Photoperiod-driven concurrent changes in hypothalamic and brainstem transcription of sleep and immune genes in migratory redheaded bunting.

The molecular regulation of sleep in avian migrants is still obscure. We thus investigated this in migratory redheaded buntings, where four life-history states (LHS; i.e. non-migratory, pre-migratory, migratory and refractory states) were induced. There was increased night-time activity (i.e. Zugunruhe) during the migratory state with reduced daytime activity. The recordings of the sleep-wake cycle in buntings showed increased night-time active wakefulness coupled with drastically reduced front and back sleep during migratory phase. Interestingly, we found the buntings to feed and drink even after lights-off during migration. Gene expression studies revealed increased hypothalamic expression of glucocorticoid receptor (nr3c1), and pro-inflammatory cytokines (il1b and il6) in pre-migratory and migratory states, respectively, whereas in brainstem Ca2+/calmodulin-dependent protein kinase 2 (camk2) was upregulated during the migratory state. This suggested a heightened pro-inflammatory state during migration which is a feature of chronic sleep loss, and a possible role of Ca2+ signalling in promoting wakefulness. In both the hypothalamus and brainstem, the expression of melatonin receptors (mel1a and mel1b) was increased in the pre-migratory state, and growth hormone-releasing hormone (ghrh, known to induce sleep) was reduced during the migratory state. The current results demonstrate key molecules involved in the regulation of sleep-wake cycle across LHS in migratory songbirds.

Photoperiodically driven transcriptome-wide changes in the hypothalamus reveal transcriptional differences between physiologically contrasting seasonal life-history states in migratory songbirds.

We investigated time course of photoperiodically driven transcriptional responses in physiologically contrasting seasonal life-history states in migratory blackheaded buntings. Birds exhibiting unstimulated winter phenotype (photosensitive state; responsive to photostimulation) under 6-h short days, and regressed summer phenotype (photorefractory state; unresponsiveness to photostimulation) under 16-h long days, were released into an extended light period up to 22 h of the day. Increased tshß and dio2, and decreased dio3 mRNA levels in hypothalamus, and low prdx4 and high il1ß mRNA levels in blood confirmed photoperiodic induction by hour 18 in photosensitive birds. Further, at hours 10, 14, 18 and 22 of light exposure, the comparison of hypothalamus RNA-Seq results revealed transcriptional differences within and between states. Particularly, we found reduced expression at hour 14 of transthyretin and proopiomelanocortin receptor, and increased expression at hour 18 of apolipoprotein A1 and carbon metabolism related genes in the photosensitive state. Similarly, valine, leucine and isoleucine degradation pathway genes and superoxide dismutase 1 were upregulated, and cocaine- and amphetamine-regulated transcript and gastrin-releasing peptide were downregulated in the photosensitive state. These results show life-history-dependent activation of hypothalamic molecular pathways involved in initiation and maintenance of key biological processes as early as on the first long day.

Hypothalamic plasticity in response to changes in photoperiod and food quality: An adaptation to support pre-migratory fattening in songbirds?

In latitudinal avian migrants, increasing photoperiods induce fat deposition and body mass increase, and subsequent night-time migratory restlessness in captive birds, but the underlying mechanisms remain poorly understood. We hypothesized that an enhanced hypothalamic neuronal plasticity was associated with the photostimulated spring migration phenotype. We tested this idea in adult migratory red-headed buntings (Emberiza bruniceps), as compared with resident Indian weaverbirds (Ploceus philippinus). Birds were exposed to a stimulatory long photoperiod (14L:10D, LP), while controls were kept on a short photoperiod (10L:14D, SP). Under both photoperiods, one half of birds also received a high calorie, protein- and fat-rich diet (SP-R, LP-R) while the other half stayed on the normal diet (SP-N, LP-N). Thirty days later, as expected, the LP had induced multiple changes in the behaviour and physiology in migratory buntings. Photostimulated buntings also developed a preference for the rich food diet. Most interestingly, the LP and the rich diet, both separately and in association, increased neurogenesis in the mediobasal hypothalamus (MBH), as measured by an increased number of cells immunoreactive for doublecortin (DCX), a marker of recently born neurons, in buntings, but not weaverbirds. This neurogenesis was associated with an increased density of fibres immunoreactive for the orexigenic neuropeptide Y (NPY). This hypothalamic plasticity observed in a migratory, but not in a non-migratory, species in response to photoperiod and food quality might represent an adaptation to the pre-migratory fattening, as required to support the extensive energy expenses that incur during the migratory flight.

Concurrent changes in photoperiod-induced seasonal phenotypes and hypothalamic CART peptide-containing systems in night-migratory redheaded buntings.

This study tested the hypothesis whether hypothalamic cocaine-and amphetamine-regulated transcript (CART)-containing systems were involved in photoperiod-induced responses associated with spring migration (hyperphagia and weight gain) and reproduction (gonadal maturation) in migratory songbirds. We specifically chose CART to examine neural mechanism(s) underlying photoperiod-induced responses, since it is a potent anorectic neuropeptide and involved in the regulation of changes in the body mass and reproduction in mammals. We first studied the distribution of CART-immunoreactivity in the hypothalamus of migratory redheaded buntings (Emberiza bruniceps). CART-immunoreactive neurons were found extensively distributed in the preoptic, lateral hypothalamic (LHN), anterior hypothalamic (AN), suprachiasmatic (SCN), paraventricular (PVN), dorsomedialis hypothalami (DMN), inferior hypothalamic (IH), and infundibular (IN) nuclei. Then, we correlated hypothalamic CART-immunoreactivity in buntings with photostimulated seasonal states, particularly winter non-migratory/non-breeding (NMB) state under short days, and spring premigratory/pre-breeding (PMB) and migratory/breeding (MB) states under long days. There were significantly increased CART-immunoreactive cells, and percent fluorescent area of CART-immunoreactivity was significantly increased in all mapped hypothalamic areas, except the SCN, PVN, AN, and DMN in photostimulated PMB and MB states, as compared to the non-stimulated NMB state. In particular, CART was richly expressed in the medial preoptic nucleus, LHN, IH and IN during MB state in which buntings showed reduced food intake and increased night-time activity. These results suggest that changes in the activity of the CART-containing system in different brain regions were associated with heightened energy needs of the photoperiod-induced seasonal responses during spring migration and reproduction in migratory songbirds.

Seasonal reproductive state determines gene expression in the hypothalamus of a latitudinal migratory songbird during the spring and autumn migration.

We investigated gonadal effects on hypothalamic transcription of genes in sham-operated and castrated redheaded buntings photostimulated into spring and autumn migratory states. RNA-Seq results showed testes-dependent differences between spring and autumn migratory states. In particular, differentially expressed genes enriched G-protein-coupled receptor and calcium-ion signaling pathways during spring and autumn states, respectively. qPCR assay showed attenuated gabra5, ttr, thra and thrb expressions, suggesting reduced GABA and thyroid hormone effects on photo-sexual response in spring. In spring castrates, reduced npy, tac1 and nrcam and increased ank3 expression suggested testicular effects on the appetite, prolactin release and neuronal functions, whereas in autumn castrates, reduced rasgrp1, grm5 and grin1, and increased mras expression suggested testicular effects on the ras, G-protein and glutamate signaling pathways. Castration-induced reciprocal switching of pomc and pdyn expressions suggested effects on the overall homeostasis in both seasons. These results demonstrate transcriptome-wide changes, with season-dependent roles of testes in songbird migration.

Difference in control between spring and autumn migration in birds: insight from seasonal changes in hypothalamic gene expression in captive buntings.

We hypothesized differences in molecular strategies for similar journeys that migrants undertake to reproduce in spring and to overwinter in autumn. We tested this in redheaded buntings (Emberiza bruniceps) photoinduced into spring and autumn migratory states, with winter and summer non-migratory states as controls. Compared with controls, buntings fattened, gained weight and showed Zugunruhe (nocturnal migratory restlessness) in the migratory state. Spring migration was associated with greater fat and body mass, and higher intensity of Zugunruhe, compared with autumn migration. Circulating corticosterone levels were higher in spring, while T3 levels were higher in autumn. Hypothalamic expression of thyroid hormone-responsive (dio2, dio3), light-responsive (per2, cry1, adcyap1) and th (tyrosine hydroxylase, involved in dopamine biosynthesis) genes showed significant changes with transition from non-migratory to the migratory state. There were significantly higher mRNA expressions in autumn, except for higher th levels in the spring. Furthermore, the expression patterns of dnmt3a (not dnmt3b) and tet2 genes suggested an epigenetic difference between the non-migrant and migrant periods, and the spring and autumn migrant periods. These results demonstrate for the first time seasonal transition in hypothalamic gene expressions, and suggest differences in regulatory strategies at the transcriptional level for spring and autumn migrations in songbirds.

Scotostimulation of reproductive neural pathways and gonadal maturation are not correlated with hypothalamic expression of deiodinases in subtropical spotted munia.

Circannual rhythm regulates the annual timing of reproduction in spotted munia, with sex differences in its relationship with the external photoperiod environment. Interestingly, munia show an atypical photosensitivity and exhibit gonadal maturation when acutely exposed to an unnatural short photoperiod (eg 3 hours of light per day; ie a long scotoperiod). The proximate mechanisms regulating scotoperiod-induced hypothalamic-pituitary-gonadal (HPG) activation are unclear. Because thyroid hormone signalling plays a central role in photoperiodic induction, we hypothesised the involvement of similar mechanism, comprising alterations in hypothalamic deiodinases, under long scotoperiod-induced HPG activation. To test this, several endpoints of cellular and molecular correlates were assayed in male and female munias after 1 and 4 weeks of exposure to an 3:21 hour light/dark cycle (3L:21D), with controls on a 21:3 hour light/dark cycle (21L:3D). We measured the hypothalamic expression of mRNA and protein of light-sensitive (neuropsin, OPN5) and reproductive (vasoactive intestinal peptide [VIP], neuropeptide Y [NPY], gonadotrophin-releasing hormone [GnRH], gonadotrophin-inhibiting hormone [GnIH]) neuropeptides by quantitative polymerase chain reaction (PCR) and immunohistochemistry, respectively. In addition, we also measured mRNA expression of types 2 (DIO2) and 3 (DIO3) deiodinases that regulate triiodothyronine-mediated GnRH release and gonadal maturation in photoperiodic species. The quantitative PCR and immunohistochemistry results were consistent. Higher OPN5 levels under 21L:3D than under 3L:21D suggested its role in sensing the length of the light period. Similarly, low VIP and high NPY expression under 3L:21D than under 21L:3D were consistent with their roles as cellular correlates of photic and nonphotic environment, respectively. High GnRH-I/low GnIH levels and gonadal recrudescence under 3L:21D, and an inverse pattern under 21L:3D, confirmed the scotostimulation of HPG axis in spotted munia. However, DIO2 and DIO3 mRNA levels did not differ between 2 scotoperiods, in contrast to their reciprocal expression pattern found during long-day photostimulation. We demonstrate for the first time sex-dependent scotostimulation of reproductive neural pathways and suggest the involvement of molecules other than hypothalamic deiodinases in the regulation of gonad development cycle in 'nonphotoperiodic' seasonally breeding vertebrates.

Temporal expression of clock genes in central and peripheral tissues of spotted munia under varying light conditions: Evidence for circadian regulation of daily physiology in a non-photoperiodic circannual songbird species.

We investigated if the duration and/or frequency of the light period affect 24-h rhythm of circadian clock genes in central and peripheral tissues of a non-photoperiodic songbird, the spotted munia (Lonchura punctulata), in which a circannual rhythm regulates the reproductive cycle. We monitored activity-rest pattern and measured 24-h mRNA oscillation of core clock (Bmal1, Clock, Per2, Cry1 and Cry2) and clock-controlled (E4bp4, Rorα and Rev-erbα) genes in the hypothalamus, retina, liver and gut of spotted munia subjected to an aberrant light-dark (LD) cycle (3.5L:3.5D; T7, T = period length of LD cycle) and continuous light (LL, 24L:0D), with controls on 24-h LD cycle (T24, 12L:12D). Munia exhibited rhythmic activity-rest pattern with period matched to T7 or T24 under an LD cycle and were arrhythmic with a scattered activity pattern and higher activity duration under LL. At the transcriptional level, both clock and clock-controlled genes showed a significant 24-h rhythm in all four tissues (except Clock in the liver) under 12L:12D, suggesting a conserved tissue-level circadian time generation in spotted munia. An exposure to 3.5L:3.5D or LL induced arrhythmicity in transcriptional oscillation of all eight genes in the hypothalamus (except Rev-erbα) and liver (except Bmal1 and Rev-erbα under T7 and Cry1 under LL). In the retina, however, all genes showed arrhythmic 24-h mRNA expression under LL, but not under T7 (except in E4bp4 and Rorα). Interestingly, unlike in the liver, Bmal1, Per2, Cry1, Rorα and Rev-erbα mRNA expressions were rhythmic in the gut under both T7 (except Rorα) and LL conditions. These results showed variable relationship of internal circadian clocks with the external light environment and suggested a weak coupling of circadian clocks between the central (hypothalamus and retina) and peripheral (liver and gut) tissues. We suggest tissue-level circadian clock regulation of daily physiology and behavior in the spotted munia.

Circannual testis and moult cycles persist under photoperiods that disrupt circadian activity and clock gene cycles in spotted munia.

We investigated whether circannual rhythms underlying annual testis maturation and moult cycles are independent of duration and frequency of the light period and circadian clock control in non-photoperiodic spotted munia. Birds were subjected to an aberrant light-dark (LD) cycle (3.5 h L:3.5 h D; T7, where T is the period length of the LD cycle) and continuous light (LL, 24 h L:0 h D), with controls on 12 h L:12 h D (T24, 24 h LD cycle). We measured the behavioural activity pattern of the birds and 24 h mRNA oscillations of circadian clock genes (bmal1, clock, per2, cry1, cry2) in the hypothalamus, the putative site of seasonal timing. Diurnal munia were rhythmic in behaviour with the period of the activity-rest cycle matched to T7 and T24, and became behaviourally arrhythmic with activity scattered throughout 24 h under LL. Similarly, exposure to 3.5 h L:3.5 h D and LL caused arrhythmicity in 24 h clock gene expression, suggesting disruption of internal circadian timing at the transcriptional level; a significant rhythm was found under 12 h L:12 h D. During an exposure of 80 weeks, munia showed two to three cycles of testis maturation and wing primaries moult under all photoperiods, although with a longer period under 12L:12D. Thus, the frequency of light period under 3.5 h L:3.5 h D or LL disrupted circadian clock gene cycles, but did not affect the generation of circannual testis and moult cycles. We conclude that the prevailing light environment and hypothalamic circadian gene cycles do not exert direct control on the timing of the annual reproductive cycle in spotted munia, suggesting independent generation of the circadian and circannual rhythms in seasonally breeding species.

Changes in brain peptides associated with reproduction and energy homeostasis in photosensitive and photorefractory migratory redheaded buntings.

Present study examined the expression of brain peptides associated with the reproduction and energy homeostasis (GnRH/GnIH, NPY/VIP), and assessed their possible functional association in the photosensitive (non-breeding, pre-breeding), photostimulated (breeding) and photorefractory (post-breeding) migratory redheaded buntings (Emberiza bruniceps), using double-labeled immunohistochemistry. Particularly, we measured immunoreactive (-ir) cell numbers, per cent cell area and cell optical density (OD) in the preoptic area (GnRH-I), midbrain (GnRH-II), paraventricular nucleus (GnIH), dorsomedial hypothalamus, DMH and infundibular complex, INc (NPY and VIP), and lateral septal organ (VIP) of buntings kept under natural photoperiods at the wintering latitude (26°55'N). There was a significant seasonal difference in GnRH-I, not GnRH-II, with reduced -ir cells in the photosensitive and photorefractory buntings, and notably with increased cell OD between the refractory and non-breeding states with no increase in testis size. Also, increased cell OD of GnIH neurons in non-breeding state indicated its role in the maintenance of small testes during the post-refractory period. Overall, seasonal changes in GnRH-I and GnIH were found consistent with their suggested roles in reproductive regulation of absolute photorefractory birds. Further, there was a significant seasonal change in cell OD of NPY neurons in DMH, not the INc. In contrast, VIP immunoreactivity was seasonally altered, with a significantly higher VIP-ir cells in breeding than the pre-breeding state. Finally, close proximity between perikarya with fibres suggested functional interactions between the GnRH and GnIH, and NPY and VIP. Thus, seasonal plasticity of brain peptides is perhaps the part of neural regulation of seasonal reproduction and associated energy homeostasis in migratory songbirds.

Pinealectomy abolishes circadian behavior and interferes with circadian clock gene oscillations in brain and liver but not retina in a migratory songbird.

In songbirds, the pineal gland is part of the multi-oscillatory circadian timing system, with participating component oscillators in the eyes and hypothalamus. This study investigated the role of the pineal gland in development of the nighttime migratory restlessness (Zugunruhe) and generation of circadian gene oscillations in the retina, brain and liver tissues in migratory redheaded buntings (Emberiza bruniceps). Pinealectomized (pinx) and sham-operated buntings entrained to short days (8h light: 16h darkness, 8L:16D) were sequentially exposed for 10days each to stimulatory long days (13L: 11D) and constant dim light (LLdim; a condition that tested circadian rhythm persistence). Whereas activity-rest pattern was monitored continuously, the mRNA expressions of clock genes (bmal1, clock, npas2, per2, cry1, rorα, reverα) were measured in the retina, hypothalamus, telencephalon, optic tectum and liver tissues at circadian times, CT, 1, 6, 13, 17 and 21 (CT 0, activity onset) on day 11 of the LLdim. The absence of the pineal gland did not affect the development of long-day induced Zugunruhe but caused decay of the circadian rhythm in Zugunruhe as well as the clock gene oscillations in the hypothalamus, but not in the retina. Further, there were variable effects of pinealectomy in the peripheral brain and liver tissue circadian gene oscillations, notably the persistence of per 2 and cry1 (optic tectum), rorα (telencephalon) and npas2 (liver) mRNA oscillations in pinx birds. We suggest the pineal gland dependence of the generation of circadian gene oscillations in the hypothalamus, not retina, and peripheral brain and liver tissues in migratory redheaded buntings.

Circadian timing in central and peripheral tissues in a migratory songbird: dependence on annual life-history states.

Predictable seasonal change in photoperiod triggers a sequential change in the daily activity-rest pattern, adaptive for migration in several bird species. The night-migratory black-headed bunting (Emberiza melanocephala) is day active under short photoperiods (8 h light:16 h dark, short day sensitive). Under long photoperiods (16 h light:8 h dark), the buntings are initially day active (long day premigratory) but subsequently become intensely night active (long day migratory) and after few weeks again return to a day active pattern (long day refractory). However, it is unclear how the daily expression of circadian genes changes during photoperiod-induced seasonal life-history states (LHSs). We measured period 2 (Per2), cryptochrome 1 (Cry1), brain and muscle arnt-like protein 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) mRNA expressions in various neural and peripheral tissues of buntings in different LHSs and discovered differences of ∼2 to 6 h in the phase and 2- to 4-fold in amplitude of circadian oscillations of Per2, Cry1, and Bmal1 between photoperiod-induced LHSs. Phase relationship in mRNA oscillations was altered between oscillator components in the circadian pacemaker system (retina, pineal, hypothalamus) as well as in the peripheral (liver, muscle) tissues. These results show for the first time altered waveforms of clock gene expressions in all tissues in parallel with behavioral shifts and suggest the involvement of circadian system in photoperiod induction of seasonal LHSs in a migratory species.

Hypothalamic gene switches control transitions between seasonal life history states in a night-migratory photoperiodic songbird.

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, tshß, 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 ß, 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 tshß 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.

Annual life history-dependent gene expression in the hypothalamus and liver of a migratory songbird: insights into the molecular regulation of seasonal metabolism.

Birds seasonally switch from one life history state (LHS) to another to maximize their fitness. Accordingly, they exhibit distinct differences in their physiological and behavioral phenotypes between seasons. Possible molecular mechanisms underlying changes through the seasons have scarcely been examined in migratory birds. The present study measured key genes suggested to be involved in the metabolic regulation of 4 photoperiodically induced seasonal LHSs in a long-distance migratory songbird, the blackheaded bunting (Emberiza melanocephala). Buntings were held under short days (8 h light:16 h darkness, 8L:16D), during which they maintained the winter nonmigratory phenotype. Then they were exposed for several weeks to long days (13L:11D). Differences in the activity-rest pattern, body fattening and weight gain, testis size, organ (heart, intestine) weights, and blood glucose and triglyceride levels confirmed that buntings sequentially exhibited spring migration-linked premigratory, migratory, and postmigratory LHSs under long days. The mRNA levels of circadian genes involved in metabolism (Bmal1, Clock, Npas2, Rorα, and Rev-erbα) and of genes that encode for proteins/enzymes involved in the regulation of glucose (Sirt1, FoxO1, Glut1, and Pygl) and lipids (Hmg-CoA; Pparα, Pparγ; Fasn and Acaca) showed LHS-dependent changes in their light-dark expression patterns in the hypothalamus and liver. These initial results on genetic regulation of metabolism in a migratory species extend the idea that the transitions between LHSs in a seasonal species are accomplished by changes at multiple regulatory levels. Thus, these findings promise new insights into the mechanism(s) of adaptation to seasons in higher vertebrates.

A photoperiodic molecular response in migratory redheaded bunting exposed to a single long day.

A long day response is triggered by the activation of EYA3 (eyes absent 3) and TSH-ß (thyroid stimulating hormone beta subunit) genes in the pars tuberalis (PT). However, protein products of these genes are not yet shown in the hypothalamus of a photoperiodic species. Therefore, using the 'first long day paradigm', EYA3 and TSH-ß along with c-FOS and GnRH peptides were immunohistochemically localized and measured in the hypothalamus of photoperiodic redheaded buntings that were maintained on short days (SD, LD 8/16) and subjected to one full long day (LD, LD 16/8). Following morning light remained turned off, and birds were sacrificed in the first hour of the day. Brains were collected and processed for immunohistochemistry of peptides. FOS-lir and GnRH-lir cells were significantly higher in the preoptic area (POA) in LD than in SD, which indicated photoperiod induced neuronal activation and downstream effects, respectively, under LD. In LD, EYA3-lir cells were significantly increased in septal lateralis (SL) with fibres extending to sub-septal organ (SSO); EYA3 fibres were very dense in median eminence. Similarly, there were significantly increased TSH-ß-lir cells in the ventricular region with much abundance in the PT and TSH-ß-lir fibres in the SSO (extending up to SL), inferior hypothalamic nucleus (IH) and infundibular nucleus (IN) in LD birds. Elevated EYA3, TSH-α and TSH-ß mRNA levels further confirmed photoperiodic induction at the transcriptional level in buntings on the first long day. These are the first results showing localization of photoperiodically induced peptides in the hypothalamus of a songbird species, the redheaded bunting.

Neural correlates of migration: activation of hypothalamic clock(s) in and out of migratory state in the blackheaded bunting (Emberiza melanocephala).

BACKGROUND: Many vertebrates distinguish between short and long day lengths using suprachiasmatic nuclei (SCN). In birds particular, the mediobasal hypothalamus (MBH) is suggested to be involved in the timing of seasonal reproduction. This study investigated the response of SCN and MBH to a single long day, and the role of MBH in induction of the migratory phenotype in night-migratory blackheaded buntings. METHODOLOGY/PRINCIPAL FINDINGS: Experiment 1 immunocytochemically measured c-fos in the SCN, and c-fos, vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) in the MBH of buntings exposed to a 20 h light period. Long light period induced significantly stronger c-fos expression, measured as number of c-fos-like immunoreactive (c-fos-lir) cells, in MBH, but not in the SCN. Within the MBH, c-fos-lir cells were significantly denser in the inferior hypothalamic nucleus (IH) and infundibular nucleus (IN), but not in the dorsomedial hypothalamus (DMH). IH and IN also had significantly increased number of VIP and NPY labeled cells. DMH had significantly increased number of VIP labeled cells only. Experiment 2 assayed c-fos, VIP and NPY immunoreactivities in the middle of day and night in the MBH of buntings, after seven long days (day active, non-migratory state) and after seven days of Zugunruhe (night active, migratory state) in long days. In the migratory state, the number of c-fos-lir cells was significantly greater only in DMH; VIP-lir cells were denser in all three MBH regions suggesting enhanced light sensitivity at night. The denser NPY-lir cells only in IN in the non-migratory state were probably due to premigratory hyperphagia. CONCLUSIONS/SIGNIFICANCE: In buntings, SCN may not be involved in the photoperiod-induced seasonal responses. MBH contains the seasonal clock sensitive to day length. VIP and NPY are parts of the neuroendocrine mechanism(s) involved, respectively, in sensing and translating the photoperiodic message in a seasonal response.

Daily expression of six clock genes in central and peripheral tissues of a night-migratory songbird: evidence for tissue-specific circadian timing.

In birds, independent circadian clocks reside in the retina, pineal, and hypothalamus, which interact with each other and produce circadian time at the functional level. However, less is known of the molecular clockwork, and of the integration between central and peripheral clocks in birds. The present study investigated this, by monitoring the timed expression of five core clock genes (Per2. Cry1. Cry2. Bmal1, and Clock) and one clock-controlled gene (E4bp4) in a night-migratory songbird, the redheaded bunting (rb; Emberiza bruniceps). The authors first partially cloned these six genes, and then measured their 24-h profiles in central (retina, hypothalamus) and peripheral (liver, heart, stomach, gut, testes) tissues, collected at six times (zeitgeber time 2 [ZT2], ZT6, ZT11, ZT13, ZT18, and ZT23; ZT0 = lights on) from birds (n = 5 per ZT) on 12 h:12 h light-dark cycle. rbPer2. rbCry1. rbBmal1, and rbClock were expressed with a significant rhythm in all the tissues, except in the retina (only rbClock) and testes. rbCry2, however, had tissue-specific expression pattern: a significant rhythm in the hypothalamus, heart, and gut, but not in the retina, liver, stomach, and testes. rbE4bp4 had a significant mRNA rhythm in all the tissues, except retina. Further, rbPer2 mRNA peak was phase aligned with lights on, whereas rbCry1. rbBmal1, and rbE4bp4 mRNA peaks were phase aligned with lights off. rbCry2 and rbClock had tissue-specific scattered peaks. For example, both rbCry2 and rbClock peaks were close to rbCry1 and rbBmal1 peaks, respectively, in the hypothalamus, but not in other tissues. The results are consistent with the autoregulatory circadian feedback loop, and indicate a conserved tissue-level circadian time generation in buntings. Variable phase relationships between gene pairs forming positive and negative limbs of the feedback loop may suggest the tissue-specific contribution of individual core circadian genes in the circadian time generation.

Biological clocks and regulation of seasonal reproduction and migration in birds.

Timekeeping is important at two levels: to time changes in physiology and behavior within each day and within each year. For the former, birds have a system of at least three independent circadian clocks present in the retina of the eyes, the pineal gland, and the hypothalamus. This differs from the situation in mammals in which the input, pacemaker, and output are localized in different structures. Each bird clock interacts with at least one other clock, and together, they appear to form a centralized clock system that keeps daily time. These clocks have a powerful endogenous component, and the daily light-dark cycle entrains them to 24 h. The timing and duration of life history stages that make up annual cycle of an individual must also be controlled by some form of timekeeping. However, evidence for the existence of an equivalent endogenous circannual clock is less clear. Environmental cues, particularly photoperiod, appear to have a more direct role than simply entraining the clock to calendar time. For example, the timing of migration is probably greatly influenced by photoperiod, but its manifestation each day, as Zugunruhe, appears to be under circadian control. Migration involves marked changes in physiology to cope with the energetic demands. There is still much that we do not know about how organisms' timekeeping systems respond to their natural environment, particularly how salient signals from the environment are perceived and then transduced into appropriately timed biological functions. However, given that changes in environmental input affects the clock, increasing human disturbance of the environment is likely to adversely affect these systems.