Constant Light in Early Life Induces Depressive-Like Behavior in Chickens Via Modulation of Hippocampal BDNF/ERK Pathway

Light management plays an important role in broiler growth and behavior. Constant light in early post hatch stage has been a common practice for improving feed intake and body weight gain in broiler chickens, while whether and how constant light in early life affects the behavior in broiler chickens is rarely reported. Results In this study, newly hatched chickens were kept in either constant light (24L:0D, LL) or natural photoperiod (12L:12D, LD) for 7 days and maintained in constant light thereafter from 8 d to 21 d of age. Constant light did not affect chicken body weight, while increased average daily feed intake (ADFI) in 7 d and 21 d and every week feed conversion ratio (FCR). Constant light exposure in early life induces depressive-like behaviors, which was associated with higher corticosterone (CORT), lower melatonin and 5-hydroxytryptamine (5-HT) plasma. Concurrently, constant light exposure increased the mRNA expression of clock related genes and suppressed the expression of antioxidative genes in the hippocampus of both 7- and 21-day-old chickens. Moreover, brain derived neurotrophic factor (BDNF)/extracellular signal-regulated kinase (ERK) pathway in hippocampus was suppressed by constant light exposure. These ndings imply that constant light exposure in early life disrupts hippocampal expression of clock genes and BDNF/ERK pathway, which contributes to depressive-like behaviors in the chicken.

Hippocampus is the key structure governing many important functions including learning and memory, as well as depressive and anxiety-like behaviors [7]. Brain derived neurotrophic factor (BDNF) is critical for the hippocampal neurogenesis and synaptic plasticity [8,9] through binding to its receptor tropomyosin receptor kinase B (TrκB) and triggering the activation of phosphatidylinositol 3-kinase (PI3K), and/or extracellular signal-regulated kinase (ERK) pathways [10]. Our previous study found that chronic constant light exposure for 3 weeks reduces hippocampal neurogenesis and impairs cognitive behaviors via suppresses BDNF/TrκB/ERK pathway [11]. The core molecular clock consists of a transcriptional-translational autoregulatory "loop" with a positive arm and a negative arm [12]. The clock and bmal1 genes and their protein products comprise the positive arm, while Per1, Per2, Per3, Cry1, Cry2, RORα, Rev-erbα genes and their protein products comprise the negative arm. Previous studies have con rmed that light exposure can in uence the clock genes in the pineal gland, hypothalamus and retina of chicks [13,14]. However, whether constant light in early life affect hippocampal neurogenesis and clock gene expression remain unclear.
Therefore, the objectives of the present study were, rstly, to elaborate the effects of constant light in early life on depressive-like behavior in chicken; secondly, to delineate the expression of BDNF/TrκB/ERK pathway and clock gene in chicken hippocampus, and to reveal their responses to constant light exposure.

Ethics statement
The experimental protocol was approved by the Animal Ethics Committee of Nanjing Agricultural University. The project number is 31972638. The sampling procedures according to the "Guidelines on Ethical Treatment of Experimental Animals' ' (2006)  were provided ad libitum. Daily feed consumption was recorded and body weight was recorded every week. By the end of the experiment, the chickens were sacri ced and hippocampus tissue quickly excised, frozen immediately in liquid nitrogen, and stored at -80°C until use.

Open eld test
Chicken was placed in the center of a 58 × 58 cm open eld apparatus with sides 70 cm high. This OF was made of white wood and the oor was marked off into 25 squares of 12 cm × 12 cm each, illuminated by a 100 W overhead bulb [15]. The following types of behaviors were analyzed for 10 min: the time of rst step, the count of grid number, defecation, steps, escapes and tweets. After testing, the oor of the OF apparatus was cleaned with towels wetted with 70% ethanol.

Balance beam test
The test was conducted using an elevated narrow balance beam (6 cm wide and 35 cm long). The balance beam was 22 cm high, so that chicks could jump down onto soft bedding without injuring themselves. The balance beam aimed at representing a novel situation (height and depth) for newly hatched chicks. Thus, we expected using this test to assess fear-related behaviors, particularly fear of heights and the respective avoidance response, similar to tests in rodents [16]. Chicks were tested once only. The test was performed as follows. Each chick was placed on the starting line at an end of the balance beam and then allowed to walk. We recorded the following parameters: (i) the time of the chick stay the beam and (ii) the distance the chick walked on the beam. If a chick stayed on the starting line or was unable to walk on the beam, it received a score of 0. Its time was recorded as 120 s.

Tonic immobility test
Chicken were captured from their home pen and carried to the adjacent room. Hens were placed on their backs in a metal cradle and restrained for 5 s by the experimenter placing one hand on the bird's chest and another over its head with the head hanging down. The single experimenter then removed their hands and stepped aside with eyes averted downwards. The test concluded after 6 min of immobilisation or when the bird righted itself after at least 10 s of immobilisation, whichever occurred rst. Duration of tonic immobility was recorded, and the restraining was repeated up to 5 times if the hen righted itself in less than 10 s. All hens were feather-scored again by the same experimenter following conclusion of the testing. control to normalize the technical variations. Data were analyzed using the method of 2 −ΔΔCT and presented relative to the CON group. All primers (Table 1) were synthesized by Suzhou GENEWIZ Biological Technology Co., Ltd (Suzhou, Jiangsu, China).

Statistical analysis
All data are presented as means ± SEM and the differences among groups were analyzed using T-Test for independent samples with SPSS 20.0 for windows. The differences were considered statistically signi cant when p < 0.05.

Effect of constant light in early life on feed conversion ratio, plasma CORT, melatonin and 5-HT concentration
Constant light did not affect (P > 0.05) chicken body weight from 1 d to 21 d ( Figure 1A), but signi cantly increased (P < 0.05) average daily feed intake (ADFI) in 7 d and 21 d ( Figure 1B). Meanwhile, constant light signi cantly increased (P < 0.05) every week feed conversion ratio (FCR) from rst week to third week ( Figure 1C). In addition, compared with LD group, 7 d and 21 d chickens plasma CORT levels ( Figure   1D) were signi cantly increased (P < 0.05) and plasma melatonin levels ( Figure 1E) were signi cantly decreased (P < 0.05) in LL group. Also, in LL group plasma 5-HT levels were signi cantly decreased (P < 0.05) in 7 d, and trend to decreased (P = 0.06) in 21 d chickens ( Figure 1F).

Effect of constant light in early life on the depression and anxiety-like behaviors in chickens
In open led test, compared with LD group, the time of rst step (Figure 2A) was signi cantly increased (P < 0.01), and the count of grid number ( Figure 2B), defecation ( Figure 2C), steps ( Figure 2D), escape ( Figure 2E) and tweets ( Figure 2F) were signi cantly decreased (P < 0.01) in LL group. In balance beam test, compared with LD group, the time stayed ( Figure 2G) was signi cantly increased (P < 0.01), and distance moved ( Figure 2H) was signi cantly decreased (P < 0.01) in LL group. In tonic immobility test, compared with LD group, the immobility time was signi cantly increased (P < 0.01) in LL group ( Figure  2I). These behavior results indicated that constant light in early life signi cantly increased depression and anxiety-like behaviors in chickens.

Discussion
In this study, we observed that constant light exposure increased ADFI and FCR, but did not affect body weight in 7 d and 21 d chickens. Similar results have been reported by Lewis, who found that compared with photoperiods in 12 h, photoperiods in 24h decreased feeding rate, increased food intake, but there was no signi cantly differences on body weight in 21 d males Cobb 500 and Ross 308 broiler [3].
However, recent study reported that compared with intermittent lighting, constant lighting in 30 lx decreased ADFI and did not affect FCR in Lingnan Yellow broiler chicks from 1-21 d [17]. The reason why these results were different from the present study may be due to the different broiler breeds and light intensity.
In the present study, behavior tests results found that constant light exposure in early life induced anxiety and depressive-like behavior in 7 d chickens. These results consistent with previous study, which found that constant light exposure increased anxiety and depressive-like behavior in mice [18]. Meanwhile, we found that constant light exposure induces higher plasma CORT levels both in 7 d and 21 d chickens.
Higher plasma CORT levels have detrimental physiological and cognitive effects, which were linked with depression-like behavior in weanling mice [19] and adult rats [20] exposed to dim light at night. CORT levels are regarded as an important indicator of depression-like behavior in rodents [21]. Thus, in this study, we speculate that constant light increased anxiety and depressive-like behavior may be directly or indirectly induced by higher plasma CORT levels in chicken.
Melatonin plays a key role in controlling circadian behavioral responses [22]. It also acts as a potent antioxidant by scavenging reactive oxygen species [23]. Previous studies reported that constant light induced depressive-like behavior in rodents is usually associated with low melatonin and high oxidative stress levels [24,25]. Indeed, we detected lower plasma melatonin and 5-HT levels, accompanied by lower hippocampal 5-HT receptor mRNA expression both in 7 d and 21 d of constant exposed chickens. Similar results have been reported by Lauber, who found that compared with photoperiods in 12 h, photoperiods in 23 h signi cantly reduced plasma melatonin in 7-week-old broiler chickens [26]. NF-E2-related factor 2 (Nrf2) and its endogenous inhibitor, Kelch-like ECH-associated protein 1 (Keap1) plays a critical role in counteract oxidative stress [27]. In this study, constant light exposure reduced Nrf2/Keap1 and superoxide dismutase (SOD) mRNA expression, as well as Nrf2 protein expression both in 7 d and 21 d chickens hippocampus. Agree with our present study, it was reported that constant light induced higher oxidative stress in rat hippocampus, cortex and cerebellum [28], and lower Nrf2 expression in rat thymus which could rescued by melatonin [29].
A number of studies indicate that constant light exposure in uences the mRNA expression of clockrelated genes, including Clock, Bmal1, Cry, and Per [30,31], which is associated with impaired hippocampal neurogenesis and depressive-like behavior [32,33] in mice. Similar with previous reported, we also found that constant light exposure signi cantly increased the mRNA expression of clock-related genes Cry1, Cry2 and Rev-erbα both in 7 d and 21 d chickens hippocampus. In mammals, Crys are important clock genes involved in the regulation of circadian rhythm [34]. Besides, Crys serve as photoactive pigments and important circadian photoreceptors for light entrainment of the circadian rhythm [35]. Mice lacking Cry1 and Cry2 genes lose completely the free-running circadian rhythmicity in wheel-running behavior [

Conclusion
In conclusion, our study shows that constant light exposure in early life induces depressive-like behaviors, accompanied by higher plasma CORT, lower plasma melatonin and 5-HT levels in the chicken.  Values are mean ± SEM (n = 40), **p < 0.01, compared with LD.