We previously used a chronic UVB exposure mouse model by shaving the dorsal hair and exposing male mice to either mock-irradiation (control) or UVB at a dose of 50 mJ/cm² daily for 10 weeks (Parikh et al., 2022). This dose is approximately equal to 25–30 min of midday ambient solar exposure in Florida on a sunny day for a person with the Fitzpatrick phototype 2–3 (D’Orazio et al., 2006; Fell et al., 2014). This dose did not cause any major skin damage as shown previously (Parikh et al., 2022). Feeding behavior is a reflection of underlying physiological and molecular processes and responses to the environment (Parikh et al., 2022), stress (Francois et al., 2022), disease (Adebakin et al., 2012), transgene expression (Newmyer et al., 2019), or drug or hormone treatment (Ghule et al., 2020; Yoon et al., 2020). To investigate feeding behavior following UVB exposure, by feeding the animals with sucrose pellets, we perform an initial calibrating experiment which demonstrate a significant higher food consumption was in the UVB-treatment group compared to the mock irradiated (control group) (Supplementary Figures S1A–C) as per the statistical t-test p value 0.0003, t-statistics (t = 3.696, df = 142), and F-statistics (F = 1.218, DFn = 71, DFd = 71). These results indicate that sucrose pellets consumption is selectively increased due to UVB exposure.

Food intake increases can be associated with homeostatic or hedonic behavior. Hedonic consumption is motivation rather than metabolism dependent (Noye Tuplin et al., 2019). To further determine whether the increase in appetite (for reward food) resulting from UVB treatment was a homeostatic or a hedonic behavior, we used the CPP test, which has been used to measure the motivational effects of objects or experiences (Tzschentke, 2007). Our experiment included control and UVB-exposed mice. Mice were habituated for 1 week, were mock-irradiated or UVB irradiated daily for 5 weeks, and were then subjected to the CPP regime for 10 days (Figure 1A). For the CPP, mice were habituated on Day 1, on Days 2–9, mice were exposed on alternate days to food (sucrose pellets) or no reward food zones, and Day 10 was the test day. We found no difference in the preference ratio for a particular side of the arena during pre-conditioning versus post-conditioning between control and UVB-treated mice (Figure 1B) as per the Two-way ANOVA (p value <0.0001), and F-statistics [F (DFn, DFd) = F (1, 86) = 21.95]. We did find, however, that UVB-treated mice consumed significantly more food during the conditioned stage of the experiment than did control mice (Figure 1C) as per the statistical t-test p value 0.0006, t-statistics (t = 3.510, df = 178), and F-statistics (F = 1.304, DFn = 91, DFd = 87), which is in line with our previous study (Parikh et al., 2022). No differences in distance traveled or total activity were observed between groups (Figures 1D,E), but Two-way ANOVA revealed that the conditioned place preference factor without the UVB factor was significant as for the distance traveled [p value <0.0001, and F-statistics (F (DFn, DFd) = F (1, 86) = 18.29)] and the total activity [p value 0.0055, and F-statistics (F (DFn, DFd) = F (1, 86) = 8.109)] (Supplementary Table S1), which ruled out the possibility that higher motor activity increased feeding behavior. Taken together, our CPP data indicate that chronic daily UVB exposure does not induce activity-related effects on food consumption but rather increases food-seeking behavior in mice.

To test whether the UVB-induced behavior changes depend on p53 activation in keratinocytes, we generated a conditional p53 knockout mouse model (Parikh et al., 2021) in which p53 deletion induced specifically in keratinocytes (p53^flox/floxK14-Cre^+/+); wild-type littermates (p53^flox/floxK14-Cre^−/−) were used as controls. These mice were exposure to UVB and subjected to the staircase test and open-field tests (Figure 2A). In the staircase test we found significantly greater food pellets consumption [p value = 0.0172, t-statistics (t = 2.624, df = 18), and F-statistics (F = 1.063, DFn = 9, DFd = 9)] with significantly less attempts of pellets uptake in the UVB treated wild-type mice (Figure 2B). Although less attempts were made by the wild-type mice, these were successful attempts, suggesting that mice spent more time in procuring the food. No significant increase in food consumption following UVB was observed in the p53 cKO animals as shown in Figure 2B [p value = 0.0766, t-statistics t = 1.878, df = 18), and F-statistics (F = 2.419, DFn = 9, DFd = 9)]. Next, to understand the food behavior in a challenging setting (bright light and open space) we implied the use of the open-field assay as previously done by us (Parikh et al., 2022). Only wild-type mice showed significant induction in the food consumption upon UVB treatment [p value 0.0414, t-statistics (t = 2.617, df = 22), and F-statistics (F = 2.351, DFn = 11, DFd = 11)] and not the cKO UVB treated mice (p value 0.3726, t-statistics (t = 0.9120, df = 20), and F-statistics (F = 1.264, DFn = 10, DFd = 10)). Our data strongly suggest that total activity [p value = 0.0416, and F-statistics (F (DFn, DFd) = F (1, 43) = 4.414)], time spent in center [p value = 0.0119, and F-statistics (F (DFn, DFd) = F (1, 43) = 6.906)], and velocity [p value = 0.0102, and F-statistics (F (DFn, DFd) = F (1, 36) = 7.345)] was significant in UVB mice and the velocity is significantly reduced in the p53-cKO animals UVB animals compared to mock (p value = 0.049) (Supplementary Table S2), indicating that there might be a modest decrease in the velocity of UVB-treated animals in open-field test when the p53 is knocked-out in keratinocytes. This compliments the food pellets consumed data, that despite of spending more time in the center zone where food is kept, the p53-cKO UVB treated mice eat less (Figure 2C). Additionally, the time spent in the peripheral areas of the open-field (non-food zone) where the UVB factor is significant [p value = 0.0016, and F-statistics (F (DFn, DFd) = F (1, 36) = 11.61] and the UVB wild-type mice spent significantly less time in periphery compared to mock wild-type mice (p value = 0.0026), suggesting that they spent more time in the center and ate more food pellets (Figure 2C). The detailed Two-way ANONA analysis with t-statistics, F-statistics, and Tukey’s multiple comparison tests appears in Supplementary Table S2. Overall, our data shows the complex yet strong interplay of the feeding and anxiety behaviors considering the UVB and the p53-cKO as two different variables. Both behavioral assays show the same trend of significant but modest effects of the UVB in wild-type mice and the effect is abrogated in the cKO UVB treated mice.

To further explore the effects of loss of p53 in keratinocytes, we subjected the mice to the open-field test without food and measured total distance traveled in the arena and total activity (Figure 3A). No significant differences between wild-type and mice lacking p53 in keratinocytes were detected (Figure 3B) as per the statistical t-test analysis for the distance traveled (p value 0.3646, t-statistics (t = 0.9278, df = 20), and F-statistics (F = 1.094, DFn = 10, DFd = 10)) and the total activity (p value 0.811, t-statistics (t = 0.2423, df = 20), and F-statistics (F = 1.919, DFn = 10, DFd = 10)), time spent in the center (p value = 0.8025, t-statistics (t = 0.2534, df = 20), and F-statistics (F = 1.130, DFn = 10, DFd = 10)), and velocity (p value = 0.3636, t-statistics (t = 0.9297, df = 20), and F-statistics (F = 1.077, DFn = 10, DFd = 10)). Since skin β-endorphin influences anxiety (Fell et al., 2014) and p53 knockout results in elevated anxiety- and depression-like behaviors (Ruan et al., 2015), we hypothesized that lack of p53 in the keratinocytes might increase anxiety-like behavior. To test this, we subjected the p53-knockout and wild-type mice to the elevated-plus maze test (Parikh et al., 2022) as shown in Figure 3A. We evaluated the number of times/visits (frequency) to the closed and open arms and total activity. A per the statistical t-test mice that lack p53 entered the closed arms of the maze more frequently than did wild-type mice (p value 0.0177, t-statistics (t = 2.585, df = 20), and F-statistics (F = 2.046, DFn = 10, DFd = 10)), although frequency of visits to open arms (p value 0.7756, t-statistics (t = 0.2890, df = 20), and F-statistics (F = 1.961, DFn = 10, DFd = 10)) and total activity (p value 0.0658, t-statistics (t = 1.946, df = 20), and F-statistics (F = 2.422, DFn = 10, DFd = 10)) did not differ (Figure 3C). Additionally, there was no significant differences in both velocity (p value = 0.1433, t-statistics (t = 1.524, df = 20), and F-statistics (F = 1.490, DFn = 10, DFd = 10)) or center time spent (p value = 0.6405, t-statistics (t = 0.4742, df = 20), and F-statistics (F = 1.619, DFn = 10, DFd = 10)) in the EPM as per the t-test. We added a sentence in the result section as shown in new Figure C and the details of the statistics are in Supplementary Table S3. This suggests that the anti-anxiety effect of skin β-endorphin is compromised when p53 is not expressed in keratinocytes.

All animal experiments were approved by the Tel Aviv University Institutional Animal Care and Use Committee (IACUC) (01–19-003). Mice were housed under a standard 12-h light/dark cycle (lights on at 20:00 pm) at constant temperature (24 ± 1°C) and humidity (50 ± 5%) with ad libitum access to food and water at all times except as mentioned. C57BL/6 J (age: 6–8 weeks old) male mice (Envigo) were used for the experiments. For generation and validation of knockout of the conditional knockout of p53 in keratinocytes, we used the same strategy as we previously reported (Parikh et al., 2021). In brief, the p53-floxed mice (a gift from Professor Eli Pikarsky, The Hebrew University of Jerusalem, Israel) were bred with mice in which the K14 promoter directs expression of Cre recombinase (a gift from Professor Ittai Ben-Porath, The Hebrew University. of Jerusalem, Israel).

Mice were shaved on the dorsal side in an area of approximately ~60% of the skin using a shaver and remaining hair were removed using depilatory cream (Veet). Mice were exposed individually to 50 mJ/cm² UVB using the standard light setting with a XX-15 stand equipped with 15-W, 302-nm UVB bulbs (Ultraviolet Products) at approximately the same time in the morning daily for 5 weeks. For the mock-irradiated control treatment, the mice underwent the same handling procedure without UVB exposure. The container was cleaned using Virusolve (Amity International) to avoid cross-contamination of odors between mice.

Mice were subjected to 1 week of habituation followed by 5 weeks of daily mock irradiation or UVB irradiation and weekly testing of food consumption. In each weekly test, the mice were food-deprived for 3 h in a home cage-like setting with one mouse per cage followed by ad libitum access to standard chow (Altromin, Germany, #1318 M) for 1 h and then to peanut-flavored reward food (TestDiet, Richmond, IN, #1811369) for 30 min. The mice were weighed before and after each weekly test. After the test was completed, the mice were housed in their home cages with ad libitum food and water. The chow was weighed before and after a mouse was placed in a cage, and chow consumed was calculated. Chow was placed in the cap of the 50 mL tube that was taped inside the cage so that the mouse could not spill the food.

The staircase test was performed as described previously (Parikh et al., 2022) except that mice were starved for 22 h prior to testing. For food deprivation, animals were fed at noon on non-testing days with water ad libitum. At the outset of testing, the mice were habituated with the sucrose pellets and then to the test boxes by placing food along the surface of the staircase steps for a 10 min session a day prior to the experimental day, to rule out the effects of neophobia. On the test day the mice were placed in the staircase chamber one mouse at a time. A mirror was used to check the pellets consumed from both the sides. The duration of the test was of 10 min, and pellets eaten, and attempts were manually scored for each animal using media recorder (Noldus Information Technology). Between trials, the staircase chamber was cleaned with Virusolve® + (Amity International).

The open-field was performed as described previously (Parikh et al., 2021) except that mice were starved for 22 h prior to testing. Time spent in the center, total distance traveled in the arena, velocity, time spent in the peripheral region (non-food zone) and activity levels were measured by video tracking using EthoVision. Between each trial, the open-field arena and the tube cap were cleaned with Virusolve (Amity International). The test session videos were scored and analyzed with EthoVision-XT software (Noldus Information Technology).

The CPP test was performed with the UVB or control-treated mice when they were under the 22-h food deprivation. The CPP conditioning chambers were translucent acrylic boxes (33 × 18 × 16 cm³) with removable clear plastic tops and retractable doors. The arena had one center zone and two side zones; the first with the reward food which was the non-textured zone, and the second without food, which was the black and white textured zone. Both the zones had a cap of a 50-mL tube which was or was not filled with the food. The experimental room was lit by normal white light (300 Lux), and behavior-related parameters were recorded by video for 15 min from the top and analyzed using EthoVision-XT software (Noldus Information Technology). Between each trial, the arena was cleaned with Virusolve (Amity International). Pellets consumed, distance traveled, and total activity were measured.

The CPP test was performed in three phases: (1) habituation (Day 0), (2) training (Days 1–8), and (3) test (Day 9). On day 0, no food was placed in either chamber. The mouse was placed in the center of the arena, the doors were retracted, and the mouse was allowed to move freely in the arena for 15 min.

During training, reward food pellets (~ 100 pellets per session) were introduced in zone 1 (the light zone), and there were no pellets in the textured zone 2 (the dark zone). Each mouse was placed with access to the reward food zone 1 (light zone) or in textured zone 2 (dark zone) on alternate days. The doors were closed to restrict access only to the zone in which the mouse was placed. The number of pellets consumed during the 15 min in which the mouse was in the zone were calculated.

On Day 9, mice were introduced into the CPP arena with doors retracted to allow free movement in the arena. No pellets were supplied. Mice were allowed to move freely for 15 min, and the duration of time spent in each zone was measured.

The test was performed using the setup described previously (Parikh et al., 2021). In short, the test was performed using EPM maze with a height of 90 cm lit by normal white light (300 Lux) in the experiment room. The maze included two opposing closed arms (arm lengths 40 cm, arm widths 5 cm, wall height 15 cm), and an open 5 × 5 cm square in the center. The experimental animals were placed in the center facing an open arm, center time spent, and velocity behavior-related parameters were measured for 5 min using the camera from the top and analyzed using EthoVision-XT software (Noldus Information Technology). Between each trial, the maze was cleaned with Virusolve® + (Amity International).

All the experimental data are shown as means and standard errors of the mean. We used a random experimental design, Student’s t-tests (two-tailed) for two-group comparisons, and ANOVAs for multiple group comparisons followed by Tukey’s multi-comparison tests. At relevant places the t-statistics (with df) and F-statistics (F, DFn, DFd) is mentioned in the text but all the related statistical details appear in Supplementary Tables S1–S3.