Molecular and Cellular Endocrinology 399 (2015) 110–121

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Hypothalamic gene switches control transitions between seasonal life history states in a night-migratory photoperiodic songbird Gaurav Majumdar a, Sangeeta Rani b, Vinod Kumar a,* a b

DST-IRHPA Center for Excellence in Biological Rhythms Research, Department of Zoology, University of Delhi, Delhi 110 007, India DST-IRHPA Center for Excellence in Biological Rhythms Research, Department of Zoology, University of Lucknow, Lucknow 226 007, India

A R T I C L E

I N F O

Article history: Received 18 June 2014 Received in revised form 15 August 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: Bunting Critical day length Circadian Genes Metabolism Opsins

A B S T R A C T

This study investigated photoperiodic plasticity in hypothalamic expression of genes implicated in the photoperiodic light perception (rhodopsin, melanopsin, neuropsin and peropsin), transduction (pax6, bmal1, clock, per2 and casr), induction (eya3, tshβ, dio2 and dio3, gnrh and gnih) and metabolism (NPY, sirtuin1, foxO1, hmgcr, citrate synthase and dehydrogenases) in photosensitive and photorefractory redheaded buntings. There was a significant increase in eya3, tsh β, dio2, pax6 and rhodopsin and decrease in dio3 mRNA expression at hour 15 and/or 19 on the day photosensitive buntings were subjected to a 13- or 16 h, but not to 8- and 11 h light exposure. Downstream reproductive and metabolic gene expression was not altered, except for an increase in those genes coding for succinate and malate dehydrogenase enzymes involved in lipogenesis. Photorefractory buntings had high dio3 mRNA expression which significantly declined after 1 short day exposure, suggesting possible involvement of dio3 in the maintenance of photorefractoriness. Positive correlation of rhodopsin on eya 3 and tshβ indicates its role in photoperiodic timing, perhaps involving the peropsin and pax6 genes. These results suggest that rapid switching of hypothalamic gene expression underlies photoperiod-induced seasonal plasticity and regulates transitions from photosensitive to photostimulated and from photorefractory to photosensitive states in migratory songbirds. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Many birds inhabiting both high and low latitudes use annual photoperiodic changes in controlling their seasonal events, particularly the processes linked with the reproduction. In long day species, increasing day lengths during the spring and summer periods initiate gonadal maturation, but post-reproduction birds become refractory to long day effects and regress their gonads (Dawson et al., 2001; Kumar, 1997; Kumar et al., 2010). Photorefractory individuals need to pass through shortening autumn and winter day lengths to become re-photosensitive to the stimulatory effects of increasing photoperiods in the forthcoming spring (Dawson et al., 2001; Kumar, 1997; Kumar et al., 2010). Light is perceived in all probability by the encephalic photoreceptors (Oliver and Bayle, 1982), which have been shown to express mRNA and/or protein of multiple photopigment molecules, viz. neuropsin (Nakane et al., 2010), rhodopsin (Wang and Wingfield, 2011), melanopsin (Chaurasia et al., 2005; Kang et al., 2010), and vertebrate ancient opsin, VA-opsin (Halford et al., 2009). The duration of perceived light is interpreted as a long day when the light

* Corresponding author. Department of Zoology, University of Delhi, Delhi 110007, India. Tel.: +911127666423; fax: +911127666564. E-mail address: [email protected]; [email protected] (V. Kumar). http://dx.doi.org/10.1016/j.mce.2014.09.020 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

period extends into the inductive phase of the entrained circadian rhythm of photoinducibility; the converse is true for the interpretation of light period as a short day (Kumar et al., 2010). A neuroendocrine response is triggered with rapid reciprocal switching between types 2 and 3 deiodinase (dio2 and dio3) mRNA expression in the hypothalamus (Yoshimura et al., 2003), with an increase and decrease in dio2 expression with the onsets of photoperiodic induction and regression and photorefractoriness, respectively; the converse is true for dio3 expression (Watanabe et al., 2007; Yasuo et al., 2005; Yoshimura et al., 2003). The deiodinase mRNA expression is further reported to be under the control of ‘gene switches’ in the pars tuberalis, PT. The inductive process is “switched on” as soon as hour 14 on the first long day with an enhanced Tshβ (thyroid stimulating hormone-beta subunit) transcription in the PT thyrotrophs (Nakao et al., 2008). Tshβ coupled with Tshα forms Tsh, which binds to its receptors in ependymal cells lining ventrolateral walls of the third ventricle and initiates dio2 transcription by hour 18 of the first long day (Nakao et al., 2008). Dio2 enzyme mediates the conversion of T4 into T3, and in turn GnRH (gonadotropin releasing hormone) secretion from the preoptic area results in the synthesis and release of pituitary gonadotropins. At hour 14, eye absent 3 (eya3) gene is also activated and acts perhaps upstream in the photoperiodic induction pathway (Majumdar et al., 2014). This molecular cascade seems to be a critical step in the photoperiodic control of seasonal cycles in birds and mammals (Dardente et al.,

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2010; Dupre et al., 2010; Masumoto et al., 2010; Nakao et al., 2008; Surbhi and Kumar, 2014). However, a study that compared molecular response with a single long day between Swedish and German populations of great tit (Parus major) found a changed mRNA expression of genes coding for deiodinase enzymes in the hypothalamus of Swedish population only, although photoperiod induced gonadotropin secretions occurred with a temporal difference in both the populations (Perfito et al., 2012). Also, Dio2, not Dio3 expression varied seasonally but was correlated neither with the seasonal photoperiodic changes nor with the GnRH levels or testis size in European starlings, Sturnus vulgaris (Bentley et al., 2013). Thus, the concept of deiodinase based mechanism for seasonal timing might not be as universally applicable as was originally envisaged. There could be real differences in the photoperiodic timing between species, or differences in results are the reflection of the experimental paradigm used to examine changes in the hypothalamic gene expression. It could also be that photoperiodic plasticity in hypothalamic gene expression, which is reported to begin with the changes in Tshβ, in fact begins with changes in the transcription of genes upstream, e.g. in the photoperiodic perception and/or transduction pathways. If yes, there would be concomitant changes in the mRNA expression of genes involved in the photoperiodic perception, transduction and induction pathways. This could be addressed by the measurement of expression pattern of both upstream and downstream genes along with deiodinase mRNA expression in more bird species, possibly inhabiting different kinds of photoperiodic environment during its annual life history, and subjected to a lighting protocol that is in some way different from the ones that have been previously used. Therefore, the goal of this study was to examine hypothalamic gene expression in long distance migratory redheaded bunting (Emberiza bruniceps), which in its travel across latitudes is exposed to consistent changes in the environmental photoperiod twice-a-year. Also, buntings rapidly respond to photostimulation as shown by increased mRNA and protein levels of genes implicated in photoperiodic induction in the first hour of day following 16 h of light exposure (Majumdar et al., 2014). Here, we exposed photosensitive buntings to a single day of photoperiods around the critical day length (CD, minimum daily light period that will induce a response in half of the test population of a species; Dunn and Sharp, 1990), and similarly photorefractory buntings to acute short days. We first measured the expression of genes that are implicated in the photoperiodic induction (eya3, tshβ, dio2, dio3, gnrh, gnih). Then, as a measure of upstream molecular response, we measured mRNA expression of genes involved in the photoperiodic light perception (rhodopsin, melanopsin, neuropsin, peropsin and rgropsin) and activation of tshβ (clock, bmal1 and per2; Unfried et al., 2009) and eya3 (pax6; Xu et al., 1997), and calcium homesostasis (e.g. gene encoding for calcium sensing receptor, casr). Finally, downstream molecular response was examined by changes in the mRNA levels of genes involved in the energy homeostasis and metabolism (glucose metabolism: neuropeptide Y, sirtuin 1, foxO1; lipid and cholesterol metabolism: hmgcr) and Kreb’s (TCA) cycle (citrate synthase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase). We expected a strong positive correlation of photoperiod length on the expression pattern of genes implicated in the photoperiodic light perception and induction in photosensitive birds. Similarly, a negative correlation was expected of photoperiod length on genes linked with regression and reproductive inactivity. Further, it was expected that photorefractory birds would show a decreased mRNA expression of genes implicated in the maintenance of gonadal regression and/ or reproductively inactive state on acute exposure to short days. In addition, there would be a parallel change in mRNA levels of genes regulating downstream seasonal physiology unless these genes were activated by a cumulative effect of one or several upstream genes over a period of photoperiodic exposure.

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2. Materials and methods 2.1. Animals and maintenance Redheaded bunting is a Palearctic-Indian latitudinal migrant songbird, which migrates in between its breeding grounds in west Asia and south-east Europe (~40°N) and overwintering grounds in India (Ali and Ripley, 1974). It is a photoperiodic species and exhibits differences in photoperiod induced cycles in fat deposition and weight gain, and gonadal maturation during long-term exposure to long day lengths (Rani et al., 2005). The present study was carried out at the University of Lucknow, Lucknow, India, as per guidelines of the Institutional Animal Ethics Committee, using male buntings that were procured from their overwintering flocks around in the late February. These birds were first acclimated to natural light and temperature conditions (NDL) for a week in an outdoor aviary (size = 3 × 2.5 × 2.5 m) and then placed indoors in a photoperiodic room (size = 2.2 × 1.8 × 2.8 m) providing programmed photoperiods and constant temperature conditions (22 ± 2 °C) until used in the experiment. 2.2. Experiment Two experiments were performed, one each with photosensitive (experiment 1) and photorefractory (experiment 2) male buntings, as per the experimental design shown in Fig. 1. 2.2.1. Experiment 1: exposure of photosensitive birds to single increasing light periods This experiment was carried out on photosensitive birds that were maintained on short days (8 h light: 16 h darkness, 8L:16D), in which buntings do not fatten (body mass = 22–24 g) and maintain small reproductively immature testes. These are called photosensitive birds, for they remain responsive to the stimulatory effects of long day

Fig. 1. Experimental design. Experiment 1: Photosensitive (Pse) redheaded buntings maintained on short days (8 h light: 16 h darkness; 8L:16D) were exposed to a single 11- 13- and 16 h light or retained on 8 h light (controls). Experiment 2: Photorefractory (LDref) buntings maintained on long days (16L:8D) were exposed to one (SD1) or seven (SD7) cycles of short days or retained in16L:8D. They (n = 4) were sampled on ZT 15 and/ or 19 for the measurement of a number of genes linked with the regulation of reproduction and metabolism (for details see Figs 2–5).

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lengths (Rani et al., 2005). We used a first day exposure experimental paradigm, which was a slightly modified form of the first day release (FDR) paradigm that has been used for photoperiod induced gene expression studies in other birds (Nakao et al., 2008; Stevenson and Ball, 2012; Visser et al., 2010). In the FDR, observations are usually made during the extended light hours on the first day. In the present study, however, we made observations on the first day of light exposure, as in the FDR, but after a period of darkness following the light exposure. This protocol, unlike the FDR, tested the effects of different photoperiod lengths simultaneously at the same time points, by restricting light period with interposed periods of darkness (see below). Short day treated photosensitive birds were singly housed in cages and placed in isolated photoperiodic chambers providing 8 h light:16 h darkness (8L:16D; L = 250 ± 5.0 lux, D = 0 lux). On day 6, birds were randomly distributed in eight groups (N = 4 each) with two groups exposed each to a single 11-, 13- or 16 h light; remaining two groups received 8 h light as on the previous day and served as controls. The same day, one group of birds from each light condition were sacrificed in between zeitgeber times (ZT0 = lights on) 14.5 and 15.5 (hour 15 group), while the second group in between ZT 18.5 and 19.5 (hour 19 group). Thus, sampling times fell after a period of 1–11 h of darkness, depending on the light exposure periods (for example, hour 19 was 11 h apart in birds that received 8 h light, and only 3 h in birds that received 16 h light), except hour 15 group programmed to receive a 16 h light period but instead received light only until the time when it was sampled in between ZT14.5 and 15.5 for mRNA expression studies. These sampling times were chosen in view of earlier findings showing induction of genes implicated in the photoperiodic induction in photosensitive quails on the first day of light exposure in two ‘waveforms’, one around ZT14 (eya3 and tshβ) and the other around ZT18 (dio2 and dio3) (see Nakao et al., 2008). 2.2.2. Experiment 2: exposure of photorefractory birds to acute short days Experiment 2 addressed molecular mechanism possibly involved in the termination of photorefractoriness. It examined mRNA expression during the first week of exposure to short days in buntings that were exposed for many weeks to a long photoperiod (16L:8D) and constant temperature conditions (22 ± 2 °C) in which they had undergone growth-regression cycles in fat deposition, body weight and testes. At this time, birds with no fat deposit, lean body mass (22–24 g) and regressed testes were photorefractory (LDref), since they stopped responding to the stimulatory effects of long day lengths (Rani et al., 2005). As in the experiment 1, birds were singly housed in cages and placed in isolated photoperiodic chambers providing 16 h light: 8 h darkness (16L:8D; L = 250 ± 5.0 lux, D = 0 lux). On day 6, buntings were distributed in three groups (N = 4 each) and exposed to 8L:16D for one (SD1) or seven cycles (SD7) of 8L:16D, or maintained on 16L (control; LDref). On the following day, with light schedule as on the previous day, birds in all three groups were sacrificed in the dark (or light, LDref group) in between ZT 14.5 and 15.5 (hour 15). 2.3. Sampling and tissue preparation Birds were sacrificed by decapitation and kept on ice. Brains were quickly removed and stored at 4 °C in the RNA later (Ambion Inc., Cat no. AM7020). Later, the hypothalamus was excised out from each brain by first slicing the brain with a surgical blade so that a thick coronal section trimmed dorsally (starting approximately at TrSm) and laterally extends from the optic chiasma to infudibular area. It was homogenized in TRIreagent (Ambion Inc., Cat no. AM9738) by bead beater, and the total RNA was extracted by chloroform precipitation and quantified, as per the manufacturer’s protocol

(Majumdar et al., 2014; Singh et al., 2013). One microgram RNA was DNase treated (Ambion Inc., Cat no. AM1906) to remove genomic DNA, if any, and single strand cDNA was prepared by MAXIMA single strand cDNA prep kit (Fermentas, Cat no. K1641). 2.4. Partial cloning and sequencing of genes We cloned portions of genes examined in this study from bunting brain cDNA using degenerate primers obtained from the conserved region of a gene sequence for other birds available in the NCBI database. RT-PCR reactions were standardized for each gene on a gradient PCR using varying primer concentration, if required, to obtain a specific band size. The band was excised out from 1% agarose gel, eluted with Qiagen gel extraction kit and the product was ligated to the pGEMT cloning vector (Promega pGEM-T Easy Vector System 1; cat no. A1360). The plasmids after transformation were confirmed by digestion, and were commercially sequenced by Eurofins Ltd. MWG, Bangalore, India. The obtained portion of a gene sequence was subjected to nBLAST (NCBI database) to confirm the identity of a gene, and submitted to NCBI gene bank with their accession numbers given in the Supplementary Table S1. 2.5. Measurement of gene expression levels (quantitative PCR, qPCR) Gene specific primers were designed using PRIMER quest (http:// www.idtdna.com/Primerquest) software (Supplementary Table S1), with stringent conditions of temperature: 60 °C, amplicon length: 75–95 bp, G + C: 50%. qPCR was performed by the LightCycler® 480 SYBR Green I Master (Roche Diagnostics, Indianapolis, IN, USA). The PCR reaction for each gene was standardized with primer concentration and total reaction volume to achieve a standard slope of −3.2 to −3.4. Melting curve analysis confirmed the primer quality. Each sample was run in duplicate along with negative RT and nontemplate controls in each plate. A 15 μl reaction volume consisted of 1 μl of cDNA (10 ng/μl), 1 μl each of forward and reverse gene specific primer (500–1000 nM), 7.5 μl of SYBR Green I master(1×) and 4.5 μl nuclease free water. β-actin was used as a reference (internal control) gene (accession no. KC874663). A fold change in the relative mRNA expression level of a gene was calculated using the formula 2−(ΔΔCt) (Livak and Schmittgen, 2001), as detailed in recent publications from our laboratory (Majumdar et al., 2014; Singh et al., 2013). Briefly, the fluorescence exceeding background levels gave the cycle threshold (Ct), which was used to calculate ΔCt(Ct[target gene] – Ct[reference gene]). Then, these Ct values were normalized against Ct value of a sample (calibrator) consisting of cDNA mix of all the samples of a group. Finally, the negative value of this powered to 2 (2−ΔΔct) was plotted. Additionally, for analyzing relative expression pattern of a gene in all the life history states, the Ct value of a pool sample from all individuals including short day photosensitive (Pse) single day photostimulated (Pst), long day refractory (LDref), 7 short day treated and photorefractory (SDref) was used as a calibrator for normalizing ΔCt values (i.e. values normalized with reference to beta-actin). 2−ΔΔct values of genes thus obtained for hour 15 group for all the treatment conditions in both the experiments were plotted to give a comparative pattern of gene expression in buntings (Fig. 7). 2.6. Statistics All statistical analyses were done by GraphPad prism software program version 5.0 (San Diego, CA, USA), unless otherwise stated (see below). One way analysis of variance (one way ANOVA) with Newman–Keuls post hoc test was used to compare mRNA expression between groups of an experiment. Unpaired Student’s t-test was used to compare values representing two time points or two groups at one time point. We determined correlation

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coefficient (r) and coefficient of determination (i.e. how close the data fit a regression line; r2) for mRNA levels of genes involved in the photoperiodic induction (eya3 and tshβ) and perception (opsin genes) from all photoperiodic conditions of the experiment 1 at hour 15. The correlation between genes was tested for significance, and p values were obtained for each correlation set. Then, p values from all correlations were subjected to Benjamini–Hochberg false discovery rate (FDR; http://www.sdmproject.com/utilities/?show = FDR) correction for multiple comparisons to give a corrected p value (= q value). Also, a non-linear regression analysis with least square fit Gaussian curve [explained by Y = Amplitude*exp (−0.5*((X-Mean)/ SD)∧2)] demonstrated gene expression trends for all the opsin genes and genes implicated in the photoperiodic induction (eya3, tshβ, dio2 and dio3). This curve gave us the timing of peak expression of a gene by defining x–y coordinates in 1000 points. This was further subjected to Kruksal–Wallis test to show a significance in difference between groups, and gave corrected p value to the Gaussian approximation. Significance was considered at p < 0.05. 3. Results 3.1. Experiment 1: exposure of photosensitive birds to single increasing light periods (i) Genes involved in photoperiodic induction (eya3, tshβ, dio2, dio3, gnrh and gnih) Eya 3 was expressed with significant differences between photoperiodic exposures both at hour 15 (F3,12 = 9.708, p = 0.0016; Fig. 2a) and hour 19 (F3,12 = 15.28, p = 0.0002; one way ANOVA) in photosensitive birds (Fig. 2b). At both times, eya3 mRNA levels were significantly higher in 13- and 16 h than in the 8- and 11 h light exposures (p < 0.05, Newman–Keul post hoc test). Also, mRNA expression at hour 19 was significantly higher in 16 h than in the 13 h light exposure (p < 0.05, Newman–Keul post hoc test; Fig. 2b). A similar significant difference occurred in tshβ mRNA expression between photoperiodic exposures at both times of day, the hour 15 (F3,12 = 7.640, p = 0.0041, Fig. 2c) and hour 19 (F3,12 = 7.166, p = 0.0052, one way ANOVA, Fig. 2d). Tshβ mRNA levels were significantly higher in groups received that 13- and 16 h light, as compared with those that received a shorter 8- or 11 h light (p < 0.05, Newman–Keul post hoc test), except that the mRNA levels did not differ between 8- and 13 h light group, perhaps due to the smaller sample size (n = 4) and relatively a larger individual variation, particularly in the 8 h group. There was a further increase in tshβ expression at hour 19 in birds that received 16 h light, and hence tshβ mRNA levels were significantly higher in 16 h than in the 13 h light treatment group (p < 0.05 Newman–Keul post hoc test). However, dio2 and dio3 mRNA expression was significantly increased and decreased, respectively, at hour 19, not hour 15, in both 13- and 16 h, as compared with the 8- and 11 h light treatment groups (p < 0.05, Newman–Keul post hoc test; Fig. 2e–h). There appears to be no effect of a single long light exposure on gnrh expression (Fig. 2i, j). Also, gnih mRNA expression occurred at high Ct values (>34 cycles) with very low levels, and so it was excluded from further analysis and presentation.

Fig. 2. Mean (±SE; n = 4) relative mRNA expression levels (2−ΔΔct value) of genes involved in photoperiodic induction of gonadal development in the hypothalamus of redheaded bunting. Photosensitive birds maintained on 8 h light per day (8L:16D) were exposed to a single 11-, 13- and 16 h light, with 8 h light as controls, and sacrificed same day in between zeitgeber times (ZT) 14.5 and 15.5 (hour 15) or 18.5 and 19.5 (hour 19; ZT 0 = lights on). Thus, the measurement of gene expression (i.e. sampling time) was done after an intervening darkness of varying durations. Note that hour 15 group that was programmed to receive 16 h of light instead received light until birds were sampled in between ZT14.5 and 15.5. Transcript levels were measured by the real time PCR for eya3 (a, b), tshβ(c, d), dio2 (e, f), dio3 (g, h), gnrh (i, j) genes in both samples (left panel, hour 15; right panel, hour 19). An asterisk on bars indicates significant difference between groups, as determined by Newman– Keul post hoc test. Significance was considered at p < 0.05.

(ii) Genes involved in photoperiodic light perception (rhodopsin, melanopsin, neuropsin, peropsin and rgropsin) Figure 3 shows expression patterns of four opsin genes in the bunting hypothalamus. Rgropsin was expressed at high Ct values (>35 cycles) with very low levels, and so it was excluded from further analysis and presentation. At hour 15, there was a significant difference between light treatment groups in the expression of rhodopsin (F3,12 = 21.86, p < 0.0001; Fig. 3a) and peropsin (F3,12 = 3.917,

p = 0.0366; one way ANOVA, Fig. 3d), but not the neuropsin (F3,12 = 0.8761, p = 0.4806; one way ANOVA, Fig. 3c). Buntings exposed to a single 13- and ~15 h (16 h group) light had significantly increased rhodopsin mRNA than those exposed to 8- and 11 h light (p < 0.05, Newman–Keul post hoc test; Fig. 3a). Also, rhodopsin mRNA levels at hour 15 were significantly higher in 16 h than in the 13 h light group (p < 0.05, Newman–Keul post hoc test; Fig. 3a).

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Newman–Keul post hoc test; Fig. 3h). With a large individual variation in expression casr levels were not different between light treatment groups (data not shown). (iv) Genes encoding enzymes involved in energy homeostasis and metabolism (NPY, sirtuin1, foxO1, hmgcr, cs, ogdh, sdhd, mdh)

Fig. 3. Mean (±SE; n = 4) relative mRNA expression levels (2−ΔΔct value) of genes involved in the photoperiodic light perception (left panel – opsin genes: rhodopsin, a; melanopsin, b; neuropsin, c; peropsin, d) and transduction of photoperiodic information (right panel – probable upstream genes; clock, e; bmal1, f; per2, g; pax6, h) in the hypothalamus of redheaded bunting (Emberiza bruniceps) at hour 15. The other details are as in Fig. 1.

Selective group comparisons showed an increased rhodopsin expression in 11 h than in the 8 h light treatment group (p = 0.0412; Student’s t-test). Similarly, peropsin mRNA levels were significantly higher in 16 h than in the 8- and 11 h treatment groups (p < 0.05; Newman–Keul post hoc test; Fig. 3d). Melanopsin expression also appeared to be significantly different between four photoperiodic exposures (F3,12 = 3.314, p = 0.0571; Fig. 3b) with a significantly higher mRNA expression in 16 h than in the 8 h light exposure (p < 0.05, Newman–Keul post hoc test; Fig. 3c). At hour 19, however, the expression levels of all the opsin genes were not different between light treatment groups, and so data are not shown. (iii) Upstream genes (clock, bmal1, per2, pax6, casr) There were no difference in the expression levels of probable upstream circadian genes at hour 15 between photoperiodic exposures (clock: F3,12 = 2.950, p = 0.0757, Fig. 3e; bmal1: F3,12 = 1.450, p = 0.2772, Fig. 3f; per2: F3,12 = 2.293, p = 0.1301; one way ANOVA, Fig. 3g). Pax6 however had photoperiod-dependent expression (F3,12 = 6.055, p = 0.0094; one way ANOVA) with a significantly higher mRNA levels in 16 h than in the 8- or 11 h treatment groups (p < 0.05,

There were variable effects of single light exposure of varying durations on the expression of genes that encode for key enzymes involved in the energy homeostasis and metabolism (Fig. 4). At hour 15, there were no significant effect of a single long photoperiods on mRNA expression of genes implicated in the glucose (NPY: F3,12 = 3.137, p = 0.0654 (hour 15), F3,12 = 1.103, p = 0.3860 (hour 19), Fig. 4a, e; sirt1: F3,12 = 2.323, p = 0.1257 (hour 15), F3,12 = 0.8177, p = 0.5086 (hour 19), Fig. 4b, f; foxO1: F3,12 = 0.01786, p = 0.9966 (hour 15), F3,12 = 1.618, p = 0.2370 (hour 19); one way ANOVA, Fig. 4c, g) and lipid and cholesterol metabolism (hmgcr: F3,12 = 3.295, p = 0.0579 (hour 15), F3,12 = 0.9157, p = 0.4625 (hour 19); one way ANOVA, Fig. 4d, h). It may be noted that difference in NPY mRNA was noticeable, and in fact birds in 16 h had a significant higher mRNA levels than in the 11 h group (p < 0.05, Newman–Keul post hoc test; Fig. 4a). Similarly, no difference was found in the expression levels of citrate synthase, cs (hour 15: F3,12 = 1.272, p = 0.3282; hour 19: F3,12 = 0.4491, p = 0.7225, Figs. 4i, m) and α-ketoglutarate dehydrogenase, ogdh (hour 15: F3,12 = 1.408, p = 0.2883; hour 19: F3,12 = 1.830, p = 0.1945, Figs. 4j, n) genes between the treatment groups. However, succinate dehydrogenase (sdhd) had differential mRNA expression between photoperiods at hour 15 (F3,12 = 9.948, p = 0.0014; Fig. 4k), but not hour 19 (F3,12 = 0.3804, p = 0.7689; one way ANOVA, Fig. 4o). There were significantly higher sdhd mRNA levels in 13- and 16 h than in the 8- or 11 h treatment groups (p < 0.05, Newman–Keul post hoc test). Similarly, malate dehydrogenase (mdh) was differentially expressed between photoperiods at both hours (hour 15: F3,12 = 7.033, p = 0.0055; hour 19: F3,12 = 5.334, p = 0.0144, one way ANOVA, Fig. 4l, p). The expression levels of mdh were significantly higher in 13- and 16 h groups than in the 8- and 11 h treatment groups at hour 15 (p < 0.05, Newman–Keul post hoc test). The levels declined at hour 19 but were still higher in 13 h as compared with the levels in the 8- and 11 h groups (p < 0.05, Newman–Keul post hoc test). 3.2. Experiment 2: exposure of photorefractory birds to acute short days Figure 5 shows gene expression levels in photorefractory birds at hour 15 after the day birds had received 1 (SD1) and 7 (SD7) short day cycles, with long day controls (LDref). There were significant differences in the expression of tshβ (F2,9 = 11.26, p = 0.0035, Fig. 5b) and dio3 (F2,9 = 5.478, p = 0.0.0278, Fig. 5d) but not eya3 (F2, 9 = 0.5143, p = 0.6145, Fig. 5a) and dio2 (F2,9 = 0.424, p = 0.6667, one way ANOVA, Fig. 5c) between the treatment groups. Reduced tshβ mRNA levels were correlated with the duration of short day exposure. The levels were significantly lower and higher in SD1 than in the LDref and SD7 birds, respectively (p < 0.05, Newman–Keul post hoc test). Similarly, dio3 was expressed at significantly higher levels in LDref than in the SD1 and SD7 groups (p < 0.05, Newman–Keul post hoc test), but the two SD groups did not differ from each other in dio3 mRNA levels. On the other hand, gnrh and gnih were expressed at high Ct values (≥35 cycles) with very low expression levels and so they were not further analyzed and presented. Among opsins, only rhodopsin expression was different between LDref and SD1 and SD7 groups (F2,9 = 4.459, p = 0.0451, one way ANOVA, Fig. 5e). The mRNA levels were significantly reduced in SD1, but not SD7, than in the LDref birds (p < 0.05, Newman–Keul post hoc test). The neuropsin, melanopsin and peropsin genes showed a trend of increasing expression with the duration of short day exposure, but their mRNA levels were not significantly different between three

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Fig. 4. Mean (±SE; n = 4) relative mRNA expression levels (2−ΔΔct value) of genes involved in the energy homeostasis and metabolism (NPY, a, e; sirtuin1, b, f; foxO1, c, g; hmgcr, d, h; citrate synthase, i, m; α-ketoglutarate dehydrogenase [ogdh], j, n; succinate dehydrogenase, k, o; malate dehydrogenase, l, p) in the hypothalamus of redheaded buntings at hour 15 and hour 19. The other details are as in Fig. 1.

treatment groups (Fig. 5f–h). Further, NPY, sirt1, foxO1 and hmgcr mRNA expression did not significantly differ between three treatment groups, although their mRNA levels were relatively higher in SD1 and SD7 than in the LDref birds (Fig. 5i–l). Similar were the cs, ogdh and sdhd mRNA levels between these three treatment groups (Fig. 5m–o). However, mdh mRNA levels were significantly increased in SD1 and SD7 than in the LDref birds (p < 0.05, Newman– Keul post hoc test Fig. 5p). 3.3. Relationship in expression patterns of genes implicated in photoperiodic light perception and induction The relationship in mRNA expression levels between genes defined by correlation coefficient (r), coefficient of determination (r2) and significance of such relationships (p and q values) are summarized in Fig. 6. There was a strong positive correlation of rhodopsin on tshβ (r = 0.7667, r2 = 0.5879, p = 0.0005; q = 0.0015) and eya3 (r = 0.8586, r2 = 0.7372, p < 0.0001; q < 0.0001) expressions. Similar

correlations were found of peropsin on tshβ (r = 0.7798, r2 = 0.6081, p = 0.0004; q = 0.0024), eya3 (r = 0.5511, r2 = 0.3037, p = 0.0269; q = 0.0538) and rhodopsin (r = 0.6314, r 2 = 0.3987, p = 0.0087; q = 0.0261) expressions. Similarly, there was a significant negative correlation of neuropsin on tshβ (r = –0.6192, r2 = 0.3834, p = 0.0105; q = 0.0635). Among upstream genes examined, there was a significant correlation of pax6 on eya3 (r = 0.5956, r2 = 0.3548, p = 0.0149; q = 0.0447) and rhodopsin (r = 0.6579, r 2 = 0.4328, p = 0.0056; q = 0.0336), but not on tshβ (r = 0.4883, r2 = 0.2384, p = 0.0550; q = 0.1099; Fig. 6a). 3.4. Relationship in gene expression patterns between photoperiod induced history states Relative expression levels with reference to a pooled cDNA sample from all four life history states gave a life history state perspective of a gene expression pattern, as analyzed by non-linear Gaussian fit curve (r2(goodness of fit)) with Kruksal–Wallis corrected p values (Fig. 7).

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Fig. 5. Mean (±SE; n = 4) relative mRNA expression levels (2−ΔΔct value) of genes involved in short day induced termination of photorefractoriness in the hypothalamus of redheaded bunting at hour 15. Photorefractory birds maintained on 16 h light per day (16L:8D) were exposed to 1 (SD1) and 7 (SD7) short days, with a control group on 16L:8D (LDref). The following day after birds had received 8 h light, they were sacrificed in between zeitgeber times (ZT) 14.5 and 15.5 (hour 15). Thus, the sampling time was intervened by 6 h of darkness in birds that received short days, but the control LDref birds were sampled in light. Transcript levels were measured by the real time PCR for genes involved in the photoperiodic induction (eya3, a; tshβ, b; dio2, c; dio3, d), light perception (rhodopsin, e; melanopsin, f; neuropsin, g; peropsin, h) and energy homeostasis and metabolism (NPY, i; sirtuin1, j; foxO1, k; hmgcr l; citrate synthase, m; α-ketoglutarate dehydrogenase [ogdh], n; succinate dehydrogenase, o; malate dehydrogenase, p). An asterisk on bars indicates significant difference between groups, as determined by Newman–Keul post hoc test. Significance was considered at p < 0.05.

There were best fit curves with significant differences in amplitudes among life history states for tshβ (r2 = 0.7119; p = 0.0051; Fig. 7b) and eya3 (r2 = 0.3709; p = 0.0311; Fig. 7a) with peak expressions in the photostimulated state. The best fit amplitude dio2 curve was almost identical to that of the tshβ (r2 = 0.8231; p = 0.0175, Fig. 7c). As expected, dio3 best fit curve (r2 = 0.6507; p = 0.0062) with peak in the photorefractory phase antiphased with dio2 expression peak (Fig. 7c, d). Among opsins, rhodopsin with r2 = 0.8575; p = 0.014 and melanopsin with r2 = 0.7332; p = 0.0049 had high bestfit amplitude curves with their peak in the Pst and SDref phases, respectively (Fig. 7e, f). However, neuropsin (r2 = 0.3526; p = 0.0468) and peropsin (r2 = 0.1547; p = 0.1829) had relatively low amplitude best fit curves and their peaks lay in the Pse and SDref phases, respectively. The expression levels of peropsin did not differ among the life history states (Fig. 7g, h).

4. Discussion 4.1. Transcriptional activation in relation to photoperiod induced life history states Our results show that transitions between seasonal life history states in the year, i.e. from photosensitive to photostimulated and from the photorefractory to photosensitive state, are associated with changes in hypothalamic gene expression. In other words, genes implicated in photoperiodic timing are activated (or inactivated) in photosensitive and photorefractory birds in response to the lengthening and shortening day lengths, respectively. These results further show molecular basis of the neuroendocrine response triggered by a daily light period longer than 11 h for photoperiodic induction in buntings. There was a linear increase in tshβ

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Fig. 6. (a) Correlation matrix showing Pearson’s correlation coefficient (r value) with significance levels (p values) of seven genes involved in photoperiodic induction and upstream pathways (light perception and transduction). (b–e) Scatter plots showing correlation, as determined by r values, of rhodopsin and neuropsin on tshβ and eya3 (b, d) and of peropsin and pax6 on eya3, tshβ and rhodopsin (c, e) mRNA expression. Lines in the scatter plot denote the linear regression (solid, eya3; broken, tshβ; dotted, rhodopsin). There was a positive correlation of rhodopsin, peropsin or pax 6, and negative correlation of neuropsin on tshβ mRNA expression.

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Fig. 7. Gene expression pattern: relationship with seasonal life history states: Data (n = 4) on relative mRNA expression levels at hour 15 (zeitgeber time 15) are plotted for selected genes in the photosensitive (Pse; short days, 8L:16D), photostimulated (Pst; single 16 h light exposure), photorefractory (LDref; long days, 16L:8D) and short day treated refractory (SDref; 7 cycles of 8L:16D) redheaded buntings. Left panel (a–d): genes involved in photoperiodic induction; eya3, tshβ, dio2 and dio3. Right panel (e–h): genes involved in photoperiodic perception; rhodopsin, melanopsin, neuropsin and peropsin. Gaussian fit curve (solid line) shows the trend of gene expression across photoperiodinduced seasonal life history states. The dotted line connects mean values. Defining points in the Gaussian curve determined peak expression shown by a solid vertical line.

expression in response to increasing light periods (mRNA levels in 11 h < 13 h < 16 h light exposure). Also, a significant rise in tshβ mRNA expression at hour 19, compared with hour 15, in 16 h (not 13 h) light exposure indicates photoperiodic adaptation of buntings to long summer days of their breeding (high) latitudes, regardless of the fact that present experiment was carried out at distant latitude (Fig. 2c, d). A parallel eya3 expression pattern further suggests tshβ and eya3 constituting the first wave of photoperiod-induced genes, with a possible functional linkage, in buntings, as suggested in other photoperiodic birds and mammals (experiment 1; Fig. 2a, b; cf. Nakao

et al., 2008; Dardente et al., 2010; Majumdar et al., 2014). However, eya3 may not be important in short day induced termination of the photorefractoriness, for eya3 mRNA expression was not altered after 1 or 7 short days in refractory buntings (Fig. 5a). Similar to the ‘first wave’, dio2 and dio3 expression pattern conformed to the pattern as genes of the second ‘waveform’ in the sense that they were reciprocally switched-on and -off, respectively, on the first day of light exposure (Majumdar et al., 2014; Nakao et al., 2008). This was despite the fact that mRNA expression at hour 19 was measured in buntings after 3- or 6 h darkness following a single 16- or 13 h

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light exposure (Fig. 2). A similar reciprocal relationship between dio2 and dio3 expression patterns was absent, however, in photorefractory buntings subjected to short days in the experiment 2. Whereas dio2 mRNA expression was not altered, dio3 mRNA expression was significantly reduced in buntings after a day of short day exposure (SD1 vs. LDref; p < 0.05, Newman–Keuls test; Fig. 5d). We suggest dio3 expression as a critical step in the maintenance of the photorefractory state, and speculate that a reduced dio3 expression in response to acute short days signaled the recovery of photoresponsivity in photorefractory buntings (cf. Fig. 5d). This is consistent with photoperiod regulated seasonal plasticity in deiodinase expression pattern in other long day breeding birds and mammals (Nakao et al., 2008; Perfito et al., 2012; Watanabe et al., 2007). A microarray study has also shown seasonal differences in cDNA expression coding for genes involved in the regulation of thyroid hormone activity and neuroplasticity in song sparrows, Melospiza melodia (Mukai et al., 2009). 4.2. Opsins and other probable upstream genes Rhodopsin appears to be a candidate molecule for photoperiodic timing in buntings, as evidenced by its photoperiod dependent correlation with eya3 (r = 0.89) and tshβ (r = 0.77). Considered together with another recent result on redheaded buntings in which eya3 protein was expressed in brain regions that are also shown to express rhodopsin in other bird species (Silver et al., 1988; Wada et al., 1998; Wang and Wingfield, 2011), it seems likely that eya3 and rhodopsin together, or independently, play a significant role in the transduction of photoperiodic information in avian brain (Majumdar et al., 2014). Perhaps melanopsin and neuropsin also contribute to the photoperiodic light perception in buntings, as evidenced by an increased melanopsin mRNA expression under long days, and by a negative correlation of neuropsin mRNA expression on tshβ mRNA levels (r = −0.6192, r2 = 0.3834, p = 0.0105; Fig. 6a, d). This is consistent with a finding in border canaries (Serinus canaria), in which neuropsin was shown inhibitory to the tshβ transcription (Stevenson and Ball, 2012). Both border canaries and redheaded buntings are photoresponsive species and exhibit absolute photorefractoriness on long day exposure (Rani et al., 2005; Storey and Nicholls, 1976). We report for the first time peropsin mRNA in a songbird brain (see Bailey and Cassone, 2004 for peropsin in chicken brain) with a significantly higher expression in buntings exposed to 16 h than to the 8 or 11 h light and with its positive correlation on rhodopsin expression (r = 0.6314, r2 = 0.3987, p = 0.0087; Fig 6a, c). Perhaps as an auxiliary opsin (Chen et al., 2001), peropsin helps in regeneration of rhodopsin in bunting hypothalamic photoreceptors. Also, with a significant correlation of pax6 on rhodopsin mRNA levels (Fig. 6a, e), we speculate the role of pax6 in the regulation of rhodopsin expression, as reported in D. melanogaster (Sheng et al., 1997). We would rule out at present the involvement of rgropsin (an auxiliary opsin; Koyanagi et al., 2002) and VA-opsin (a light sensing molecule reported in chicken brain; Halford et al., 2009) in the photoperiodic light perception in buntings since these genes were not amplified (rgropsin) or cloned and sequenced (VA opsin) from bunting hypothalamus. Although sampling limited to two times per day in this study are inadequate to derive a conclusion about circadian genes, the absence of a significant difference in mRNA expression between photoperiods (experiment 1), in which birds exposed had identical entrainment with short days prior to a single light exposure, does not support a direct role of per 2, clock or bmal1 in triggering a neuroendocrine response in buntings. This does not weaken the evidence for circadian basis of photoperiodic time measurement in birds including buntings, however (see Kumar et al., 2010). It may be noted that the circadian clock(s) regulating photoperiodism may be separate from the ones regulating other daily functions, like activity behavior and

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melatonin secretion in birds (for references see Kumar et al., 2006, 2010). If so, we may need to examine changes in circadian genes in specific cells rather than in the whole hypothalamus, as we did in the present study. Further, an increased pax6 expression after a 16 h light exposure with a significant correlation on eya3 mRNA levels may suggest its involvement in the photoperiodic induction pathway via eya3 gene activation in buntings (Fig. 6a, e), consistent with reported involvement of pax6 in the activation of eya family of genes (Xu et al., 1997). Similarly, the absence of photoperiod dependent expression of gene coding CaSR argues against the involvement of calcium sensing neurons, which have been suggested to act as photoreceptors in the chicken lateral septum (Li et al., 2004). However, calcium could possibly still contribute to the photoperiodic signaling cascade via a different hitherto an unrecognized pathway. 4.3. Downstream reproductive and metabolic genes A single long light exposure did not activate the transcription of both reproductive (e.g. gnrh) and metabolic (NPY, hmgcr, foxO1 or sirt1) genes, as was indicated by no change in their mRNA expression after a long light exposure. Either hour 19 was perhaps too early a time for the measurement of reproductive and metabolic effects (Fig. 4e–h), or a downstream molecular response results from a cumulative effect of stimulatory photoperiods over several days of exposure. Interestingly, genes coding for TCA cycle enzymes selectively responded to the light exposure, with photoperiod dependent and independent mRNA expression of mdh/sdhd and cs/ ogdh genes, respectively (Fig. 4k, l, o, p). We speculate that this was due to difference in their activation pathways. For example, enhanced mdh activation involves a thyroid hormone based mechanism, since T3 administration is known to elevate mdh enzyme levels in the rat liver (Tarentino et al., 1966). An increased mdh mRNA expression on the first long day may also signal an early initiation of lipogenesis in buntings, for mdh gene is directly linked with lipogenesis (Ghosh and Ray, 1994; Tepperman and Tepperman, 1965). Results from a feeding–fasting experiment on rats also show association of the liver and epididymal lipogenesis with mdh levels (Pande et al., 1964). Enhanced mdh mRNA expression may also indicate an initiated lipogenesis via malic acid activity in parallel with the sdhd enzyme activity (Vitorica et al., 1981). It may be cautioned however, that present results would require validity by results on physiological measures (e.g. hormones, enzymes and/or metabolites) of reproductive and metabolic effects, which could not be done in this study. 4.4. Gene expression pattern: relationship with seasonal life history states Clearly, gene expression patterns were associated with photoperiodic timing of seasonal life history states in buntings. Eya3, tsh β and dio2 mRNA levels were low in the photosensitive unstimulated and photorefractory birds. Contrarily, dio3 expression was high in these birds with higher mRNA levels in photorefractory birds (Fig. 7), consistent with high dio3 expression in short day refractory Siberian hamsters, Photodopus sungorus (Stevenson and Prendergast, 2013). Thus, with other findings on few birds and mammals (Nakao et al., 2008; Perfito et al., 2012; Watanabe et al., 2007, Stevenson and Prendergast, 2013), the present results implicate dio3 as an important candidate molecule in the regulation of photoperiodic timing in buntings. In addition to deiodinases, the expression of genes possibly involved upstream in the photoperiodic pathways in buntings appears associated with seasons (cf. Fig. 5; Fig. 7e–h). Particularly, rhodopsin and neuropsin expression seems to be closely linked with transitions in the photoperiod-induced seasonal life history states, i.e. from photosensitive to photostimulated and from photorefractory to

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photosensitive states. There was a photoperiod-dependent expression of rhodopsin in photosensitive buntings exposed to photoperiods > CD (Figs 3a, 7e), consistent with the photoperiodic effects on rhodopsin expression in the salmon fish (Nakane et al., 2013) and house sparrows (Wang, 2009). Similarly, high neuropsin expression under short days in photorefractory buntings compared with its expression in photosensitive birds subjected to a single long day (cf. Figs 5, 7g). An increasing trend in the melanopsin and peropsin expression was also found under short days in photorefractory buntings (cf. Figs 5, 7f, h). 5. Conclusions The present results provide a molecular basis of transitions in photoperiod-induced phenologies with seasons in redheaded buntings. They support the idea that a photoperiodic species consistently responds at gene levels to the changes in annual photoperiodic cycle. Photoperiod dependent expression of upstream genes suggests eya3, rhodopsin and neuropsin as possible candidate molecules, mainly involved in the relay of photoperiodic information to the neuroendocrine system in birds. Downstream effects on seasonal physiology are likely to be the result of a summated effect of upstream molecular interactions over several days, as determined by external photoperiods. This may be an adaptive strategy in a photoperiodic species to safeguard the initiation of energy demanding processes, particularly linked with development of migration and reproduction, in a situation of a sudden drastic change in the environment. Overall, the present study for the first time shows that molecular response to a photoperiodic stimulus on the first day persists even if it was immediately followed by a period of darkness. It suggests that the activation of hypothalamic ‘gene switches’ involved at different levels (photoperiodic perception, transduction and induction) distinguish between non-stimulatory and stimulatory effects of a photoperiod. Thus, photoperiodic timing mechanism in songbirds may be more complex than is generally assumed. An extensive investigation is therefore needed in species inhabiting both the low and high latitudes to present a more generalized view of the brain molecular switches that regulate seasonality in birds, in particular, and vertebrates, in general. Acknowledgments Generous financial support by research grants from the Science and Engineering Research Board (Department of Science and Technology), New Delhi, through IRHPA and regular projects (IR/SO/LU02/2005(G); SR/SO/AS-50/2010) and Department of Biotechnology, New Delhi (BT/PR4984/MED/30/752/2012) and DU-DST PURSE (Dean(R)/2012/1477-seasonal clocks) grant are gratefully acknowledged. GM received a SRF from Council and Scientific and Industrial Research, New Delhi. Appendix: Supplementary Material Supplementary data to this article can be found online at doi:10.1016/j.mce.2014.09.020. References Ali, S., Ripley, S.D., 1974. Handbooks of the Birds of India and Pakistan, vol. 10. Oxford University Press, Bombay/London/ New York. Bailey, M.J., Cassone, V.M., 2004. Opsin photoisomerases in the chick retina and pineal gland: characterization, localization, and circadian regulation. Invest. Ophthalmol. Vis. Sci. 45, 769–775. Bentley, G.E., Tucker, S., Chou, H., Hau, M., Perfito, N., 2013. Testicular growth and regression are not correlated with Dio2 expression in a wild male songbird, sturnus vulgaris, exposed to natural changes in photoperiod. Endocrinology 154, 1813–1819.

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