552323
research-article2014
JBRXXX10.1177/0748730414552323Journal Of Biological RhythmsAshley et al. / Circadian Rhythms in Arctic Songbirds
Revealing a Circadian Clock in Captive Arctic-Breeding Songbirds, Lapland Longspurs (Calcarius lapponicus), under Constant Illumination Noah T. Ashley,*,1 Takayoshi Ubuka,† Ingrid Schwabl,‡ Wolfgang Goymann,‡ Brady M. Salli,§ George E. Bentley,|| and C. Loren Buck§ *Department of Biology, Western Kentucky University, Bowling Green, Kentucky, USA, †Department of Biology, Waseda University, Shinjuku, Tokyo, Japan, ‡Abteilung für Verhaltensneurobiologie, Max-Planck-Institut für Ornithologie, Seewiesen, Germany, §Department of Biological Sciences, University of Alaska Anchorage, Alaska, USA, and ||Department of Integrative Biology, Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA Abstract Most organisms in temperate or tropic regions employ the light-dark (LD) cycle as the primary Zeitgeber to synchronize circadian rhythms. At higher latitudes (>66°33′), continuous illumination during the summer presents a significant time-keeping dilemma for polar-adapted species. Lapland longspurs (Calcarius lapponicus), arctic-breeding migratory songbirds, are one of the few recorded species maintaining an intact diel rhythm in activity and plasma melatonin titers during polar summer. However, it is unknown whether rhythms are endogenous and entrain to low-amplitude polar Zeitgeber signals, such as daily variations in light intensity and the spectral composition of the sun (as measured by color temperature). Wild-caught male and female longspurs were brought into captivity, and locomotor activity was assessed using infrared detection. To examine if rhythms were endogenous, birds were exposed to constant bright light (LL; 1300 lux) or constant darkness (DD; 0.1 lux). All birds exhibited freerunning activity rhythms in LL and DD, suggesting the presence of a functional circadian clock. Mean periods in LL (22.86 h) were significantly shorter than those in DD (23.5 h), in accordance with Aschoff’s rule. No birds entrained to diel changes in light intensity, color temperature, or both. To examine endogenous molecular clock function, the Per2 gene was partially cloned in longspurs (llPer2) and transcripts were measured in hypothalamic tissue punches, eye, and liver using competitive polymerase chain reaction. Ocular llPer2 gene expression was periodic in LL and elevated at ZT24 (CT24) for LD or constant conditions (LL and DD), but llPer2 rhythmicity was not detected in hypothalamus or liver. Plasma melatonin was significantly lower in LL compared with LD or DD. In conclusion, rhythmic ocular Per2 expression and melatonin secretion may maintain the circadian activity rhythm across the polar day. Keywords circadian rhythm, clock genes, Lapland longspur, melatonin, Per2
1. To whom all correspondence should be addressed: Noah T. Ashley, Department of Biology, Western Kentucky University, 1906 College Heights Blvd. #11080, Bowling Green, KY 42101-1080, USA; e-mail: [email protected]. JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 29 No. 6, December 2014 456–469 DOI: 10.1177/0748730414552323 © 2014 The Author(s)
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The primary environmental cue, or Zeitgeber, that synchronizes daily rhythms of most tropical- and temperate-dwelling organisms is the solar light-dark (LD) cycle (Pittendrigh, 1981; Aschoff, 1989). In Antarctica or north of the Arctic Circle (>66°33′ latitude), this Zeitgeber is noticeably lacking for much of the summer and winter months, where continuous light (polar day) or darkness (polar night), respectively, prevails (Pielou, 1994). The seasonal absence of the LD cycle poses a unique dilemma to the regulation of circadian rhythms among polar-adapted organisms. Several adaptations of the biological clock have evolved to cope with continuous polar day or night. Some species, such as reindeer (Rangifer tarandus) and ptarmigan (Lagopus mutus), completely abandon circadian organization during winter and summer and resort to ultradian rhythms (Stokkan et al., 1994; Eloranta et al., 1995; Reierth and Stokkan, 1998; van Oort et al., 2005; Bloch et al., 2013). Among high-arctic shorebirds, there is a diversity of time-keeping approaches depending on species, sex, and breeding stage. During the preincubation stage, birds are arrhythmic but switch at incubation to either a freerunning rhythm that is synchronized among pairs (semipalmated sandpiper [Calidris pusilla]), a robust 24-h rhythm (caregiving sex of uniparental shorebirds), or remain arrhythmic (competitive sex of polygamous shorebirds; Steiger et al., 2013). Lastly, several taxa, such as migratory songbirds (Hau et al., 2002; Silverin et al., 2009; Ashley et al., 2013; Steiger et al., 2013), arctic ground squirrels (Uroceitelllus parryii; Swade and Pittendrigh, 1967; Long et al., 2005; Williams et al., 2012), and bumblebees (Bombus spp.; Stelzer and Chittka, 2010), exhibit consistent intact rhythms in behavior and physiology throughout the polar summer. These observations suggest entrainment to alternative environment cues that may include subtle daily variations in light intensity, spectral composition of light, or ambient temperature, but experimental evidence is lacking (Swade and Pittendrigh, 1967; Krüll, 1985; Pohl, 1999). Presumably, persistence of diel rhythms is necessary in these particular species to capitalize upon diel fluctuations in food supply, predators, and/or thermoregulatory requirements. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) serves as the major pacemaker that regulates daily cycles in physiology and behavior (Buhr and Takahashi, 2013). However, in birds, the pineal gland, the retinae of the eyes, and hypothalamus act as independent oscillators that are mutually coupled (Gwinner and Brandstätter, 2001; Cassone, 2014). Within the hypothalamus, 2 nuclei are involved with circadian organization in birds: the medial suprachiasmatic nucleus (mSCN) and visual
suprachiasmatic nucleus (vSCN; Cassone and Moore, 1987; Yoshimura et al., 2001). On a molecular level, these oscillators are controlled by a highly conserved mechanism that involves rhythmic transcription and translation of clock genes. The major components of the clock machinery involve the interplay between positive elements clock and bmal1 and negative elements Period 1 (Per1), Per2, Per3, and cryptochrome 1 (cry1) and cry2 (Tei et al., 1997; Zheng et al., 1999a; Zheng et al., 1999b; Yoshimura et al., 2000; Yasuo et al., 2002; Yasuo et al., 2004ab; Helfer et al., 2006). Per1 expression is absent in birds (Yoshimura et al., 2000; Yasuo et al., 2002). Avian homologues of Per2 have been cloned in a variety of bird species (e.g., Japanese Quail [Coturnix coturnix japonica], chicken [Gallus domesticus], house sparrow [Passer domesticus], blue tit [Cyanistes caeruleus], and redheaded bunting [Emberiza bruniceps]; Yoshimura et al., 2000; Brandstätter et al., 2001; Doi et al., 2001; Steinmeyer et al., 2012; Singh et al., 2013). Circadian expression of Per2 has been demonstrated in the eye and pineal gland (Yoshimura et al., 2000; Doi et al., 2001). In addition, several clock genes are expressed rhythmically in the mSCN of quail (Yasuo et al., 2002), whereas house sparrows generate independent pPer2 rhythms in the mSCN and vSCN (Abraham et al., 2002; Abraham et al., 2003). Lapland longspurs (Calcarius lapponicus), songbirds that migrate to Alaska and northern Canada to breed every year from wintering areas in temperate regions, maintain diel rhythms in activity during polar day with a quiescence period of only 4 to 5 h (Ashley et al., 2013; Steiger et al., 2013). Plasma melatonin is also rhythmic (Hau et al., 2002; Ashley et al., 2013) and negatively correlated with daily fluctuations in light intensity and ambient temperature (Ashley et al., 2013). The spectral composition of sunlight varies over the polar day (measured by color temperature [K]; Pohl, 1999), and birds possess photoreceptors that are sensitive to differing wavelengths of light (Hart, 2001). Thus, it is possible that diel fluctuations in the spectral qualities of sunlight could synchronize rhythms. At this juncture, it is unclear whether these low-amplitude photic and thermal cues act as alternative zeitgeber signals (Swade and Pittendrigh, 1967; Krüll, 1976b, c; Pohl, 1999) or simply mask overt activity rhythms (Aschoff and von Goetz, 1989). The aims of this study were 3-fold. We sought to determine (1) whether longspurs maintain an endogenous circadian rhythm when exposed to constant light (LL) or darkness (DD) compared with LD, (2) whether manipulations of light intensity (quantity) and color temperature (quality) can induce entrainment, and (3) whether circadian clock gene expression, specifically Per2, exhibits rhythmicity similar to
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patterns observed in house sparrows (Brandstätter et al., 2001; Abraham et al., 2002; Helfer et al., 2006) and other avian species (Yoshimura et al., 2000; Doi et al., 2001; Singh et al., 2013). Per2 mRNA levels peak early in the day and are light sensitive (Abraham et al., 2002; Yasuo et al., 2003; Helfer et al., 2006; Singh et al., 2013). We hypothesized that longspurs would possess a functioning circadian clock and therefore would display free-running rhythms in LL and DD compared with LD. We further predicted that experimental alterations in light intensity and color temperature would entrain behavioral rhythms and that Per2 expression in hypothalamus, eye, and liver would exhibit rhythmicity and specifically increase during subjective day in LD and constant conditions.
Materials and Methods Animals Twenty-eight male and 20 female Lapland longspurs were captured in Barrow, Alaska (71°N, 156°W) in the summer of 2009 (for study site details, see Ashley et al., 2013) and held in temporary indoor aviaries before transport by plane to the University of Alaska Anchorage (UAA; 61°N, 150°W) in modified pet carriers on July 1. Birds were then housed indoors within the UAA vivarium in 2 indoor flight aviaries on a light cycle representative of short day lengths (8L:16D) at a temperature of 20 °C ± 1 °C. Birds were provided with food (bird seed mix) and water ad libitum and given weekly protein supplements of moistened egg food (Quiko) and live mealworms. Procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the UAA Institutional Animal Care and Use Committee, as well as municipal, state, tribal, and federal authorities. Experimental Settings After birds finished their prebasic molt (October 3), subjects were randomly assigned to 1 of 3 rooms of identical dimensions that contained individual bird cages (36.2 × 41.3 × 43.8 cm) on a stainless-steel rack (4 cages per row). After random assignment, we attempted to balance sex ratio across each group: room 1 (9 males, 7 females), room 2 (9 males, 7 females), room 3 (10 males, 6 females). To provide a measure of general activity, each cage was outfitted with infrared motion detectors (model PK IS215T; Honeywell, Morristown, NJ) to measure beam breaks. Each detector was placed about 10 cm above the
upper perch of the cage. The detector produced a downward, cone-shaped beam that permitted detection of movement within all areas of the cage. To eliminate interference with other detectors, cardboard dividers were placed between adjacent cages. This manipulation also visually isolated birds from each other. Each detector was wired to an interface box that could accommodate up to 7 different channels; each interface box (7 total) was connected to a 56-channel hub directly plugged into a PC computer in an outer room running ClockLab (Actimetrics, Wilmette, IL). The number of beam breaks was recorded for each channel and binned into 5-min intervals. Lighting To ensure that birds were exposed to similar lighting conditions and to manipulate light intensity and color temperature, each room was outfitted with custom-built light sources. On a separate metal rack, 224cm (8-ft) fluorescent bulbs (32 W, 6500 K color temperature; Sylvania, Danvers, MA) were mounted in quadruplet vertically. In total, there were 3 quadruplets of bulbs spaced evenly. Within each quadruplet, only 2 identical bulbs were turned on at once. This arrangement allowed for instantaneous switching between different light sources that differed in light intensity, color temperature, or both. We quantified light intensity (lux) using a light meter at each bird’s cage, and the distance from the light source to the cages was standardized such that each cage received about 1300 lux (subjective “day” light setting). This value approximates the lower threshold of light intensities recorded in polar summer (Arendt, 2012; Ashley et al., 2013). Light schedules (timing of on/off) were controlled by computer using Clocklab’s integrated light control module. A separate “night” light that emitted only blue light was continuously on (plugged into an outlet on the opposing wall relative to birds); dim blue light is often employed in avian studies to prevent photostimulation because its shorter wavelengths fail to penetrate the avian skull to activate deep encephalic photoreceptors compared with the longer wavelengths of red light (Oishi and Lauber, 1973). When fluorescent lights were switched off (referred to as “dark”), the light intensity was about 0.1 lux. Experiments Experiment 1. From October 3 to 16, birds were exposed to a 16L:8D photoperiod with lights-on at 0700 h. This photoperiod was chosen to acclimate birds to long days before continuous exposure to LL. All birds entrained to 16L:8D. To examine activity
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rhythms of birds to constant conditions (LL and DD) relative to LD, birds were exposed to the same order of light treatments—(1) LL (October 16-22), (2) 12L:12D (October 22-27), (3) DD (October 27-29)— instead of counterbalancing treatments across rooms. This ensured that birds from separate rooms were not differentially photostimulated, which could have an effect on rhythms when the experiment was repeated (see below). Activity rhythms were recorded throughout these periods. The LD treatment (lights-on at 0700 h) was deliberately placed after the LL treatment to reentrain birds that displayed free-running rhythms. The DD treatment had to be shortened to 3 d because some birds were losing body mass because little feeding occurred in darkness. Activity onsets and offsets were estimated using the automated detection function in Clocklab. Free-running periods were calculated by periodogram analysis using Clocklab software for ≥3 d of light treatment. Experiment 2. From October 27 to November 10, a separate set of experiments was conducted to examine whether alterations in either light intensity or color temperature were sufficient to entrain these birds. For these studies, birds were placed in 3 d of DD to induce free-running rhythms and then exposed to various light exposures. In room 1, we tested whether a 55% reduction in light intensity could lead to entrainment. To manipulate light intensity without altering color temperature, a 2-stop 0.5 neutral density filter was used (Rosco, Stamford, CT) to reduce light intensity by 55%. In this group, birds received 12 h of 780 lux (reduced from 1300 lux by disconnecting 1 of 2 lights per quadruplet) followed by 12 h of 350 lux (same bulbs with filter). This latter value represents lower light intensity that falls just below minimum light intensities recorded in the field in Barrow, Alaska, during polar summer (Ashley et al., 2013). Color temperature remained the same (6500 K) and was verified by a precision colormeter (Cal-COLOR 400; Cooke Corp., Romulus, MI). In room 2, color temperature was manipulated without altering light intensity by using bulbs with the same wattage. In this group, birds received 12 h of 6500 K at 780 lux (blue light) and then 12 h of 3500 K at 780 lux (red light). Again, this alteration in spectral composition is similar to the range of natural light spectra encountered in northern Alaska (Ashley et al., 2013). In room 3, birds were exposed to 12 h L (780 lux, 6500 K) and then 12 h D. After 6 d (November 5), all birds were exposed to DD for 3 d. To investigate whether simultaneous reductions in light intensity and color temperature entrained activity rhythms, birds in rooms 2 and 3 were exposed to 12 h of 780 lux at 6500 K and then 12 h of 350 lux at 3500 K for 3 d (November 7-10). Birds in room 1
Figure 1. Experimental time line. (A) Male and female longspurs were maintained on a nonbreeding photoperiod of 8L:16D for approximately 3 mo and then transferred to individual cages. Birds were exposed to 16L:8D for 2 wk. (B) Longspurs were sequentially exposed to LL (1300 lux, 6500 K), 12L (1300 lux, 6500 K):12D, and DD treatments and activity rhythms were measured. (C) Light intensity (lux) or color temperature (K) were reduced by 55% and 46%, respectively, for the second 12 h of the daily 24-h cycle, and entrainment of activity rhythms was assessed: group 1, 12L (780 lux, 6500 K, white):12L (350 lux, 6500 K; gray); group 2, 12L (780 lux, 6500 K):12L (780 lux, 3500 K; red). These treatments followed exposure to DD (0.1 lux) for 3 d, which typically induced free-running rhythms. An additional manipulation that involved simultaneous reductions in light intensity and color temperature was conducted on 2 groups of longspurs to assess entrainment of rhythms: groups 2 and 3, 12L (780 lux, 6500 K):12L (350 lux, 3500 K; pink). Throughout these experiments, a third group was exposed to 12L:12D to serve as a control. (D) Birds were entrained to 12L:12D; exposed to either LD, DD, or LL; and then euthanized at 4 different time points to assess Per2 expression in hypothalamus, eye, and liver.
received the control treatment of 12 h of L (1300 lux, 6500 K) followed by 12 h of darkness for 3 d (Fig. 1). Experiment 3. To examine patterns of Per2 gene expression in the brain, eye, and liver in relation to LD, LL, and LD groupings, birds were first exposed to 12L (1300 lux, 6500 K):12D for 6 d (November 11-18; Fig. 1). Phasing of entrainment was staggered across rooms such that lights turned on in rooms 1, 2, and 3 at 1000, 0900, and 1100 h, respectively, and all birds synchronized activity rhythms. A staggered design
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prevented tissue collection from occurring at the same time in all rooms. On November 17, rooms 1, 2, and 3 were switched to DD, LL, and LD at 1000, 0900, and 1100, respectively. We followed a similar sampling protocol used to examine pper2 expression in house sparrows that involved sampling of tissue over a 48-h period (Abraham et al., 2002). Starting at circadian time (CT) 6 or zeitgeber time (ZT) 6 (midday), 2 Lapland longspurs were taken from their cage and blood samples were obtained (see specific methods below). Birds were then immediately exposed to isoflurane vapors using a vaporizer (5% induction) for 0.17). Given the results, the response of longspurs to light intensity or color temperature treatments (free-run or arrhythmia) differed significantly from those exposed to LD (entrainment; Fisher exact tests, all p < 0.0001). Experiment 3 All birds entrained to the 12L:12D photoperiod before starting the experiment. For llPer2 expression of hypothalamic punches, there were no significant effects of light treatment (2-way ANOVA; F2,35 = 2.29, p = 0.12), sampling time (F3,35 = 0.84, p = 0.48), or their interaction (F6,35 = 1.12, p = 0.37; Fig. 5A). Moreover,
Figure 4. Representative actograms (double-plotted) of longspur activity rhythms subjected to 12L:12D, 3 d of DD, and then (A) 12L (780 lux, 6500 K; horizontal white bar):12 DD (black bar; control), (B) 12 L (780 lux, 6500 K):12L (780 lux, 3500 K; lower gray bar designated as “red”), or (C) 12 L (780 lux, 6500 K):12L (380 lux, 6500 K, gray bar). Horizontal bars on the top of graphs represent light-dark schedule (black depicts dark phase) of initial 12L:12D treatment. Horizontal bars on the bottom of graphs represent experimental light regimes. B shows a tendency toward a freerunning rhythm, whereas C displays arrhythmia.
hypothalamic llPer2 expression did not display a periodic relationship with time of day (harmonic regression, all p’s > 0.05). However, there was a significant effect of sampling time on llPer2 expression in the eye (2-way ANOVA; F3,36 = 5.17, p = 0.005) but no significant effects of light treatment (F2,36 = 0.98, p = 0.38) or its interaction with sampling time (F6,36 = 1.20, p = 0.33). More specifically, ocular llPer2 expression was higher at 24 h compared with 6, 12, and 18 h (Fisher PLSD, all p < 0.048) for LD and LL birds (Fig. 5B). These findings were further corroborated by significant circadian periodicity identified in ocular llPer2 expression of LL birds (harmonic regression, F2,13 = 4.48, p = 0.03, R2 = 0.408) but not LD birds.
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Ashley et al. / CIRCADIAN RHYTHMS IN ARCTIC SONGBIRDS 463
Figure 5. Per2 mRNA concentration in (A) hypothalamus (zmol/mg total RNA), (B) eye (amol/mg total RNA), and (C) liver (amol/mg total RNA) and (D) plasma melatonin concentration (pg/mL) at 4 different time points in LD, LL, and DD. Bars represent mean ± SEM. Horizontal bars on top of graphs represent light-dark schedule (black depicts dark phase). ZT18 and CT18 are double-plotted for better visualization of rhythms. Within-group comparisons that share letters indicate no significant differences between time points. n = 4 per group, except for the hypothalamus, ZT24/0 group (n = 3).
For llPer2 expression in liver, there was a significant effect of lighting treatment (2-way ANOVA, F2,36 = 7.39, p = 0.002). Neither sampling time (F3,36 = 1.70, p = 0.19) nor its interaction with lighting treatment
(F6,36 = 1.48, p = 0.22) was statistically significant. On average, llPer2 expression in liver was significantly higher in LD birds compared with LL and DD longspurs (Fisher PLSD, all p < 0.004; Fig. 5C). Liver llPer2
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expression was not associated with time of day (harmonic regression, all p’s > 0.05). Lighting treatment (F2,36 = 6.97, p = 0.003) and sampling time (F3,36 = 17.06, p < 0.0001) had significant effects on plasma melatonin concentration, but there was no significant interaction effect between the 2 factors (F6,36 = 0.82, p = 0.56). Irrespective of time, average melatonin concentration was suppressed in LL longspurs compared with LD and DD birds (Fig. 5D; Fisher PLSD, all p < 0.014). Irrespective of treatment, melatonin concentration was significantly elevated at 18 h relative to 6-, 12-, and 24-h sampling periods (Fisher PLSD, all p < 0.001). Furthermore, a significant melatonin rhythm in relation to the 24-h cycle was identified in LD birds (harmonic regression; F2.13 = 5.33, p = 0.02, R2 = 0.45) but not in LL or DD birds.
Discussion Free-Running Rhythms in Constant LL and DD Male and female longspurs exhibited period lengths that were significantly less than 24 h when exposed to LL or DD, indicating the presence of an endogenous clock(s) that regulates locomotor activity. These findings also support the hypothesis that free-living longspurs are likely synchronizing rhythms to low-amplitude polar Zeitgeber cues because our data indicate that an environment lacking these potential entraining agents, namely, constant captive conditions, induces free-running activity rhythms. Although exposure to bright LL (2000 lux) can induce rapid arrhythmia in some species (e.g., pigeons, Columba livia; Yamada et al., 1988), house sparrows will initially exhibit a short free-running period for >7 d when exposed to LL (100 lux), but rhythms eventually become arrhythmic (Cassone et al., 2008). Even though our study used lighting of higher intensity (1300 lux) to attempt to mimic the lower threshold of polar-day light intensities, longspurs still expressed free-running rhythms. Because activity rhythms were assessed for a short period of time (
research-article2014
JBRXXX10.1177/0748730414552323Journal Of Biological RhythmsAshley et al. / Circadian Rhythms in Arctic Songbirds
Revealing a Circadian Clock in Captive Arctic-Breeding Songbirds, Lapland Longspurs (Calcarius lapponicus), under Constant Illumination Noah T. Ashley,*,1 Takayoshi Ubuka,† Ingrid Schwabl,‡ Wolfgang Goymann,‡ Brady M. Salli,§ George E. Bentley,|| and C. Loren Buck§ *Department of Biology, Western Kentucky University, Bowling Green, Kentucky, USA, †Department of Biology, Waseda University, Shinjuku, Tokyo, Japan, ‡Abteilung für Verhaltensneurobiologie, Max-Planck-Institut für Ornithologie, Seewiesen, Germany, §Department of Biological Sciences, University of Alaska Anchorage, Alaska, USA, and ||Department of Integrative Biology, Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA Abstract Most organisms in temperate or tropic regions employ the light-dark (LD) cycle as the primary Zeitgeber to synchronize circadian rhythms. At higher latitudes (>66°33′), continuous illumination during the summer presents a significant time-keeping dilemma for polar-adapted species. Lapland longspurs (Calcarius lapponicus), arctic-breeding migratory songbirds, are one of the few recorded species maintaining an intact diel rhythm in activity and plasma melatonin titers during polar summer. However, it is unknown whether rhythms are endogenous and entrain to low-amplitude polar Zeitgeber signals, such as daily variations in light intensity and the spectral composition of the sun (as measured by color temperature). Wild-caught male and female longspurs were brought into captivity, and locomotor activity was assessed using infrared detection. To examine if rhythms were endogenous, birds were exposed to constant bright light (LL; 1300 lux) or constant darkness (DD; 0.1 lux). All birds exhibited freerunning activity rhythms in LL and DD, suggesting the presence of a functional circadian clock. Mean periods in LL (22.86 h) were significantly shorter than those in DD (23.5 h), in accordance with Aschoff’s rule. No birds entrained to diel changes in light intensity, color temperature, or both. To examine endogenous molecular clock function, the Per2 gene was partially cloned in longspurs (llPer2) and transcripts were measured in hypothalamic tissue punches, eye, and liver using competitive polymerase chain reaction. Ocular llPer2 gene expression was periodic in LL and elevated at ZT24 (CT24) for LD or constant conditions (LL and DD), but llPer2 rhythmicity was not detected in hypothalamus or liver. Plasma melatonin was significantly lower in LL compared with LD or DD. In conclusion, rhythmic ocular Per2 expression and melatonin secretion may maintain the circadian activity rhythm across the polar day. Keywords circadian rhythm, clock genes, Lapland longspur, melatonin, Per2
1. To whom all correspondence should be addressed: Noah T. Ashley, Department of Biology, Western Kentucky University, 1906 College Heights Blvd. #11080, Bowling Green, KY 42101-1080, USA; e-mail: [email protected]. JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 29 No. 6, December 2014 456–469 DOI: 10.1177/0748730414552323 © 2014 The Author(s)
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The primary environmental cue, or Zeitgeber, that synchronizes daily rhythms of most tropical- and temperate-dwelling organisms is the solar light-dark (LD) cycle (Pittendrigh, 1981; Aschoff, 1989). In Antarctica or north of the Arctic Circle (>66°33′ latitude), this Zeitgeber is noticeably lacking for much of the summer and winter months, where continuous light (polar day) or darkness (polar night), respectively, prevails (Pielou, 1994). The seasonal absence of the LD cycle poses a unique dilemma to the regulation of circadian rhythms among polar-adapted organisms. Several adaptations of the biological clock have evolved to cope with continuous polar day or night. Some species, such as reindeer (Rangifer tarandus) and ptarmigan (Lagopus mutus), completely abandon circadian organization during winter and summer and resort to ultradian rhythms (Stokkan et al., 1994; Eloranta et al., 1995; Reierth and Stokkan, 1998; van Oort et al., 2005; Bloch et al., 2013). Among high-arctic shorebirds, there is a diversity of time-keeping approaches depending on species, sex, and breeding stage. During the preincubation stage, birds are arrhythmic but switch at incubation to either a freerunning rhythm that is synchronized among pairs (semipalmated sandpiper [Calidris pusilla]), a robust 24-h rhythm (caregiving sex of uniparental shorebirds), or remain arrhythmic (competitive sex of polygamous shorebirds; Steiger et al., 2013). Lastly, several taxa, such as migratory songbirds (Hau et al., 2002; Silverin et al., 2009; Ashley et al., 2013; Steiger et al., 2013), arctic ground squirrels (Uroceitelllus parryii; Swade and Pittendrigh, 1967; Long et al., 2005; Williams et al., 2012), and bumblebees (Bombus spp.; Stelzer and Chittka, 2010), exhibit consistent intact rhythms in behavior and physiology throughout the polar summer. These observations suggest entrainment to alternative environment cues that may include subtle daily variations in light intensity, spectral composition of light, or ambient temperature, but experimental evidence is lacking (Swade and Pittendrigh, 1967; Krüll, 1985; Pohl, 1999). Presumably, persistence of diel rhythms is necessary in these particular species to capitalize upon diel fluctuations in food supply, predators, and/or thermoregulatory requirements. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) serves as the major pacemaker that regulates daily cycles in physiology and behavior (Buhr and Takahashi, 2013). However, in birds, the pineal gland, the retinae of the eyes, and hypothalamus act as independent oscillators that are mutually coupled (Gwinner and Brandstätter, 2001; Cassone, 2014). Within the hypothalamus, 2 nuclei are involved with circadian organization in birds: the medial suprachiasmatic nucleus (mSCN) and visual
suprachiasmatic nucleus (vSCN; Cassone and Moore, 1987; Yoshimura et al., 2001). On a molecular level, these oscillators are controlled by a highly conserved mechanism that involves rhythmic transcription and translation of clock genes. The major components of the clock machinery involve the interplay between positive elements clock and bmal1 and negative elements Period 1 (Per1), Per2, Per3, and cryptochrome 1 (cry1) and cry2 (Tei et al., 1997; Zheng et al., 1999a; Zheng et al., 1999b; Yoshimura et al., 2000; Yasuo et al., 2002; Yasuo et al., 2004ab; Helfer et al., 2006). Per1 expression is absent in birds (Yoshimura et al., 2000; Yasuo et al., 2002). Avian homologues of Per2 have been cloned in a variety of bird species (e.g., Japanese Quail [Coturnix coturnix japonica], chicken [Gallus domesticus], house sparrow [Passer domesticus], blue tit [Cyanistes caeruleus], and redheaded bunting [Emberiza bruniceps]; Yoshimura et al., 2000; Brandstätter et al., 2001; Doi et al., 2001; Steinmeyer et al., 2012; Singh et al., 2013). Circadian expression of Per2 has been demonstrated in the eye and pineal gland (Yoshimura et al., 2000; Doi et al., 2001). In addition, several clock genes are expressed rhythmically in the mSCN of quail (Yasuo et al., 2002), whereas house sparrows generate independent pPer2 rhythms in the mSCN and vSCN (Abraham et al., 2002; Abraham et al., 2003). Lapland longspurs (Calcarius lapponicus), songbirds that migrate to Alaska and northern Canada to breed every year from wintering areas in temperate regions, maintain diel rhythms in activity during polar day with a quiescence period of only 4 to 5 h (Ashley et al., 2013; Steiger et al., 2013). Plasma melatonin is also rhythmic (Hau et al., 2002; Ashley et al., 2013) and negatively correlated with daily fluctuations in light intensity and ambient temperature (Ashley et al., 2013). The spectral composition of sunlight varies over the polar day (measured by color temperature [K]; Pohl, 1999), and birds possess photoreceptors that are sensitive to differing wavelengths of light (Hart, 2001). Thus, it is possible that diel fluctuations in the spectral qualities of sunlight could synchronize rhythms. At this juncture, it is unclear whether these low-amplitude photic and thermal cues act as alternative zeitgeber signals (Swade and Pittendrigh, 1967; Krüll, 1976b, c; Pohl, 1999) or simply mask overt activity rhythms (Aschoff and von Goetz, 1989). The aims of this study were 3-fold. We sought to determine (1) whether longspurs maintain an endogenous circadian rhythm when exposed to constant light (LL) or darkness (DD) compared with LD, (2) whether manipulations of light intensity (quantity) and color temperature (quality) can induce entrainment, and (3) whether circadian clock gene expression, specifically Per2, exhibits rhythmicity similar to
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patterns observed in house sparrows (Brandstätter et al., 2001; Abraham et al., 2002; Helfer et al., 2006) and other avian species (Yoshimura et al., 2000; Doi et al., 2001; Singh et al., 2013). Per2 mRNA levels peak early in the day and are light sensitive (Abraham et al., 2002; Yasuo et al., 2003; Helfer et al., 2006; Singh et al., 2013). We hypothesized that longspurs would possess a functioning circadian clock and therefore would display free-running rhythms in LL and DD compared with LD. We further predicted that experimental alterations in light intensity and color temperature would entrain behavioral rhythms and that Per2 expression in hypothalamus, eye, and liver would exhibit rhythmicity and specifically increase during subjective day in LD and constant conditions.
Materials and Methods Animals Twenty-eight male and 20 female Lapland longspurs were captured in Barrow, Alaska (71°N, 156°W) in the summer of 2009 (for study site details, see Ashley et al., 2013) and held in temporary indoor aviaries before transport by plane to the University of Alaska Anchorage (UAA; 61°N, 150°W) in modified pet carriers on July 1. Birds were then housed indoors within the UAA vivarium in 2 indoor flight aviaries on a light cycle representative of short day lengths (8L:16D) at a temperature of 20 °C ± 1 °C. Birds were provided with food (bird seed mix) and water ad libitum and given weekly protein supplements of moistened egg food (Quiko) and live mealworms. Procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the UAA Institutional Animal Care and Use Committee, as well as municipal, state, tribal, and federal authorities. Experimental Settings After birds finished their prebasic molt (October 3), subjects were randomly assigned to 1 of 3 rooms of identical dimensions that contained individual bird cages (36.2 × 41.3 × 43.8 cm) on a stainless-steel rack (4 cages per row). After random assignment, we attempted to balance sex ratio across each group: room 1 (9 males, 7 females), room 2 (9 males, 7 females), room 3 (10 males, 6 females). To provide a measure of general activity, each cage was outfitted with infrared motion detectors (model PK IS215T; Honeywell, Morristown, NJ) to measure beam breaks. Each detector was placed about 10 cm above the
upper perch of the cage. The detector produced a downward, cone-shaped beam that permitted detection of movement within all areas of the cage. To eliminate interference with other detectors, cardboard dividers were placed between adjacent cages. This manipulation also visually isolated birds from each other. Each detector was wired to an interface box that could accommodate up to 7 different channels; each interface box (7 total) was connected to a 56-channel hub directly plugged into a PC computer in an outer room running ClockLab (Actimetrics, Wilmette, IL). The number of beam breaks was recorded for each channel and binned into 5-min intervals. Lighting To ensure that birds were exposed to similar lighting conditions and to manipulate light intensity and color temperature, each room was outfitted with custom-built light sources. On a separate metal rack, 224cm (8-ft) fluorescent bulbs (32 W, 6500 K color temperature; Sylvania, Danvers, MA) were mounted in quadruplet vertically. In total, there were 3 quadruplets of bulbs spaced evenly. Within each quadruplet, only 2 identical bulbs were turned on at once. This arrangement allowed for instantaneous switching between different light sources that differed in light intensity, color temperature, or both. We quantified light intensity (lux) using a light meter at each bird’s cage, and the distance from the light source to the cages was standardized such that each cage received about 1300 lux (subjective “day” light setting). This value approximates the lower threshold of light intensities recorded in polar summer (Arendt, 2012; Ashley et al., 2013). Light schedules (timing of on/off) were controlled by computer using Clocklab’s integrated light control module. A separate “night” light that emitted only blue light was continuously on (plugged into an outlet on the opposing wall relative to birds); dim blue light is often employed in avian studies to prevent photostimulation because its shorter wavelengths fail to penetrate the avian skull to activate deep encephalic photoreceptors compared with the longer wavelengths of red light (Oishi and Lauber, 1973). When fluorescent lights were switched off (referred to as “dark”), the light intensity was about 0.1 lux. Experiments Experiment 1. From October 3 to 16, birds were exposed to a 16L:8D photoperiod with lights-on at 0700 h. This photoperiod was chosen to acclimate birds to long days before continuous exposure to LL. All birds entrained to 16L:8D. To examine activity
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rhythms of birds to constant conditions (LL and DD) relative to LD, birds were exposed to the same order of light treatments—(1) LL (October 16-22), (2) 12L:12D (October 22-27), (3) DD (October 27-29)— instead of counterbalancing treatments across rooms. This ensured that birds from separate rooms were not differentially photostimulated, which could have an effect on rhythms when the experiment was repeated (see below). Activity rhythms were recorded throughout these periods. The LD treatment (lights-on at 0700 h) was deliberately placed after the LL treatment to reentrain birds that displayed free-running rhythms. The DD treatment had to be shortened to 3 d because some birds were losing body mass because little feeding occurred in darkness. Activity onsets and offsets were estimated using the automated detection function in Clocklab. Free-running periods were calculated by periodogram analysis using Clocklab software for ≥3 d of light treatment. Experiment 2. From October 27 to November 10, a separate set of experiments was conducted to examine whether alterations in either light intensity or color temperature were sufficient to entrain these birds. For these studies, birds were placed in 3 d of DD to induce free-running rhythms and then exposed to various light exposures. In room 1, we tested whether a 55% reduction in light intensity could lead to entrainment. To manipulate light intensity without altering color temperature, a 2-stop 0.5 neutral density filter was used (Rosco, Stamford, CT) to reduce light intensity by 55%. In this group, birds received 12 h of 780 lux (reduced from 1300 lux by disconnecting 1 of 2 lights per quadruplet) followed by 12 h of 350 lux (same bulbs with filter). This latter value represents lower light intensity that falls just below minimum light intensities recorded in the field in Barrow, Alaska, during polar summer (Ashley et al., 2013). Color temperature remained the same (6500 K) and was verified by a precision colormeter (Cal-COLOR 400; Cooke Corp., Romulus, MI). In room 2, color temperature was manipulated without altering light intensity by using bulbs with the same wattage. In this group, birds received 12 h of 6500 K at 780 lux (blue light) and then 12 h of 3500 K at 780 lux (red light). Again, this alteration in spectral composition is similar to the range of natural light spectra encountered in northern Alaska (Ashley et al., 2013). In room 3, birds were exposed to 12 h L (780 lux, 6500 K) and then 12 h D. After 6 d (November 5), all birds were exposed to DD for 3 d. To investigate whether simultaneous reductions in light intensity and color temperature entrained activity rhythms, birds in rooms 2 and 3 were exposed to 12 h of 780 lux at 6500 K and then 12 h of 350 lux at 3500 K for 3 d (November 7-10). Birds in room 1
Figure 1. Experimental time line. (A) Male and female longspurs were maintained on a nonbreeding photoperiod of 8L:16D for approximately 3 mo and then transferred to individual cages. Birds were exposed to 16L:8D for 2 wk. (B) Longspurs were sequentially exposed to LL (1300 lux, 6500 K), 12L (1300 lux, 6500 K):12D, and DD treatments and activity rhythms were measured. (C) Light intensity (lux) or color temperature (K) were reduced by 55% and 46%, respectively, for the second 12 h of the daily 24-h cycle, and entrainment of activity rhythms was assessed: group 1, 12L (780 lux, 6500 K, white):12L (350 lux, 6500 K; gray); group 2, 12L (780 lux, 6500 K):12L (780 lux, 3500 K; red). These treatments followed exposure to DD (0.1 lux) for 3 d, which typically induced free-running rhythms. An additional manipulation that involved simultaneous reductions in light intensity and color temperature was conducted on 2 groups of longspurs to assess entrainment of rhythms: groups 2 and 3, 12L (780 lux, 6500 K):12L (350 lux, 3500 K; pink). Throughout these experiments, a third group was exposed to 12L:12D to serve as a control. (D) Birds were entrained to 12L:12D; exposed to either LD, DD, or LL; and then euthanized at 4 different time points to assess Per2 expression in hypothalamus, eye, and liver.
received the control treatment of 12 h of L (1300 lux, 6500 K) followed by 12 h of darkness for 3 d (Fig. 1). Experiment 3. To examine patterns of Per2 gene expression in the brain, eye, and liver in relation to LD, LL, and LD groupings, birds were first exposed to 12L (1300 lux, 6500 K):12D for 6 d (November 11-18; Fig. 1). Phasing of entrainment was staggered across rooms such that lights turned on in rooms 1, 2, and 3 at 1000, 0900, and 1100 h, respectively, and all birds synchronized activity rhythms. A staggered design
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prevented tissue collection from occurring at the same time in all rooms. On November 17, rooms 1, 2, and 3 were switched to DD, LL, and LD at 1000, 0900, and 1100, respectively. We followed a similar sampling protocol used to examine pper2 expression in house sparrows that involved sampling of tissue over a 48-h period (Abraham et al., 2002). Starting at circadian time (CT) 6 or zeitgeber time (ZT) 6 (midday), 2 Lapland longspurs were taken from their cage and blood samples were obtained (see specific methods below). Birds were then immediately exposed to isoflurane vapors using a vaporizer (5% induction) for 0.17). Given the results, the response of longspurs to light intensity or color temperature treatments (free-run or arrhythmia) differed significantly from those exposed to LD (entrainment; Fisher exact tests, all p < 0.0001). Experiment 3 All birds entrained to the 12L:12D photoperiod before starting the experiment. For llPer2 expression of hypothalamic punches, there were no significant effects of light treatment (2-way ANOVA; F2,35 = 2.29, p = 0.12), sampling time (F3,35 = 0.84, p = 0.48), or their interaction (F6,35 = 1.12, p = 0.37; Fig. 5A). Moreover,
Figure 4. Representative actograms (double-plotted) of longspur activity rhythms subjected to 12L:12D, 3 d of DD, and then (A) 12L (780 lux, 6500 K; horizontal white bar):12 DD (black bar; control), (B) 12 L (780 lux, 6500 K):12L (780 lux, 3500 K; lower gray bar designated as “red”), or (C) 12 L (780 lux, 6500 K):12L (380 lux, 6500 K, gray bar). Horizontal bars on the top of graphs represent light-dark schedule (black depicts dark phase) of initial 12L:12D treatment. Horizontal bars on the bottom of graphs represent experimental light regimes. B shows a tendency toward a freerunning rhythm, whereas C displays arrhythmia.
hypothalamic llPer2 expression did not display a periodic relationship with time of day (harmonic regression, all p’s > 0.05). However, there was a significant effect of sampling time on llPer2 expression in the eye (2-way ANOVA; F3,36 = 5.17, p = 0.005) but no significant effects of light treatment (F2,36 = 0.98, p = 0.38) or its interaction with sampling time (F6,36 = 1.20, p = 0.33). More specifically, ocular llPer2 expression was higher at 24 h compared with 6, 12, and 18 h (Fisher PLSD, all p < 0.048) for LD and LL birds (Fig. 5B). These findings were further corroborated by significant circadian periodicity identified in ocular llPer2 expression of LL birds (harmonic regression, F2,13 = 4.48, p = 0.03, R2 = 0.408) but not LD birds.
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Figure 5. Per2 mRNA concentration in (A) hypothalamus (zmol/mg total RNA), (B) eye (amol/mg total RNA), and (C) liver (amol/mg total RNA) and (D) plasma melatonin concentration (pg/mL) at 4 different time points in LD, LL, and DD. Bars represent mean ± SEM. Horizontal bars on top of graphs represent light-dark schedule (black depicts dark phase). ZT18 and CT18 are double-plotted for better visualization of rhythms. Within-group comparisons that share letters indicate no significant differences between time points. n = 4 per group, except for the hypothalamus, ZT24/0 group (n = 3).
For llPer2 expression in liver, there was a significant effect of lighting treatment (2-way ANOVA, F2,36 = 7.39, p = 0.002). Neither sampling time (F3,36 = 1.70, p = 0.19) nor its interaction with lighting treatment
(F6,36 = 1.48, p = 0.22) was statistically significant. On average, llPer2 expression in liver was significantly higher in LD birds compared with LL and DD longspurs (Fisher PLSD, all p < 0.004; Fig. 5C). Liver llPer2
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expression was not associated with time of day (harmonic regression, all p’s > 0.05). Lighting treatment (F2,36 = 6.97, p = 0.003) and sampling time (F3,36 = 17.06, p < 0.0001) had significant effects on plasma melatonin concentration, but there was no significant interaction effect between the 2 factors (F6,36 = 0.82, p = 0.56). Irrespective of time, average melatonin concentration was suppressed in LL longspurs compared with LD and DD birds (Fig. 5D; Fisher PLSD, all p < 0.014). Irrespective of treatment, melatonin concentration was significantly elevated at 18 h relative to 6-, 12-, and 24-h sampling periods (Fisher PLSD, all p < 0.001). Furthermore, a significant melatonin rhythm in relation to the 24-h cycle was identified in LD birds (harmonic regression; F2.13 = 5.33, p = 0.02, R2 = 0.45) but not in LL or DD birds.
Discussion Free-Running Rhythms in Constant LL and DD Male and female longspurs exhibited period lengths that were significantly less than 24 h when exposed to LL or DD, indicating the presence of an endogenous clock(s) that regulates locomotor activity. These findings also support the hypothesis that free-living longspurs are likely synchronizing rhythms to low-amplitude polar Zeitgeber cues because our data indicate that an environment lacking these potential entraining agents, namely, constant captive conditions, induces free-running activity rhythms. Although exposure to bright LL (2000 lux) can induce rapid arrhythmia in some species (e.g., pigeons, Columba livia; Yamada et al., 1988), house sparrows will initially exhibit a short free-running period for >7 d when exposed to LL (100 lux), but rhythms eventually become arrhythmic (Cassone et al., 2008). Even though our study used lighting of higher intensity (1300 lux) to attempt to mimic the lower threshold of polar-day light intensities, longspurs still expressed free-running rhythms. Because activity rhythms were assessed for a short period of time (
