessing wheel-running activity, one for light pulse experiment and one for novel object recognition and active avoidance tests.

Mice (post-natal day 20) were individually housed in running wheel–equipped cages, placed in light-proof circadian cabinets under controlled light intensity (∼600 lux) whose timing was controlled by an external timer. After a 10-day acclimatization, adolescent mice (post-natal day 30) were exposed to 4 weeks of either 12 L:12D (control) or LCD (19 L:5D;12 L:12D). Locomotor activity was monitored by continuously recording wheel revolutions and data was collected and analyzed using ClockLab software version 6 (Actimetrics, United States). The last 3 weeks of the experiment were analyzed for each condition. We calculated circadian activity variables including total wheel-running activity, daily rhythm amplitude, rhythm period, and wheel running activity onset. The total activity was calculated as the average number of wheel revolutions per 24 h over 3 weeks. Total and weekly period and total amplitude were calculated using a chi-square periodogram, activity was obtained with the daily countings and expressed as total activity and weekdays/weekend activity, and onset was also calculated for weekdays and weekends. Non-parametric circadian rhythm analysis were also performed including interdaily stability, which quantifies the invariability between the days, which gives an indication of the rhythm fragmentation, and relative amplitude, which quantifies the robustness of the rhythm (Van Someren et al., 1999). In addition, a detailed analysis of activity bouts was also done, with the term bout defined as a sustained period of activity. Specifically, we calculated the total number of activity bouts, the number of activity bouts per day, the average length of the activity bouts and the average peak rate of activity (defined as the maximum number of counts per minute).

After 4 weeks of either LCD or control conditions, mice were placed in 12 L:12D for 5 days and tested on the novel object recognition task. The novel object recognition task is divided in 3 days: On day 1, mice were placed in an open arena (40 cm × 40 cm × 40 cm) and were allowed to explore for 5 min before being returned to their home cage. On day 2, mice were placed in the same open arena with two identical quadratic yellow blocks placed on the diagonal at an equal distance from the walls and were allowed to freely explore the objects for 10 min. Mice were then returned to their home cage. On day 3, mice were placed in the arena with the familiar object and a novel object which consisted of a quadratic red block and were allowed to explore both objects for 10 min. At the end of each test, the arena and objects were sanitized with 70% v/v ethanol. The habituation and the test days were conducted at ZT9-11. Mice were recorded using Ethovision software, and the time exploring the familiar and the novel object was calculated as the percentage of time spent with the novel versus the familiar object. We also analyzed the discrimination index (DI), calculated as the time spent exploring the novel object minus the time spent exploring the familiar object divided by total exploration time. A value below zero describes subjects exploring the familiar object more than the novel object. A value above zero describes animals exploring the novel object more than the familiar object.

After 4 weeks of either LCD or control conditions, mice were placed in 12 L:12D for 5 days and transferred to a shuttle box (San Diego Instruments, San Diego, CA, United States) at ZT9-11. Mice received 30 electric foot shocks through the grid floor. During each test shock (0.10 mA, maximum duration 30 s) the gate between the two compartments remained open, and mice had a chance to escape the shock by crossing to the adjacent compartment. The schedule in trials #1–5 was fixed ratio (FR)-1 (crossing the gate once in order to escape the shock). In the remaining trials #6–30, the schedule was changed to FR-2 (crossing the gate twice in order to escape the shock). The shock was omitted when the animals crossed the gate the appropriate number of times. The number of escape failures and the escape latency were used as a measure of the avoidance responses, as previously described (López-Moraga et al., 2022). Only the C57BL/6 J mice were used for this test since the foot shock intensity protocol is based on C57BL/6 J sensitivity (Landgraf et al., 2015).

After 4 weeks of either LCD or control conditions, mice were placed in 12 L:12D for 5 days and then sacrificed at four different ZTs: (in reference to the daily light cycle): ZT2, ZT8, ZT16, and ZT22. Mice were deeply anesthetized with isoflurane and transcardially perfused with 1% phosphate-buffered saline (PBS) solution immediately followed by 4% paraformaldehyde solution in PBS (pH = 7.4) for tissue fixation. Then, the brains were collected, post-fixed in 4% PFA for 24 h, cryoprotected in 30% sucrose solution, and stored at 4°C until use. Frozen brains were sectioned (30 μm) with a standard Leica microtome (SM2010R) and stored in cryoprotectant solution at −20°C until use. Six to seven coronal sections from each brain encompassing the SCN, MeA, and hippocampus were processed for immunohistochemistry (IHC) analysis. Immunofluorescent labeling was performed in free-floating slices that were first blocked for 1 h in 0.1 M phosphate buffer (PB) containing 3% normal horse serum (NHS) and then permeabilized in 0.2% Triton X-100. Slices were then incubated with antibodies: rabbit anti-SST (1:1000; PA5-85759, Invitrogen, United States), goat anti–c-FOS (1:500; SC-52-G Santa Cruz, USA) in 1% PBS, 3% NHS, and 0.2% Triton X-100 for 24 or 48 h at 4°C with constant shaking. After three washes in PBS, slices were incubated for 1 h at room temperature with fluorescent-tagged secondary antibodies, which were all used at 1:1,000 dilution. (1:100; AlexaFluor anti-rabbit 647 nm, anti-goat pig 555 nm). Brain sections were washed, mounted in gelatin on glass slides, counter-stained, and cover-slipped with Dapi Fluoromount-G™ (Electron Microscopy Sciences, United States). Images were taken with a Leica TCS SP8 multiphoton confocal microscopy system (Leica Microsystems, Germany) and LASX software (Leica Microsystems, Germany). Neuronal quantifications were performed blind with ImageJ software. Quantification data were plotted either as the average or as the total number of positive neurons per nucleus per animal.

For each mouse, two to three slices encompassing the SCN, MeA and hippocampus were processed for RNAscope in situ hybridization [Multiplex Fluorescent Reagent Kit version 2, Advanced Cell Diagnostics (ACD)] following the manufacturer’s instructions for detection of VGAT (ACD, catalog #319191), VGLUT2 (ACD, catalog #319171), SST (ACD, catalog #404631), Per1 (ACD, catalog #438751), Clock (ACD, catalog #492401) and c-FOS (ACD, catalog #316921) mRNA at 4 different ZTs: ZT2, ZT8, ZT16, and ZT22. Sections were washed in PBS, treated with hydrogen peroxide and mounted in glass slides with 1% PBS. Later, brain sections were incubated in the retrieval buffer and dehydrated in ethanol, previously to a protease treatment. Then, sections were incubated with the specific probes for 2 h at 40°C, and the amplification steps were followed as indicated by the manufacturers. Finally, sections were counter-stained, and cover-slipped with Dapi Fluoromount-G™ (Electron Microscopy Sciences, United States). Images were taken with a Leica TCS SP8 multiphoton confocal microscopy system (Leica Microsystems, Germany) and LASX software (Leica Microsystems, Germany). Quantifications were performed blind with Fiji ImageJ software. Semiquantitative histological scoring methodology was used to quantify clock genes Period1 (Per1) and Clock. A score of 0 means no staining or less than 1 dot for every neuron, whereas a score of 4 means greater than 15 dots per neuron/cell or > 10% dots in clusters.

Light pulse experiments were performed as previously described (Fernandez et al., 2018). Adolescent mice housed under 12 L:12D conditions were kept in constant darkness (DD) for 24 h to avoid any light exposure effect. Mice were exposed to a 30 min, 600 lux light pulse light at the beginning of the subjective night (CT14) and then transferred back in DD for an additional 45 min. Mice were then perfused with 4% paraformaldehyde, and brains were collected as described in the Immunohistochemistry paragraph. Control mice were kept in DD and sacrificed at the same CT.

Investigators who participated in end point analyses were blinded to the light protocol. Statistical analysis was carried out using GraphPad Prism (GraphPad Software, La Jolla, CA, United States). For rhythmicity analysis, data were fitted to a sinusoidal curve using the nonlinear least-squares regression comprising both sine and cosine waveforms (constrained period of 24 h). The coefficient of determination (R²) was used as a proxy of goodness-of-fit. Additionally, the empirical p value was calculated using CircWave v.1.4. Outliers were identified and removed by the ROUT method provided by Graph Pad Prism Software, and normality was assessed (Shapiro–Wilk, Kolmogorov–Smirnov, D’Agostino and Pearson, and Anderson–Darling tests) before performing the corresponding statistical analyses. For normally distributed data, a parametric test was used one-way or two-way analysis of variance (ANOVA) or Student’s t-test and p-value less than 0.05 was considered significant, and was indicated as followed: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The statistical tests performed for each experiment are indicated in the figure legends.

We first evaluated the effect of LCD exposure on the circadian locomotor activity rhythms using voluntary wheel-running activity (Figure 2). Running wheel activity of singly housed mice was recorded in control and LCD condition for 4 weeks, as shown in Figures 2A,B. Wheel-running analysis revealed that control and LCD mice exhibited similar period (h) (Figure 2C), amplitude (Figure 2D), relative amplitude (Figure 2E) and total activity (Figure 2F). Analysis also revealed similar activity onsets and total activity within the weekdays and weekends (Figures 2G,H respectively). We further analyzed the total activity from ZT17 to ZT24 in LCD mice (Figure 2I) and found no significant differences when comparing the effect of 7 h of light exposure on weekdays versus the dark phase during the weekends. The activity bouts were analyzed to evaluate the fragmentation of the activity during the day. No significant differences were found in the total number of bouts (Figure 2J), bouts per day (Figure 2K), average bout length (Figure 2L) and average peak rate (Figure 2M) between light conditions. Finally, non-parametric analysis showed no significant differences in the intradaily variability (Figure 2N) and interdaily stability (Figure 2O) between control and LCD mice. Altogether, these results indicate that exposure to LCD has no effect on the circadian wheel-running activity in adolescent mice.

Exhaustive research previously showed that altered light environments disrupt the molecular circadian clock in the SCN along with the circadian locomotor activity (Shuboni and Yan, 2010; Fonken et al., 2013; Ikeno and Yan, 2016). Although our wheel-running activity analysis did not reveal any effect of the LCD on the circadian locomotor rhythms, we evaluated whether LCD exposure altered the expression patterns of Clock and Per1 genes in the SCN. Thus, adolescent mice exposed to either control or LCD for 4 weeks were sacrificed at 4 different ZTs: ZT2, ZT8, ZT16, and ZT22. Brain sections encompassing the SCN were then assessed for Clock and Per1 expression using in situ hybridization (ISH) RNAscope. Both Clock and Per1 showed significant rhythmicity in control and LCD mice (Supplementary Figures S1A,B). We found significantly increased expression of Clock gene at ZT2 and ZT16 in LCD mice compared to control (Figures 3A,B), with no change in the daily expression patterns. No differences were observed in Per1 expression across the 24 h cycle (Figures 3C,D). To determine the effect of LCD on neuronal activity rhythm in the SCN, c-FOS, a proxy for neuronal activity, was quantified at ZT2, ZT8, ZT16, and ZT22. We found that c-FOS expression shows significant rhythmicity in control and LCD mice in the SCN (Supplementary Figure S1C). Interestingly, LCD mice show a significant reduction in the number of c-FOS positive cells during the light phase at ZT2, and ZT8 (Figures 3E,F). Together, these results revealed that LCD exposure during adolescence does not alter circadian clock expression and neuronal activity rhythms in the SCN.

Given recent evidence showing impaired memory and cognition in rodents after exposure to altered light environments (Liu et al., 2022; Sangma and Trivedi, 2023), we evaluated whether exposure to LCD alters long-term memory using the NOR task (Lueptow, 2017) (Figure 4A). We found that mice exposed to the LCD showed decreased time exploring the novel object (Figure 4B) and increased time exploring the familiar object (Figure 4C) compared to the control group. Less exploration of the novel object and more exploration of the familiar object led to a significantly lower discrimination index for the LCD mice in comparison to the control group (Figure 4D). Altogether, these results show that exposure to LCD during adolescence impairs object recognition long-term memory.

The dentate gyrus (DG) of the hippocampus regulates object recognition in rodents (Jessberger et al., 2009; Dees and Kesner, 2013; Jiang et al., 2023) and exposure to altered light environments induces memory impairment in rats (Sangma and Trivedi, 2023). Furthermore, the DG displays clock genes rhythmicity regulating memory (Jilg et al., 2010; Gonzalez et al., 2023). Hence, we analyzed Clock and Per1 expression patterns and neuronal activity rhythms using c-FOS in the DG at ZT2, ZT78, ZT16, and ZT22. No differences were found in both Clock and Per1 daily expression patterns and rhythmicity (Figures 5A–D; Supplementary Figures S1D,E). However, we found that LCD mice showed a significant increase in Per1 expression at ZT8 compared to control (Figures 5C,D). Immunofluorescence analysis of c-FOS expression in the DG revealed no significant rhythmicity in the neuronal activity in LCD or in control mice (Supplementary Figure S1F). Nevertheless, we observed a phase advance in neuronal activity rhythm as shown by increased c-FOS expression at ZT8 in LCD mice compared to control mice (Figures 5E,F). These data show that despite small alterations in clock genes’ expression patterns, LCD altered the neuronal activity rhythm in the granule layer of the DG.

Previous investigations showed altered affective behaviors following aberrant light exposure (LeGates et al., 2012; Fernandez et al., 2018). Here, we studied the effect of exposure to LCD during adolescence on avoidance behavior using an active avoidance test (Figure 6A). We found that LCD mice showed a significant increase in the latency (Figure 6B) and in the number of failures (Figure 6C) to escape the foot shock compared to control mice. These data suggest that LCD exposure during adolescence increases avoidance responses, such as freezing behavior in response to an aversive stimulus.

Although the MeA receives direct light inputs (Hattar et al., 2006) and is involved in the regulation of avoidance behaviors (Miller et al., 2019), no study has been conducted to unveil alterations in the MeA induced by altered light environments. Therefore, we first assessed the effect of LCD exposure on the MeA molecular clock by quantifying Clock and Per1 mRNA expression at ZT2, ZT8, ZT16, and ZT22 using RNAscope. Both Clock and Per1 exhibit significant rhythmicity in the MeA of control and LCD mice (Supplementary Figures S1G,H). However, we found that LCD significantly altered Clock and Per1 expression in the MeA, as shown by their antiphase expression patterns compared to control mice (Figures 7B,D). Specifically, LCD induced a significant reduction in Clock expression during the dark phase at ZT16 and ZT22 (Figures 7A,B) and a significant increase of Per1 expression at ZT16 compared to control (Figures 7C,D). To test the effect of LCD on neuronal activity rhythms in the MeA, we analyzed c-FOS expression at 4 ZT times. We found that control and LCD mice show significant neuronal activity rhythmicity (Supplementary Figure S1I), and that LCD induced a phase advance in neuronal activity rhythm compared to control (Figures 7E,F). Thus, while control mice display a peak of c-FOS expression at ZT16, mice exposed to LCD show a peak of c-FOS at ZT8 followed by a reduction at ZT16 (Figures 7E,F). Altogether, these data show that LCD exposure during adolescence disrupts the MeA molecular clock and the neuronal activity rhythm.

To confirm photoresponsiveness of MeA neurons during adolescence, adolescent mice housed in 12 L:12D condition were placed in DD for 24 h and then exposed to a single 30-min light pulse at the beginning of their subjective dark phase (Circadian time CT14) and then sacrificed. MeA photoresponsiveness was evaluated by analyzing Per1 and c-FOS expression using RNAscope and immunofluorescence. We found that a 30-min light pulse significantly increased Per1 and c-FOS expression in the MeA (Figures 8A–D). Since the MeA is composed of heterogeneous neuronal subpopulations (Wu et al., 2017), we specifically analyzed the acute effect of light at night in glutamatergic and GABAergic neurons. We found that a 30-min light pulse did not affect Per1 and c-fos expression in glutamatergic (Figures 8E–H) nor in GABAergic populations in the MeA (Figures 8I–L). Considering the role of somatostatin (SST) in the amygdala in the circadian modulation of anxiety (Albrecht et al., 2013), we assessed Per1 and c-FOS expression in SST neurons in the MeA. We found that a 30-min light pulse at night induced a significant increase of Per1 and c-FOS expression in the SST neuronal subpopulation in the MeA (Figures 8M–P) compared to control mice. Our analysis revealed for the first time that selective subtypes of MeA neurons are affected by acute exposure to light at night.

Given that SST in the amygdala plays a crucial role in mediating avoidance behavior (Yu et al., 2016) we hypothesized that LCD influences clock gene expression and neuronal activity rhythms in SST neurons, consequently impacting associated behaviors. Thus, we investigated the effect of LCD exposure on Clock and Per1 expressions at ZT2, ZT8, ZT16, and ZT22 in SST neurons. We found that LCD exposure disrupted Clock rhythmicity (Supplementary Figure S1J) and increased the overall daily Clock expression with a significant increase at ZT2 compared to control mice (Figures 9A,B). Mice exposed to LCD displayed anti-phasic Per1 expression patterns compared to control mice (Figures 9C,D), showing significant differences at ZT2 with no changes in rhythmicity (Supplementary Figure S1K). We further evaluated SST neuronal activity rhythm by measuring c-FOS expression by immunofluorescence. We found that control and LCD mice show significant SST neuronal activity rhythmicity (Supplementary Figure S1L) and that LCD induced a phase advance in SST neuronal activity rhythm compared to control, showing a peak of c-FOS at ZT8 and a significant reduction at ZT16, while control mice showed a peak at ZT16 (Figures 9E,F). Considering the role of SST expression in the amygdala on avoidance behavior and in modulation of anxiety (Albrecht et al., 2013; Ahrens et al., 2018; Sun et al., 2020), we evaluated the effect of LCD exposure on SST expression in the MeA by RNAscope and immunofluorescence at ZT2, ZT8, ZT16, and ZT22. We found no difference in the SST expression patterns neither at the mRNA nor at the protein levels between control and LCD mice (Figures 10A–C). Overall, these data suggest that LCD disrupts molecular clock and neuronal activity patterns in the SST neurons in the MeA while not affecting daily SST expression.

The data presented in the study are deposited in the Mendeley repository doi: 10.17632/s4gxwzhmfk.1↗.