Effect of lethal yellow ( AY ) mutation and photoperiod alterations on mouse behavior

Decrease in natural illumination in fall/winter months causes depressive-like seasonal affective disorders in vulnerable individuals. Obesity is another risk factor of depression. The lethal yellow (AY ) mutation causes ectopic expression of agouti protein in the brain. Mice heterozygous for AY mutation (AY/a) are obese compared to their wild-type littermates (a/a). The main aims of the study were to investigate the effects of AY mutation, photoperiod and the interaction between these factors on daily activity dynamics, feeding, locomotor and exploratory activities, anxiety-related and depressive-like behaviors in mild stress condition. Six weeks old mouse males of AY/a and a/a lines were divided into four groups eight animals each and exposed to long(14 h light and 10 h darkness) or short(4 h light and 20 h darkness) day conditions for 28 days. Then the behavior of these mice was successively investigated in the home cage, open field, elevated plus-maze and forced swim tests. We did not observed any effect of AY mutation on the general activity, water and food consumption in the home cage; locomotion and exploration in the open field test; anxiety-related behavior in the open field and elevated plus-maze tests. At the same time, AY mutation increased depressive-like immobility time in the forced swim test (F1.28 = 20.03, p = 0.00012). Shortday conditions decreased nocturnal activity in the home cage, as well as locomotion (F1.28 = 16.33, p = 0.0004) and exploration (F1.28 = 16.24, p < 0.0004) in the open field test. Moreover, short-day exposition decreased time spent in the center of the open field (F1.28 = 6.57, p = 0.016) and in the open arms of the elevated plus-maze (F1.28 = 12.08, p = 0.0017) tests and increased immobility time in the forced swim test (F1.28 = 9.95, p = 0.0038). However, no effect of the interaction between AY mutation and photoperiod on immobility time in the forced swim test was observed. Therefore, short-day photoperiod and AY mutation increased depressive-like behavior in the forced swim test by means of different mechanisms.

Decrease in natural illumination in fall/winter months causes depressive-like seasonal affective disorders in vulnerable individuals.Obesity is another risk factor of depression.The lethal yellow (A Y ) mutation causes ectopic expression of agouti protein in the brain.Mice heterozygous for A Y mutation (A Y /a) are obese compared to their wild-type littermates (a/a).The main aims of the study were to investigate the effects of A Y mutation, photoperiod and the interaction between these factors on daily activity dynamics, feeding, locomotor and exploratory activities, anxiety-related and depressive-like behaviors in mild stress condition.Six weeks old mouse males of A Y /a and a/a lines were divided into four groups eight animals each and exposed to long-(14 h light and 10 h darkness) or short-(4 h light and 20 h darkness) day conditions for 28 days.Then the behavior of these mice was successively investigated in the home cage, open field, elevated plus-maze and forced swim tests.We did not observed any effect of A Y mutation on the general activity, water and food consumption in the home cage; locomotion and exploration in the open field test; anxiety-related behavior in the open field and elevated plus-maze tests.At the same time, A Y mutation increased depressive-like immobility time in the forced swim test (F 1.28 = 20.03,p = 0.00012).Shortday conditions decreased nocturnal activity in the home cage, as well as locomotion (F 1.28 = 16.33,p = 0.0004) and exploration (F 1.28 = 16.24,p < 0.0004) in the open field test.Moreover, short-day exposition decreased time spent in the center of the open field (F 1.28 = 6.57, p = 0.016) and in the open arms of the elevated plus-maze (F 1.28 = 12.08, p = 0.0017) tests and increased immobility time in the forced swim test (F 1.28 = 9.95, p = 0.0038).However, no effect of the interaction between A Y mutation and photoperiod on immobility time in the forced swim test was observed.Therefore, short-day photoperiod and A Y mutation increased depressive-like behavior in the forced swim test by means of different mechanisms.Key words: lethal yellow; photoperiod; activity; anxiety; depressive-like behavior; mice.

Introduction
Seasonal alterations of natural illumination in high or moderate latitudes trigger numerous adaptive changes in the nervous system and behavior of all wild animals.Although humans mainly live at constant illumination, its decrease during fall/ winter months can cause seasonal affective disorders (SAD) or subsyndromal SAD characterized by carbohydrate crav ing, overeating, weight gain, decreased libido, hypersomnia and prominent fatigue (Levitan, 2007) in some vulnerable individuals.SAD and subsyndromal SAD are observed in 11-21 % of individuals and are a considerable social and eco nomic problem due to high risk of disability (Miller, 2005).Laboratory mice have been proposed as an animal model of SAD (Otsuka et al., 2014).However, the reported data about effect of photoperiod alteration on mouse behavior are contradictory (Otsuka et al., 2014;Young et al., 2018).
The main aims of the study were the following: to inves tigate the effects of (1) A Y mutation, (2) short/long photope riod and (3) these factors interaction on brain monoamines and behavior.Here we compared: the effect of the long and shortday conditions on the daily activity in the home cage, the locomotor, exploratory activities, anxietyrelated, depressive like behavior in heterozygous A Y /a mice and their wildtype littermates (a/a).

Materials and methods
Animals and experiments.The study was conducted at the Center for Genetic Resources of Laboratory Animals at the Institute of Cytology and Genetics Siberian Branch, Russian Academy of Sciences (Novosibirsk, Russia) using equipment supported by the Russian Ministry of Education and Science (project No. RFMEFI62117X0015).Experiments were carried out on SPFstate mouse males C57BL/6A Y (A Y /a, n = 16) and their wild type littermates C57BL/6 (a/a, n = 16).These mouse genotypes were bred by crossing a/a females and A Y /a males.Such breeding results only in obtaining A Y /a and a/a mice (50 : 50) which have similar genetic background, are born and nurtured by the mothers of the a/a genotype.The genotype of mice is assayed by the fur color which is yellow in A Y /a mice (Boston et al., 1997) and black in a/a mice.At the beginning of the experiment the A Y /a and a/a mice were 6 weeks old, weighed 20.9 ± 0.3 g and 20.0 ± 0.3 g (F 1.30 = 5.03, p = 0.032), respectively.The males of the same genotype (A Y /a or a/a) were kept for 28 days in groups of four in individually ventilated cages (Optimice, Animal Care Systems, Inc.) in rooms with 20fold air exchange at temperature 24 ± 2 °C, humidity 45-50 %, and at long (14 h of light : 10 h of darkness, 14L : 10D, 40 lx) or short (4 h of light : 20 h of darkness, 4L : 20D, 40 lx) photoperiods with daybreak at 01:00 (14L : 10D) or 11:00 (4L : 20D) and sunset at 15:00 (14L : 10D and 4L : 20D).The 14L : 10D photoperiod is the standard photoperiod in our SPFvivarium.Food and litter were autoclaved at 121 °C before use.Animals were pro vided with deionized water (produced in a Millipore device) with Severyanka (Ekoproekt, St. Pe ters burg, Russia) mineral supplement and feed ad libitum.There were four experimental groups of eight animals: (1) a/a kept at 14L : 10D, (2) a/a kept at 4L : 20D, (3) A Y /a kept at 14L : 10D and (4) A Y /a kept at 4L : 20D.Two days before the tests, the animals were isolated in the same cages to reduce group effect.The animals were kept at the same photoperiods during the tests.First, the dynamics of locomotion, sleep, water and food consumption in the home cage was tested using PhenoMaster (TSE, Germany).Then, the mice were tested for three Home cage activity.Daily dynamics of locomotor acti vity, sleep duration, water and food consumption were in vesti gated in PhenoMaster (TSE, Germany) according to the manufac turer's instruction.The device consists of eight individual cages equipped with infrared sensors those tracing the animal movements.Drinking bowls and feeders were also equipped with sensors, allowing the accurate measurement of water and food consumption.The data from the sensors were re corded each minute and processed by the software from the manufacturer.The animals learned to use drinking bowls and feeders for two successive days and then they were isolated in PhenoMaster cages and their locomotion, sleep duration, water and food consumed were recorded for 48 h.The first 24hour period (1-24 h) was considered as an adaptive one and was not taken into account.Therefore, the baseline animal activity was assessed only on the basis of data recorded for the second 24hour period (25-48 h) (Khotskin et al., 2017).According to the manufacturer's instructions, the software determined the state of sleep as lack of mobility for 40 s or more.This sleep estimation correlates with the EEG estimation of the sleep state (Pack et al., 2007;Fisher et al., 2012;Bais et al., 2015).The dynamics of locomotor activity and sleep duration were respectively evaluated by the distance travelled (m) and accumulated sleep time (min) during one hour, while the water and food consumption were measured as water (ml) and food (g) quantity consumed during 24 h.Other behavioral tests were held between 15:00 and 18:00 in the dark time.The EthoStudio software was applied for automatic tracking of mouse behavior in OF, EPM and FS tests (Kulikov et al., 2008(Kulikov et al., , 2010(Kulikov et al., , 2014)).In OF and FS tests we used the transmitted (inverted) light for automatic tracking of mouse behavior when the light was transmitted through the arena to a WEB camera placed at 80 cm above the arena and connected to a computer via a USB 3.0 port.Since a mouse regardless of its color (white, agouti or black) is opaque, it contrasts with background in the transmitted light (Kulikov et al., 2008).
Open field test (OF).The OF test was carried out on a brightly illuminated white plastic arena of 55 cm in diameter with the wall of 30 cm in height.The 25 % zone in the center of the arena was selected as the center.The arena was made of opaque polyvinyl chloride and placed on a semitransparent platform.The arena was brightly illuminated (300 lx) with two halogen lamps (35 W) placed 40 cm below the platform.A mouse was place at the wall of the arena and its movement was automatically tracked for 5 min.The EthoStudio software automatically calculates two behavioral traits: the time spent in the center (%) is calculated as the ratio of mouseassociated pixels in the center to the total number of mouseassociated pixels and the distance travelled during the test (m), while the number of vertical postures were recorded by an experienced rater blind to experiment (Kulikov et al., 2008).The time in the OF center is negatively associated with fear (Carol et al., 2002;Prut, Belzung, 2003), while the numbers of vertical postures are associated with exploration (Crusio, 2001;Alves et al., 2012).The apparatus was cleaned with wet and dry napkins after each test.
Elevated plus-maze test (EPM).The EPM test was car ried out in the apparatus made of gray plastic including two closed and two open arms (30 cm in length × 5 cm in width).The close arms were framed by plastic walls of 30 cm in height.The apparatus was elevated at 60 cm above the floor and dimly illuminated with diffuse light (≈100 lx) of a halo gen lamp (25 W) placed a meter above.Newly developed Microsoft Kinect 1 3-D sensor was applied for the first time for automatic tracking of mouse behavior in the EPM.This sensor was earlier successfully applied to track pig behavior (Kulikov et al., 2014).Microsoft Kinect 1 3D sensor was placed 60 cm above the surface of the EPM and connected to a computer via a USB port.In contrast to standard digital video tracking systems based only on color or brightness of pixels, the present version of the EthoStudio software uses also the depth data provided by the 3D sensor.Depth data contains the distance (m) from each pixel of the animal and arena to the sensor.The height threshold algorithm marks pixels higher or lower the threshold (1.5 cm in height) as associated with animal (1) or background (0), respectively (Kulikov et al., 2014).The main advantage of the 3D sensor over standard digital video is that the former can track animal of any color in open and closed arms of the EPM.
A mouse was placed in the center of the EPM with the head targeted to a closed arm and its movement was auto matically tracked for 5 min.Since the 3D sensor can track animal both in open and closed arms the EthoStudio software can evaluate the following traits: the times (%) spent in the center, closed and open arms based on the ratio of the number of mouseassociated pixels in these parts of the maze to the total number of mouseassociated pixels.The time spent in the center and in the closed arms is, respectively, negatively and positively associated with fear (Carol et al., 2002;Prut, Belzung, 2003).The apparatus was cleaned with wet and dry napkins after each test.
Forced swim test (FS).Mice were placed for 6 min into a clear plastic tank (18 cm in diameter and 30 cm in height) filled with water at temperature 25 °C for 2/3 of the volume (20 cm).The water tank was placed on a semitransparent platform and illuminated (150 lx) with one halogen lamp (35 W) placed 40 cm below the platform (Kulikov et al., 2010).Behavior in the FS test was automatically evaluated for the last four minutes of the test using EthoStudio software by immobility time (s) and rate of alteration of the animal silhouette (%).The last parameter is the ratio of the number of mouseassociated pixels that have changed their position between two adjacent frames to the mean number of mouseassociated pixels in these frames (%).We used the mean rate calculated for the last four minutes of the test as the index of active resistance.The immobility time was calculated as the time (s) when the rate of the animal silhouette alteration was less than 17 %.
Statistics.The data were presented as the mean ± SEM.The mean values were compared using two way ANOVA ("ge notype" and "photoperiod" factors).Daily dynamics of the locomotor activity and sleep in the home cage was com pared using repeated measure ANOVA with "genotype" and "photoperiod" as the between and the "day time" as the within variables.The differences between groups were determined using the Fisher LSD post hoc multiple pairwise comparisons.Statistical significance was set at p < 0.05.

Results
Photoperiodic changes in weight of a/a and A Y /a mice.The effect of the "genotype" factor on weight was observed: at the end of experiment mice of A Y /a genotype (32.4 ± 0.4 g) were heavier than of a/a genotype (26.7 ± 0.4 g; F 1.28 = 110.81,p < 0.0001).At the same time, no effect of the "photoperiod" factor (F 1.28 = 2.03, n. s.) or the "genotype" × "photoperiod" interaction (F 1.28 < 1, n. s.) on animals' weight was found.

Photoperiodic changes in locomotor activity, anxietyrelated and depression-like behavior in a/a and A Y /a mice.
In the OF test we revealed effects of the "photoperiod" factor on the traveled distance (F 1.28 = 16.33,p = 0.0004), time spent in the center (F 1.28 = 6.57, p = 0.016) and the numbers of verti cal postures (F 1.28 = 16.24,p < 0.0004).However, no effect of the "genotype" factor (F 1.28 < 1, n. s.) or the interaction "genotype" × "photoperiod" (F 1.28 < 1, n. s.) on these traits was observed.Shortday conditions decreased horizontal (travel ed distance) (a/a, p = 0.0016; A Y /a, p = 0.035) and vertical (vertical postures) (a/a, p = 0.0053; A Y /a, p = 0.012) activities, while anxietyrelated time in the center was decreased only in a/a mice ( p = 0.035), but not in A Y /a ones ( p = 0.17) (Fig. 2).Blue and red bars under the panel (a) show the dark time for long-and short-day exposition, correspondingly.Since no effect of the "genotype" factor on the locomotor activity and sleep time is observed, the points of the corresponding curves are the means of values for a/a and A Y /a mice exposed to the same photoperiod.**p < 0.01 difference between long-and short-day conditions.In the EPM test we did not observed any effect of the "geno type" factor (F 1.28 < 1, n. s.) and the interaction "genotype" × × "photoperiod" (F 1.28 < 1, n. s.) on time spent in the center, open and closed arms.At the same time, significant effects of the "photoperiod" factor on the time spent in the center (F 1.28 = 27.04,p = 0.000016), open (F 1.28 = 12.08, p = 0.0017) and closed (F 1.28 = 25.02,p = 0.000026) arms were revealed.Mice of both genotypes exposed to shortday spent less time in the center (A Y /a, p = 0.0006; a/a, p = 0.0016) and open arms (A Y /a, p = 0.001; a/a, p = 0.21, n. s.), but spent more time in the closed arms (A Y /a, p = 0.0001; a/a, p = 0.014) of the EPM compared to mice exposed to longday (Fig. 3).
In the FS test the influences of factors "genotype" (rate of silhouette alteration, F 1.28 = 21.54,p = 0.00007; immobility time, F 1.28 = 20.03,p = 0.00012) and "photoperiod" (rate of silhouette alteration, F 1.28 = 13.36,p = 0.0011; immobility time, F 1.28 = 9.95, p = 0.0038), but not the factors' interaction (F 1.28 < 1, n. s. for both traits) on the rate of silhouette altera tion and immobility time were revealed.The rate of silhouette alteration was lower ( p = 0.0005) and immobility time was higher ( p = 0.0004) in A Y /a mice exposed to the longday conditions compared to a/a animals exposed to the same photoperiod (Fig. 4).Short photoperiod decreased the rate of silhouette alteration ( p = 0.003) and increased immobility time ( p = 0.0045) in a/a, but not in A Y /a mice (see Fig. 4).

Discussion
In the present study, new information about the effects of the "genotype" (A Y mutation), "photoperiod" and "genotype" × × "pho toperiod" interaction was obtained.
The A Y mutation results from large deletion in the promo tor of the mouse agouti gene that puts the agouti gene under control of the promotor of an ubiquitously expressed Raly gene (Perry et al., 1994).Although, normally, agouty protein is expressed only in hair follicles, this deletion causes ectopic expression of agouti protein in many tissues including the brain, adipose and other tissues (Boston et al., 1997).Agouti protein inhibits melanocortin4 receptors (Lu et al., 1994) in volved in the regulation of total metabolism, feeding, anxiety and depressivelike behavior (Caruso et al., 2014;Gragnoli, 2014).However, since its creation, A Y /a mice are mainly used to study the effect of obesity and type 2 diabetes on the peripheral control of metabolism, immunity, reproduction etc.This is the first systematic study of the effect of the A Y muta tion on the mouse behavior as well as on the vulnerability to altered photoperiod.
In the present study we did not find any effect of the "genotype" factor (A Y mutation) on the general activity, sleep duration and feeding in the home cage; locomotor and explora tory activity in the OF test, anxietyrelated behavior at mild stress conditions in the OF and EPM tests.At the same time, the A Y mutation dramatically reduced active escape behavior (evaluated by the rate of silhouette alteration) and increased depressivelike immobility in the FS test.There are two pos sible mechanisms of the depressantlike effect of A Y mutation: a direct via blockade of melanocortin mechanism of mood regulation or an indirect via obesity.The first hypothesis does not seem to be true, since melanocortin4 receptor inhibitors show antidepressant effect (Chaki, Okuyama, 2005;Chaki et al., 2005;Chaki, Okubo, 2007).The second hypothesis seems to be more correct, since the leptin receptor deficiency results in obesity and increased depressivelike behavior in the FS test in Lepr db /Lepr db mice (Sharma et al., 2010).
We first showed marked effect of photoperiod on daily dy namics of mouse activity in the home cage.Mice are noc turnal animals and under "normal", longday conditions, they showed "normal" daily dynamics: they were more active in the darkness.At the same time, no such daily dynamics was observed in mice exposed to shortday condition: they did not increase their locomotor activity in the darkness.There are contradictory data about the effect of photoperiod alterations on anxietyrelated and depressivelike behavior.Some authors reported that exposition of C57BL/6 mice for 21 days to short-day condition (8L : 16D) increased anxiety-re lated and depressivelike immobility in the EPM and FS tests, respectively (Otsuka et al., 2014).Other authors reported that exposition of C57BL/6 mice for 14 days to longday con ditions (19L : 5D) increased anxiety-related and depressive-like immo bility in the EPM and FS tests, respectively (Young et al., 2018).
Here we first showed that exposition to short-day conditions reduced locomotion (travelled distance), exploration (numbers of vertical postures), time spent in the center in the OF test, time spent in the center and open arms in the EPM test, as well as increased time spent in the closed arms in the EPM test.These findings can be interpreted as increase of anxiety-related behavior.We also found that the prolonged exposition to short photoperiod increased depressivelike behavior evaluated by decrease in the rate of silhouette alteration and increase in immobility time in the FS test in mice exposed to shortday conditions.Therefore, our results indicate that prolonged exposition to shortday condition increased anxietyrelated and depressivelike behavior in mice.This results and con clusion agree with those of T. Otsuka with coauthors ( 2014), but contradict to those of J.W. Young and coauthors (2018).We think that these discrepancies result from the difference in experimental protocols.We and T. Otsuka with coworkers (2014) used a mild lighting conditions for longday exposi tion 14 h, 40 lx and 16 h, 50 lx, respectively.At the same time, J.W. Young and coauthors (2018) used more intensive lighting, 19 h, 130 lum (≈150 lx) which is stressful for mice and frequently used as a stressor in the chronic unpredict able stress model (Monteiro et al., 2015) and in the OF test.Therefore, the protocol of J.W. Young and coauthors (2018) is a model for study the effect of intensive lighting rather than photoperiod alteration.
A key problem of the present study was to study the effect of genetic obesity on the vulnerability to shortday photoperiod.We did not find any effect of the "genotype" × "photoperiod" interaction on the studied behavioral traits in the home cage, OF, EPM and FS tests.Therefore, genetic obesity cause by A Y mutation does not seem to increase vulnerability to shortday photoperiod.Although exposition to short day conditions and A Y mutation separately increase immobility time in the FS test, they increase this depressivelike behavior by means of different mechanisms.

Conclusions
One practical conclusion can be based on the results.Obese A Y /a and Lepr db /Lepr db mice show similar elevated depres sivelike behavior in the FS test.At the same time, breeding of A Y /a mice is more simple than that of Lepr db /Lepr db mice.Moreover, being a dominant mutation that can be easily de termined by the fur color of its carriers, A Y mutation is a very useful tool for studying the interaction between hereditary obesity and other neurological mutations.

For citation :
Bazhenova E.Y., Fursenko D.V., Khotskin N.V., Sorokin I.E., Kulikov A.V. Effect of lethal yellow (A Y ) mutation and photoperiod alterations on mouse behavior.Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding.2019;23(1):55-61.DOI 10.18699/VJ19.461Влияние мутации lethal yellow (A Y ) и изменений фотопериода на поведение мыши successive days in open field (OF), elevated plusmaze (EPM) and forced swim (FS) tests.The maintenance of mice was supported by the basic re search project No. 032420180016.All procedures comply with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and were approved by the Committee on the Ethics of Animal Experiments of the Russian National Center of Genetic Resources of Laboratory Animals based in SPFvivarium of Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia (protocol No. 32 of March 18, 2016).
Е.Ю.Баженова, Д.В.Фурсенко Н.В.Хоцкин, И.Е.Сорокин, А.В Photoperiodic changes in weight of a/a and A Y /a mice.The effect of the "genotype" factor on weight was observed: at the end of experiment mice of A Y /a genotype (32.4 ± 0.4 g) were heavier than of a/a genotype (26.7 ± 0.4 g; F 1.28 = 110.81,p < 0.0001).At the same time, no effect of the "photoperiod" factor (F 1.28 = 2.03, n. s.) or the "genotype" × "photoperiod" interaction (F 1.28 < 1, n. s.) on animals' weight was found.Photoperiodic changes in home cage behavior in a/a and A Y /a mice.No effect of the factors "genotype" (locomotion, F 1.24 < 1, n. s.; sleep duration, F 1.24 = 1.2, n. s.), "photoperiod" (locomotion, F 1.24 = 2.7, n. s.; sleep duration, F 1.24 < 1, n. s.) and the "genotype" × "photoperiod" interaction (F 1.24 < 1, n. s., for both traits) on locomotion and sleep duration was observ ed.At the same time, the effects of the "day time" factor on locomotion (F 23.552 = 8.05, p < 0.00001) and sleep duration (F 23.552 = 14.83, p < 0.00001) were observed: in mice of both genotypes.The effects of "photoperiod" × "day time" inter action on the daily dynamics of locomotion (F 23.552 = 3.42, p < 0.00001) and sleep duration (F 23.552 = 6.65, p < 0.00001) were also observed.This indicates an influence of photoperiod on the daily dynamics of mouse activity.Mice exposed to longday conditions were more active(199.54± 36.27  m/h in darkness vs 96.69 ± 21.01 m/h in light, p = 0.000018) in the darktime period, while activity mice exposed to shortday conditions did not differ in the dark and lighttime periods (81.98 ± 36.27 m/h in darkness vs 33.89 ± 21.01 m/h in light, n. s.) (Fig.1, a).At the same time, mice exposed to longday (9.05 ± 1.44 min/h in darkness vs 26.20 ± 3.04 min/h in light,

Fig 1 .
Fig 1. Daily dynamics of locomotor activity (distance travelled, m/h) (a), sleep duration (min/h) (b) as well as the daily consumption of water (ml) (c) and food (g) (d ) in the home cage in a/a and A Y /a mice exposed to long-and short-day conditions.

Fig. 2 .
Fig. 2. Travelled distance (m), number of vertical postures and time spent in the center (%) in the OF test in a/a and A Y /a mice exposed to long-and short-day conditions.*p < 0.05; **p < 0.01 vs mice of the same genotype exposed to long-day conditions.a/a a/a a/a A Y /a A Y /a A Y /a

Fig. 3 .
Fig. 3. Time spent (%) in the center, open and closed arms in the EPM test in a/a and A Y /a mice exposed to long-and short-day conditions.*p < 0.05; **p < 0.01; ***p < 0.001 vs mice of the same genotype exposed to long-day conditions.

Fig 4 .
Fig 4. Rate of mouse silhouette alteration (%) and immobility time (s) for the last 4 min in the FS test in a/a and A Y /a mice exposed to long-and short-day conditions.**p< 0.01 vs mice of the same genotype exposed to long-day conditions; ### p < 0.001 vs a/a mice exposed to long-day conditions.