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.

Photoperiodic modulation of melatonin receptor and immune genes in migratory redheaded bunting.

The transcriptional regulation of innate immune function across annual life history states (LHS) remains obscure in avian migrants. We, therefore, investigated this in a migratory passerine songbird, redheaded bunting (Emberiza bruniceps), which exhibits long-distance vernal migration from India to Central Asia. We exposed the birds (N = 10) to differential photoperiodic conditions to induce a non-migratory (NM), pre-migratory (PM), migratory (MIG), and refractory (REF) state, and performed gene expression assays of melatonin receptors (MEL1A and MEL1B), and innate immunity-linked genes (IL1B, IL6, TLR4, and NFKB) in spleen and blood. We found a significant reduction in splenic mass and volume, and a parallel increase in fat accumulation, and testicular growth in birds under migratory state. The gene expression assay revealed an upregulation of MEL1A and MEL1B mRNA levels in both the tissues in MIG. Additionally, we found a nocturnal increase of splenic IL1B expression, and IL1B, IL6, and TLR4 expression in the blood. The mRNA expression of melatonin receptors and proinflammatory cytokine showed a positive correlation. These results suggest that melatonin relays the photoperiodic signal to peripheral immune organs, which shows LHS-dependent changes in mRNA expression of immune genes.

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.

Ambient temperature affects multiple drivers of physiology and behaviour: adaptation for timely departure of obligate spring migrants.

We investigated the role of ambient temperature in departure from wintering areas of migratory black-headed buntings in spring. Birds transferred at 22 and 35°C to long days were compared with one another and with controls held on short days for indices of readiness to migrate (Zugunruhe, fattening, mass gain), levels of testosterone and gonadal recrudescence. Temperature affected the development of migratory behaviour and physiology: buntings under long days at 35°C, compared with those at 22°C, showed altered migratory behaviour (daily activity and Zugunruhe onset), and enhanced muscle growth and plasma testosterone levels, but showed no effect on testis growth. Temperature was perceived at both peripheral and central levels, and affected multiple molecular drivers culminating into the migratory phenotype. This was evidenced by post-mortem comparison of the expression of 13 genes with known functions in the skin (temperature-sensitive TRP channels: trpv4 and trpm8), hypothalamus and/or midbrain (migration-linked genes: th, ddc, adcyap1 and vps13a) and flight muscles (muscle growth associated genes: ar, srd5a3, pvalb, mtor, myod, mstn and hif1a). In photostimulated birds, the expression of trpv4 in skin, th in the hypothalamus and midbrain, and srd5a3, ar, pvalb and mtor in flight muscle, in parallel with testosterone levels, was greater at 35°C than at 22°C. These results demonstrate the role of ambient temperature in development of the spring migration phenotype, and suggest that transcriptional responsiveness to temperature is a component of the overall adaptive strategy in latitudinal songbird migrants for timely departure from wintering areas in spring.

Neuromorphometric changes associated with photostimulated migratory phenotype in the Palaearctic-Indian male redheaded bunting.

Neural substrates, including brain areas, differential gene expression and neuroendocrine basis, of migration are known. However, very little is known about structural changes in the brain that underlie the development and cessation of migration in long-distance avian migrants. Towards this, we investigated neuromorphological changes in the higher-order associative areas in male redheaded bunting (Emberiza bruniceps), which is a Palaearctic-Indian night migrant with wintering grounds in India. Photosensitive birds (8L:16D; SD) were exposed to stimulatory long days (16L:8D; LD), with controls retained on non-stimulatory short days. LD birds depicted shifts to, and sustained night-time activity as recorded by actograms. LD birds demonstrated increased body mass, fat deposition and testicular volume in keeping with the migratory phenotype. When LD birds had exhibited 10.0 ± 2.4 cycles of Zugunruhe (intense nighttime activity in captives, akin to night migratory flight in the wild), bird brains were fixed by transcardial perfusion, and changes in the neuronal morphometry of pallial, sub-pallial and hypothalamic brain regions studied using rapid Golgi technique with modifications, as used and validated in our laboratory. There were significant differences in both area and perimeter of soma in the visual hyperpallium apicale implicated in migratory orientation and the neuroendocrine control region for timing of migration, the mediobasal hypothalamus. We attribute these neuromorphometric changes in the soma area and perimeter to the photostimulated changes associated with the development of migration and reproductive phenotypes in redheaded buntings. It is suggested that changes in the neuronal plasticity in brain control regions parallel photoperiod-induced physiological responses.

Development of vernal migration in redheaded buntings: concurrent behavioral, physiological and neural changes under stimulatory photoperiods.

We investigated changes in behavior, physiology and selected brain regions during the development of vernal migration and reproduction phenotypes in migratory redheaded buntings. We monitored 24 h activity-rest pattern and measured food intake, fat deposition, and body mass of buntings exposed for 12 weeks to short (SP, 8L : 16D) and long (LP, 13L : 11D) photoperiods at 22 ± 2 °C temperature. Under LP, not SP, buntings exhibited a photostimulated spring migration phenotype (hyperphagia, fat deposition and body mass gain). However, there were sex differences in the development of vernal migration, as shown by faster and earlier induction of Zugunruhe (nocturnal migratory restlessness) in males than in females. In the next experiment, increasing photoperiods over 12 weeks following the vernal equinox induced behavioural and physiological changes associated with vernal migration phenotypes in both male and female buntings, but in a sex-dependent manner. In a subsequent experiment over 8 weeks corresponding to the spring migration period we found an increased expression of CART, not NPY, in INc, and decreased expression of GnRH-I in POA in the brain by week 6 of the observation under increasing photoperiods. There was also an increased expression of doublecortin (a marker of neuronal incorporation) in the olfactory bulb and song control nuclei (Area X and HVC, higher vocal centre) in male birds. These results demonstrate changes in the brain peptides and neuronal recruitment along with changes in the behaviour and physiology, and give insights into the concurrent photoperiodic induction of the seasonal response at multiple levels in migratory songbirds.

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.

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.

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.

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.

Nocturnal melatonin levels decode daily light environment and reflect seasonal states in night-migratory blackheaded bunting (Emberiza melanocephala).

We proposed two perhaps overlapping hypotheses. Hypothesis 1 examined whether daily light information was transduced by a change in the pattern of daily melatonin secretion. Hypothesis 2 tested whether the melatonin amplitude peak was contingent upon seasonal states. To test these hypotheses, we performed three experiments on night migratory blackheaded buntings (Emberiza melanocephala). The first two experiments measured plasma melatonin levels in buntings exposed to light-dark (LD) cycles, with white and/or blue (450 nm) and red (640 nm) light periods differing by about 10-12 fold in the level of illuminance. In birds exposed to a 12 h day (white light) at 0.66 W m(-2) (dim) and 7.85 W m(-2) (bright) light intensities (experiment 1), night melatonin levels were significantly affected by the end of 8-week exposure in the dim, not bright, day with buntings showing a bimodal nocturnal melatonin peak. Similarly, in birds on 13 h days in white, blue and red light periods at 0.028 W m(-2) (dim) and 0.28 (bright) W m(-2) intensities (experiment 2), mid night melatonin levels were significantly higher in red than in the blue or white light periods after 4.5 weeks of exposure. The third experiment measured plasma melatonin levels in buntings that were held under natural light conditions (NDL, 27°N; experiment 3A), or exposed to LD cycles (experiment 3B). There were slightly more elevated melatonin levels early at night during the spring (photosensitive) than during the autumn (photorefractory) migration season, without a difference in nocturnal melatonin peak between these two times providing almost similar light hours. Similarly, there was no difference in mid night melatonin levels between photosensitive and photorefractory buntings subjected to a skeleton photoperiod (1L : 11D : 1L : 11D), although melatonin levels were higher in the first than in the second 11 h dark phase in photosensitive birds. Overall, these results show that (i) nocturnal melatonin levels decode the intensity and wavelength of the daily light environment, and (ii) the daily melatonin secretion pattern subtly reflects seasonal states in the migratory blackheaded bunting.

Molecular basis of photoperiodic control of reproductive cycle in a subtropical songbird, the Indian weaver bird (Ploceus philippinus).

Less is known about genetic basis of photoperiodic regulation of reproductive cycle in subtropical birds. This study measured the expression levels of DIO2, DIO3, GnRH, and GnIH genes in Indian weaver birds subjected to short days (8h light:16h darkness, 8L:16D) and long days (16L:8D) for 48weeks. Whereas small, reproductively inactive testes were maintained under short days, weaver birds underwent testis recrudescence - regression cycle under long days. Relative expression levels of DIO2, DIO3, GnRH and GnIH genes were quantified by the real-time PCR (qPCR) in hypothalamus of birds (n=4) sampled at the beginning of the experiments, and after 10 and 48weeks of short and long day exposures. These sample times represented photosensitive unstimulated (day 0), and under long days the recrudescence (photostimulated, after 10weeks) and regression (photorefractory, after 48weeks) testicular phases. Birds under short days served as controls. The expression pattern of these genes corresponded with testicular phases. High and low GnRH and DIO2 levels were found in birds with large and small testes, respectively. By-and-large the converse was true for GnIH and DIO3 expression levels. Thus, after 10weeks of exposure, there was a significant difference in the mRNA levels between short and long day birds, with small and large testes, respectively. The results also suggest for a possible rapid switching between DIO2 and DIO3 and GnRH and GnIH expressions during testis maturation - regression cycle in Indian weaver birds.

Duration of melatonin regulates seasonal plasticity in subtropical Indian weaver bird, Ploceus philippinus.

Day length regulates seasonal plasticity connected with reproduction in birds. Rhythmic pineal melatonin secretion is a reliable indicator of the night length, hence day length. Removal of rhythmic melatonin secretion by exposure to constant bright light (LLbright) or by pinealectomy renders several species of songbirds including Indian weaver bird (Ploceus philippinus) arrhythmic. Present study investigated whether rhythmic melatonin is involved in the regulation of key reproductive neuropeptides (GnRH I and GnIH) and reproduction linked neural changes, viz. song control nuclei, in Indian weaver birds. Two experiments were performed using birds in an arrhythmic condition with low (under LLbright) or no (in the absence of pineal gland) endogenous melatonin. In experiment I, three groups of birds (n=5 each) entrained to 12L:12D were exposed to LLbright (25lux) for two weeks. Beginning on day 15 of LLbright, a control group received vehicle for 16h and two treatment groups were given melatonin in drinking water for 8h or 16h. In experiment II, one group of sham-operated and three groups of pinealectomized birds (n=5 each) entrained to 12L:12D were exposed to constant dim light (LLdim, 0.5lux). Beginning on day 15 of LLdim, three groups received similar treatment as in experiment I. Birds were perfused after thirty cycles of the melatonin treatment, and brain sections were immunohistochemically double-labeled for GnRH I and GnIH or Nissl stained. Activity was recorded throughout the experiments, while body mass and testes were measured at the beginning and end of the experiment. Birds were synchronized with melatonin cycles and measured the duration of melatonin as "night". Pinealectomized birds that received 16h of melatonin had significantly higher GnIH-ir cells than those received 8h melatonin; there was no difference in the GnRH I immunoreactivity between two treatment groups however. Intact birds that received long duration melatonin cycles exhibited small song control nuclei, specifically the high vocal center (HVC) and the robust nucleus of the arcopallium (RA), while birds that received short duration melatonin or no melatonin exhibited large HVC and RA. Thus, melatonin possibly regulates seasonal reproduction via GnIH secretion, and also controls seasonal neuroplasticity in the song control system in songbirds.

Adaptation of oxidative phosphorylation to photoperiod-induced seasonal metabolic states in migratory songbirds.

Eukaryotic cells produce chemical energy in the form of ATP by oxidative phosphorylation of metabolic fuels via a series of enzyme mediated biochemical reactions. We propose that the rates of these reactions are altered, as per energy needs of the seasonal metabolic states in avian migrants. To investigate this, blackheaded buntings were photoperiodically induced with non-migratory, premigratory, migratory and post-migratory phenotypes. High plasma levels of free fatty acids, citrate (an intermediate that begins the TCA cycle) and malate dehydrogenase (mdh, an enzyme involved at the end of the TCA cycle) confirmed increased availability of metabolic reserves and substrates to the TCA cycle during the premigratory and migratory states, respectively. Further, daily expression pattern of genes coding for enzymes involved in the oxidative decarboxylation of pyruvate to acetyl-CoA (pdc and pdk) and oxidative phosphorylation in the TCA cycle (cs, odgh, sdhd and mdh) was monitored in the hypothalamus and liver. Reciprocal relationship between pdc and pdk expressions conformed with the altered requirements of acetyl-CoA for the TCA cycle in different metabolic states. Except for pdk, all genes had a daily expression pattern, with high mRNA expression during the day in the premigratory/migratory phenotypes, and at night (cs, odhg, sdhd and mdh) in the nonmigratory phenotype. Differences in mRNA expression patterns of pdc, sdhd and mdh, but not of pdk, cs and odgh, between the hypothalamus and liver indicated a tissue dependent metabolism in buntings. These results suggest the adaptation of oxidative phosphorylation pathway(s) at gene levels to the seasonal alternations in metabolism in migratory songbirds.

Intermittent Food Absence Motivates Reallocation of Locomotion and Feeding in Spotted Munia (Lonchura punctulata).

BACKGROUND: Daily feeding and locomotion are interrelated behaviours. The time spent in feeding and rate of food intake depends on food availability. In low food condition, the birds would show intense movement (locomotion) for a longer time throughout the day however during abundant food supply they may chose higher activity and food intake in the morning and evening only. In the present study we hypothesized that in Spotted Munia (Lonchura punctulata), intermittent food availability during day would reallocate their interrelated behaviors, the feeding (food intake) and locomotor activity patterns. METHODS: Two groups of birds (N = 6 each) were kept individually in activity cages under 12L:12D. Group 1 (Control; C) had ad libitum food but group 2 (Treatment; T) had food for 6 hours only (2 h presence followed by 2 h absence; 2P:2A) during 12 hour light period. In the first week, group 2 received food with 'lights on' (TI; ZT 0-2, 4-6 and 8-10; where ZT 0= zeitgeber time 0, time of lights ON). In the following week, the food was given 2 hours after 'lights on' (TII; ZT 2-4, 6-8, 10-12). The food intake and locomotor activity under each condition were observed. RESULTS: The results showed that locomotor activity was induced during food deprivation and suppressed during food availability. Also the food deprivation led to increased food intake. CONCLUSION: Our results suggest that intermittent food availability/deprivation reallocates the locomotor activity and food intake in Spotted Munia.

Circadian rhythms are not involved in the regulation of circannual reproductive cycles in a sub-tropical bird, the spotted munia.

Circannual rhythms regulate seasonal reproduction in many vertebrates. The present study investigated whether circannual reproductive phenotypes (rhythms in growth of gonads and molt) were generated independently of the circadian clocks in the subtropical non-photoperiodic spotted munia (Lonchura punctulata). Birds were subjected to light:dark (LD) cycles with identical light but varying dark hours, such that the period of LD cycle (T) equaled 16 h (T16; 12 h L:4 h D), 21 h (T21; 12 h L:9 h D), 24 h (T24; 12 h L:12 h D) and 27 h (T27; 12 h L:15 h D), or to continuous light (LL, 24 h L:0 h D) at ~18°C. During the ~21 month exposure, munia underwent at least two cycles of gonadal development and molt; changes in body mass were not rhythmic. This was similar to the occurrence of annual cycles in reproduction and molt observed in wild birds. A greater asynchrony between circannual cycles of gonad development and molt indicated their independent regulation. Females showed reproductive rhythms with similar circannual periods, whilst in males, circannual periods measured between peak gonadal size were longer in T21 and T24 than in T16 or T27. This suggested a sex-dependent timing of annual reproduction in the spotted munia. Also, food availability periods may not influence the circannual timing of reproduction, as shown by the results on the rhythm in gonadal growth and regression in munia under T-photocycles and LL that provided differential light (feeding) hours. Further, a short-term experiment revealed that activity-rest patterns in munia were synchronized with T-photocycles, but were arrhythmic under LL. We conclude that circadian rhythms are not involved in the timing of the annual reproductive cycle in the spotted munia.

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.

Photoperiodic regulation of seasonal reproduction in higher vertebrates.

Long-lived animals such as birds and mammals adapt readily to seasonal changes in their environment. They integrate environmental cues with their internal clocks to prepare and time seasonal physiological changes. This is reflected in several seasonal phenotypes, particularly in those linked with migration, hibernation, pelage growth, reproduction and molt. The two endocrine secretions that play key roles in regulating the seasonal physiology are melatonin and thyroid hormone. Whereas, melatonin is used as an endocrine index of day length (and consequently duration of night), the seasonal up- and down-regulation of thyroid hormone affects the physiology, perhaps by influencing different pathways. Both of these hormones are shown to act via a 'photoperiodic axis' constituted by the photoreceptors, hypothalamus and pituitary. Recent studies have revealed that the pars tuberalis that connects hypothalamus and pituitary, locally synthesizes the thyroid stimulating hormone (TSH) in response to light (birds) or melatonin (mammals). The levels of TSH regulate the DIO2 and DIO3 synthesis in the ependymal cells in hypothalamus, and in turn affect the release of gonadotropin releasing hormone. This review mainly focuses on the current understanding of the mechanisms of photoperiodic regulation of seasonal responses in the higher vertebrates.

Photoperiodic effects on activity behaviour in the spiny eel (Macrognathus pancalus).

The study focused on the characteristics of circadian locomotor activity in the spiny eel, M. pancalus, kept under different photoperiodic conditions. Two experiments were conducted. Experiment 1 tested the light intensity dependent effect on circadian rhythmicity of the locomotor activity in spiny eel. Three groups of fish were entrained to 12L:12D conditions for 10 days. Thereafter, they were released to constant conditions for 15 days as indicated below: group 1-DD (0 lux), group 2- LL(dim) (-1 lux) and group 3-LL(bright) (-500 lux). The locomotor activity of the fish, housed singly in an aquarium, was recorded continuously with infrared sensors connected to a computer. More than 90% activity of the eels was confined to the dark hours suggesting nocturnal habit. Under constant conditions, the activity in 7/9 fish in group 1, 4/8 in group 2 and 3/8 in group 3, started free running with a mean circadian period of 24.48 +/- 0.17 h, 23.21 +/- 0.47 h and 25.54 +/- 1.13 h in respective groups. Remaining fish in each group became arrhythmic. This suggests that spiny eel can be synchronised to LD cycle and under constant conditions they free run with a circadian period. However, their activity under LL is light intensity dependent; higher the intensity, more disruption in circadian locomotor activity. Experiment 2 was conducted to study the effect of decreasing night length (increasing photoperiod) on circadian locomotor activity. The fish were sequentially exposed to 16D (8L:16D), 12D (12L:12D), 8D (16L:8D), 4D (20L:4D) and 2D (22L:2D) for 10 days in each condition, thereafter, they were released in constant dark (DD= 0lux). The results showed that the duration of night length affects both, the amplitude and duration of locomotor activity. It can be concluded that the spiny eels are nocturnal and that their locomotor activity is under the circadian control and may be influenced by the photoperiod.

Light wavelength dependent circadian and seasonal responses in blackheaded bunting.

Animals in the wild are exposed to daily variations in sun light, viz. duration, intensity and spectrum. Photosensitive blackheaded buntings (Emberiza melanocephala) were exposed to photoperiods differing in the length of light period, wavelengths and intensity. The effects of such light changes were measured on locomotor activity rhythm as well as seasonal responses like development of migratory restlessness: Zugunruhe, body mass and gonadal growth. The results show that the buntings are differentially responsive to light wavelengths and intensities and are indicative of a phase-dependent action of light on the circadian photoperiodic system. These birds seem to use changes in the light variables of the solar environment to regulate their circadian and seasonal responses.