Christopher C. Chabot

Effects of physiological cycles of infused melatonin on circadian rhythmicity in pigeons

SummaryThe role of the hormone melatonin in the circadian system of pigeons (Columba livia) was investigated. Using an automatic infusion system, melatoni at physiological levels was delivered for 10 h each day to cannulated, pinealectomized (P-X) pigeons in constant darkness. These cyclic infusions of melatonin entrained feeding rhythms in P-X pigeons while vehicle infusions were ineffective entraining agents. When the retinae of P-X pigeons were removed (E-X), feeding rhythms were abolished in constant darkness. When cyclic melatonin infusions were delivered to these birds (E-X and P-X), feeding rhythmicity was restored whereas vehicle infusions alone did not restore rhythmicity. When melatonin infusions were terminated in E-X/P-X pigeons, feeding rhythms persisted for several days but eventually decayed. Blood melatonin levels were measured in both P-X and E-X/P-X birds infused cyclically with exogenous melatonin and were found to be within the physiological range both in level and pattern. These results strongly suggest that endogenous melatonin, released by the pineal gland and the retinae, regulates the timing of feeding rhythms by entraining other oscillators in the circadian system of the pigeon.

Circadian Modulation of the Rat Acoustic Startle Response

The acoustic startle response (ASR) of male rats was measured during several sessions over a 24-hr period in both a light-dark cycle and a constant-dark condition. Each session consisted of 10 trials each at 80, 90, 100, 110, and 120 dB white noise. The results indicate robust daily and circadian modulation of ASR amplitude that consist of an approximately twofold nocturnal increase at eliciting-stimuli intensities above 80 dB. Similar results were observed in female rats in constant-dark conditions. To determine whether daily changes in auditory thresholds were responsible for the observed modulation, ASR reflex modification procedures were used. These procedures were designed to measure auditory thresholds at frequencies of 10 and 40 kHz at several times of day. The results suggest a lack of significant circadian differences in auditory thresholds at these frequencies. This study demonstrates a novel role of the rat circadian system in the modulation of ASR amplitude.

Circadian feeding and locomotor rhythms in pigeons and house sparrows.

Feeding and locomotor activities were measured simultaneously in homing pigeons (Columba livia) and house sparrows (Passer domesticus). Feeding, as well as locomotor activity, was found to be regulated by a circadian clock in both of these species. Implantation of melatonin-filled capsules or exposure to constant light abolished feeding and locomotor rhythms in both species. Removal of the pineal gland from pigeons did not abolish either rhythm, whereas pinealectomy abolished both feeding and locomotor rhythms in house sparrows. Although feeding rhythms were generally more robust than locomotor rhythms in both of these species, different feeding and locomotor free-running periods were not observed within any individual pigeon or house sparrow. These results are consistent with the hypothesis that each of these species has a single pacemaker that regulates the timing of feeding and locomotor activity, but they do not rule out the possibility that separate clocks regulate these behaviors.

Circatidal and Circadian Rhythms of Locomotion in Limulus polyphemus

The nocturnal increases in the sensitivity of the lateral eye of Limulus polyphemus, the species of horseshoe crab found along the Atlantic coast, have been firmly established as being controlled by an endogenous circadian clock (1, 2, 3) located in the brain (4). Virtually nothing is known, however, about the control of the animal’s behavioral rhythms of mating and spawning that are observed in the intertidal zone during high tides in late spring (5, 6, 7). Many other marine species, especially intertidal crabs, exhibit similar rhythmic behaviors that have been demonstrated to be under the control of endogenous clocks that are circatidal (8, 9, 10, 11, 12), circadian (10, 12), or both. While there is some evidence that the activity of juvenile horseshoe crabs is primarily nocturnal (13, 14), and possibly controlled by a circadian clock (14), we know of no published work showing that locomotor activity in the adult is endogenously controlled on either a 12.4-h (circatidal) or 24-h (circadian) basis. We report here that locomotor activity in adult individuals of L. polyphemus is endogenously modulated on both a circatidal and a circadian basis and that when the animals are subjected to a light-dark (LD) cycle, most activity occurs at night. The locomotor activity of individual adult horseshoe crabs was recorded using activity chambers located in recirculating aquaria. Animals were exposed to three conditions: a 12:12 LD cycle, at 11–14 °C (“fall” conditions, LD1), a 14:12 LD cycle, at 17–21 °C (“summer” conditions, LD2), and constant darkness (DD). Typical records of the locomotor activity of three horseshoe crabs exposed to these three different photoperiods are presented in Figure 1. Circatidal rhythms were observed in all animals. While significant activity rhythms (15) in the tidal range (12.4 h) were found in only 3 of 6 animals (tau 12.83 0.78 h [mean SEM]) during LD1, in LD2, significant tidal rhythms (12.2 0.1 h) were observed in all animals. In some cases in LD2 (4 of 6 animals), clear free-running rhythms were sometimes apparent, (Fig. 1; middle, bottom panels), while in other cases the activity appeared to synchronize to the LD cycles (Fig. 1; top). In DD, circatidal rhythms (12.6 0.2 h) were found in 5 of 6 animals (Fig. 1; all panels). Most animals (5 of 6 in LD1; 6 of 6 in LD2) exhibited significant rhythms in the circadian range (tau 24.29 0.14 h). Periodogram analyses (15) and visual inspection indicated that 5 of the 6 animals tested synchronized their activity to the initial 12:12 LD cycle (LD1). The single animal that did not thus synchronize had a very low level of activity. Significantly more activity occurred during the dark phase than the light phase in 4 of 6 animals (Fig. 1; top and bottom [but not middle] panels). The average period (tau) for these animals in the daily (24-h) range in LD1 was 24.12 0.09 h. Upon subsequent exposure to “summer” conditions (LD2), 3 (of 6) animals remained synchronized to the LD cycle (Fig. 1; top panel). In others (2 of 6), this apparent synchronization was not stable (Fig. 1; middle, days 10–18 and days 29–42) and, in still another animal, the synchronization, if any, was unclear (Fig. 1; bottom). Animals that both synchronized and showed a clear onset of activity initiated their activity a significant amount of time (1.7 0.1 h; P 0.005) before the lights went out during LD2 but not LD1 (1.1 0.5 h; P 0.15). Significantly more activity occurred during D versus L periods in 3 of 6 animals (Fig. 1; top panel only). In constant darkness (DD), all animals also expressed significant circadian rhythms (25.27 0.69 h; Fig. 1, all panels). In addition, the activity patterns of 3 of 6 animals in DD exhibited evidence of entrainment based on the similarity of phasing with the previous LD cycle (Fig. 1; top, middle). L. polyphemus was significantly more active overall during LD2 than during LD1 and DD (P 0.03). Neither circatidal (P 0.78) nor Received 12 February 2004; accepted 7 June 2004. * To whom all correspondence should be addressed. E-mail: chrisc@mail.plymouth.edu Reference: Biol. Bull. 207: 72–75. (August 2004)

Daily rhythmicity of the rat acoustic startle response

We have measured the acoustic startle response (ASR) amplitude and latency in rats housed in a 12:12 light:dark (LD) cycle. The response amplitudes to eliciting stimuli (ES) of 110 dB or 120 dB (white noise) were significantly higher (nearly two-fold) during D than during L. Similar, but nonsignificant, trends were also observed at ES intensities of 90 dB or 100 dB. While some significant LD ASR latency differences were observed, we cannot ascribe them to the photoperiodic phase at this time. These findings conclusively demonstrate that the mammalian ASR amplitude exhibits daily rhythmicity.

The Relationship Between Small- and Large-Scale Movements of Horseshoe Crabs in the Great Bay Estuary and Limulus Behavior in the Laboratory

The overall goal of our research program is to determine the short- and long-term patterns of horseshoe crab (Limulus polyphemus) movements in the Great Bay estuary and then seek an understanding of the endogenous and exogenous processes that give rise to these patterns. Small- and large-scale movement data were obtained from 27 horseshoe crabs tracked using ultrasonic telemetry for at least a year. During mating season animals were most active during high tides, but they did not increase their activity or approach mating beaches during every high tide. During the remainder of the year tidal or daily patterns of activity were less evident, and the extent of their movements gradually decreased as water temperatures dropped in the late fall and winter. During the spring, when water temperatures exceeded 10°C, tagged animals moved several km up into the estuary into shallow water (< 4 m) 1 month prior to spawning. A similar temperature threshold was also evident in laboratory experiments, with little rhythmic behavior expressed at temperatures below 11°C. Mating activity lasted approximately 1 month and was followed by a period of high activity. In the fall, most animals moved downriver into deeper water, where they remained during the colder months. Thus, the majority of Limulus exhibited a seasonal pattern of movement, remaining within a 3 km stretch of the estuary. In the laboratory, animals expressed both daily and tidal rhythms of locomotion. Those with daily rhythms were more likely to be diurnal than nocturnal, but both tendencies were evident. The clock involved in modulating their locomotory activity appears to be separate from the clock controlling their circadian rhythm of visual sensitivity. When animals were exposed to “artificial tides”, created by changing water depth every 12.4 hours, they expressed clear tidal rhythms of activity that were synchronized to the imposed tides. Similar data were obtained from horseshoe crabs in running wheels placed in the estuary. However, if the running wheels were attached to a floating dock, so water depth did not change with the tides, the horseshoe crabs were primarily diurnal. Thus, while endogenous biological clocks are capable of controlling many aspects of horseshoe crab locomotion, the actual patterns manifested in the field are strongly influenced by the water depth changes associated with the tides, as well as light levels and seasonal changes in water temperature.

Behavioral avoidance of fluoranthene by fathead minnows (Pimephales promelas)

A monitoring system was used to examine the behavioral response of fathead minnows (Pimephales promelas) to plumes of the polycyclic aromatic hydrocarbon fluoranthene. Previously unexposed fish and fish surviving acute exposure to fluoranthene were presented with three different concentrations of fluoranthene. Both groups of fish avoided fluoranthene. Pre-exposure did not enhance or diminish avoidance of fluoranthene. The lowest concentration of fluoranthene which produced an avoidance response was 14.7 mu/l, and the concentration of fluoranthene which did not produce an avoidance response was 8.6 micrograms/l. These results were comparable to the LOEC for survival in 7-day fathead embryo-larval growth and survival tests for fluoranthene. Thus, a fathead minnow could escape from areas highly contaminated with fluoranthene and have a better opportunity to survive, whereas fish would fail to avoid areas where fluoranthene concentrations are below 8.6 micrograms/l and suffer further toxicosis.

Rhythms of Locomotion Expressed by Limulus polyphemus, the American Horseshoe Crab: I. Synchronization by Artificial Tides

Limulus polyphemus, the American horseshoe crab, has an endogenous clock that drives circatidal rhythms of locomotor activity. In this study, we examined the ability of artificial tides to entrain the locomotor rhythms of Limulus in the laboratory. In experiments one and two, the activity of 16 individuals of L. polyphemus was monitored with activity boxes and “running wheels.” When the crabs were exposed to artificial tides created by changes in water depth, circatidal rhythms were observed in animals exposed to 12.4-h “tidal” cycles of either water depth changes (8 of 8 animals) or inundation (7 of 8 animals). In experiment three, an additional 8 animals were exposed to water depth changes under cyclic conditions of light and dark and then monitored for 10 days with no imposed artificial tides. Most animals (5) clearly synchronized their activity to the imposed artificial tidal cycles, and 3 of these animals showed clear evidence of entrainment after the artificial tides were terminated. Overall, these results demonstrate that the endogenous tidal clock that influences locomotion in Limulus can be entrained by imposed artificial tides. In the laboratory, these tidal cues override the influence of light/dark cycles. In their natural habitat, where both tidal and photoperiod inputs are typically always present, their activity rhythms are likely to be much more complex.

Rhythms of Locomotion Expressed by Limulus polyphemus, the American Horseshoe Crab: II. Relationship to Circadian Rhythms of Visual Sensitivity

In the laboratory, horseshoe crabs express a circadian rhythm of visual sensitivity as well as daily and circatidal rhythms of locomotion. The major goal of this investigation was to determine whether the circadian clock underlying changes in visual sensitivity also modulates locomotion. To address this question, we developed a method for simultaneously recording changes in visual sensitivity and locomotion. Although every animal (24) expressed consistent circadian rhythms of visual sensitivity, rhythms of locomotion were more variable: 44% expressed a tidal rhythm, 28% were most active at night, and the rest lacked statistically significant rhythms. When exposed to artificial tides, 8 of 16 animals expressed circatidal rhythms of locomotion that continued after tidal cycles were stopped. However, rhythms of visual sensitivity remained stable and showed no tendency to be influenced by the imposed tides or locomotor activity. These results indicate that horseshoe crabs possess at least two biological clocks: one circadian clock primarily used for modulating visual sensitivity, and one or more clocks that control patterns of locomotion. This arrangement allows horseshoe crabs to see quite well while mating during both daytime and nighttime high tides.

The effects of water pressure, temperature, and current cycles on circatidal rhythms expressed by the American horseshoe crab, Limulus polyphemus

The American horseshoe crab, Limulus polyphemus, expresses tidal rhythms of locomotion that can be entrained to cyclic fluctuations in water depth, but the ability of other tidal cues to entrain locomotor activity has not been assessed. In this study, tidal inundation cycles of ∼12.4 h delivered in the laboratory clearly entrained the locomotor patterns of most animals. However, smaller amplitude water level fluctuations and large amplitude temperature cycles (10°C) were less successful while small-amplitude temperature fluctuations (3°C) and current cycles (0.3 m s−1) were unsuccessful. In the field, animals confined to modified running wheels expressed rhythms of locomotor activity that were clearly synchronized with the tides, but only if they were allowed to experience water level changes. Overall, these results suggest a hierarchy among potential tidal entrainment cues: inundation and water level changes are of primary importance, while other factors such as current and temperature changes appear to play secondary roles.