Mirror imaging phase response curves obtained for the circadian rhythm of a bat with single steps of light and darkness∗

Abstract

The circadian rhythm in the flight activity of a tropical microchiropteran bat Taphozous melanopogon responds at all phases with delay phase shifts to single light‐on steps (DD/LL transfers). The circadian rhythm responds at all phases with advance phase shifts to single light‐off steps (LL/DD transfers). Phase shifts were measured from the delays or advances of the onsets of flight activity on days following DD/LL and LL/DD transfers relative to the temporal course of the onsets of activity in controls. The magnitude of the phase shifts was a function of the phases in which the transfers were made. The On‐PRC and Off‐PRC plotted from such data are mirror‐images in their time‐course and wave‐form.

The phase shifts of the circadian rhythm in either direction were accompanied by changes in period (for the duration of our recordings after die transfer). The period lengthened following a delay shift and it shortened following an advance shift. The phase shifts are abrupt and discernible in the first cycle after perturbation. There are no transients.     

Single prenatal injections of melatonin or the D1-dopamine receptor agonist SKF 38393 to pregnant hamsters sets the offsprings' circadian rhythms to phases 180° apart

Pregnant Syrian hamsters with lesions of the suprachiasmatic nucleus (SCN) received single injections of melatonin or the D1-dopamine receptor agonist, SKF 38393 on day 15 of gestation (1 day before birth). Pups were weaned on postnatal day 20 and their freerunning activity rhythms recorded for 3–4 weeks. The pups' phases on the day of weaning were significantly clustered in both of the treatment groups, but the average phases differed by approximately 180°. The results demonstrate that a single prenatal stimulus is sufficient to set the phases of the hamsters' rhythms and that the phase established depends on the stimulus. Both c-fos mRNA and Fos protein were expressed in the fetal SCN after SKF 38393 injection but neither were expressed after melatonin injection. Simulations showed that a single stimulus could produce the observed synchrony from a population of uniformally distributed phases if the phase shifts were three to four times the magnitude of the adult hamster light phase response curve (PRC). A light pulse PRC mimicked the effect of an SKF 38393 injection and a dark-pulse PRC mimicked the effects of a melatonin injection. Together these results suggest that dopamine and melatonin either are, or mimic, maternal entraining signals that represent day and night. Accepted: 12 November 1996     

Responses of Odonata chromatophores to environmental stimuli

Responses to change in temperature and light intensity were studied in three species of Australian Odonata using time-lapse photography. In each species, responses to temperature are dependent on both the instantaneous temperature and the direction of temperature change. At temperatures below those which produce unstable colour phases, the change to dark phase takes about 9 hr and is constant in rate. The reverse change is directly temperature dependent and can be much more rapid. Responses to change in light intensity are attributable to the heating effect of light rather than to true light sensitivity. All colour changes show wide individual variation in both rate and amount. They are slightly affected by temperature acclimation but are unaffected by prevailing weather, time of year, geographic location, or age.     

Biomass plug development and propagation in porous media

Exopolymer-producing bacteria can be used to modify soil profiles for enhanced oil recovery or bioremediation. Understanding the mechanisms associated with biomass plug development and propagation is needed for successful application of this technology. These mechanisms were determined from packed-bed and micromodel experiments that simulate plugging in porous media. Leuconostoc mesenteroides was used, because production of dextran, a water-insoluble exopolymer, can be controlled by using different carbon sources. As dextran was produced, the pressure drop across the porous media increased and began to oscillate. Three pressure phases were identified under exopolymer-producing conditions: the exopolymer-induction phase, the plugging phase, and the plug-propagation phase. The exopolymer-induction phase extended from the time that exopolymer-producing conditions were induced until there was a measurable increase in pressure drop across the porous media. The plugging phase extended from the first increase in pressure drop until a maximum pressure drop was reached. Changes in pressure drop in these two phases were directly related to biomass distribution. Specifically, flow channels within the porous media filled with biomass creating a plugged region where convective flow occurred only in water channels within the biofilm. These water channels were more restrictive to flow causing the pressure drop to increase. At a maximum pressure drop across the porous media, the biomass yielded much like a Bingham plastic, and a flow channel was formed. This behavior marked the onset of the plug-propagation phase which was characterized by sequential development and breakthrough of biomass plugs. This development and breakthrough propagated the biomass plug in the direction of nutrient flow. The dominant mechanism associated with all three phases of plugging in porous media was exopolymer production; yield stress is an additional mechanism in the plug-propagation phase.     

The photobiology of Hydra’s periodic activity

Hydra’s response to a light pulse is a phase shift of the state of bioelectric activity correlated with the periodic shortening-elongation behaviour. The direction and absolute value of a phase shift depend on intensity, direction, application phase (along the periodic activity state), and wavelength of the light pulse. Repetitive pulses entrain the behavioural cycle. The period of the behavioural cycle depends on intensity and wavelength of steady background illumination; however, the light effect is not exerted isotropically along all the phases of the behavioural cycle. Inferences are drawn on light influence on the behaviour pacemaking mechanism. By using polyclonal antibodies against squid rhodopsin, an opsin-like protein has been identified in the ectodermal layer, presumably in sensory cells.     

Melatonin enhances the sensitivity of circadian pacemakers to light in the nocturnal field mouse Mus booduga

The effect of exogenous melatonin (1 mg/kg) on light pulse (LP) induced phase shifts of the circadian locomotor activity rhythm was studied in the nocturnal field mouse Mus booduga. Three phase response curves (PRCs: LP, control, and experimental) were constructed to study the effect of co-administration of light and melatonin at various circadian times (CTs). The LP PRC was constructed by exposing animals free-running in constant darkness (DD) to LPs of 100-lux intensity and 15-min duration, at various CTs. The control and experimental PRCs were constructed by using a single injection of either 50% DMSO or melatonin (1 mg/kg dissolved in 50% DMSO), respectively, administered 5 min before LPs, to animals free-running in DD. A single dose of melatonin significantly modified the waveform of the LP PRC. The experimental PRC had significantly larger areas under advance and delay regions of the PRC compared to the control PRC. This was also confirmed when the phase shifts obtained at various CTs were compared between the three PRCs. The phase delays at three phases (CT12, CT14, and CT16) of the experimental PRCs were significantly greater than those of the control and the LP PRCs. Based on these results we conclude that phase shifting effects of melatonin and light add up to produce larger responses.     

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.     

Population analysis of a commercial Saccharomyces cerevisiae wine yeast in a batch culture by electric particle analysis, light diffraction and flow cytometry

Data from electric particle analysis, light diffraction and flow cytometry analysis provide information on changes in cell morphology. Here, we report analyses of Saccharomyces cerevisiae populations growing in a batch culture using these techniques. The size distributions were determined by electric particle analysis and by light diffraction in order to compare their outcomes. Flow cytometry parameters forward (related to cell size) and side (related to cell granularity) scatter were also determined to complement this information. These distributions of yeast properties were analysed statistically and by a complexity index. The cell size of Saccharomyces at the lag phase was smaller than that at the beginning of the exponential phase, whereas during the stationary phase, the cell size converged with the values observed during the lag phase. These experimental techniques, when used together, allow us to distinguish among and characterize the cell size, cell granularity and the structure of the yeast population through the three growth phases. Flow cytometry patterns are better than light diffraction and electric particle analysis in showing the existence of subpopulations during the different phases, especially during the stationary phase. The use of a complexity index in this context helped to differentiate these phases and confirmed the yeast cell heterogeneity.     

Phase Resetting of the Human Circadian Pacemaker with Use of a Single Pulse of Bright Light

Since the initial studies reporting that light can alter the phase position of the human circadian system, there has been increasing interest in the use of bright light as a tool for manipulating the phase position of the circadian pacemaker. Exposure protocols typically require subjects to receive 2-5 h of exposure over several circadian cycles. As a consequence, bright light treatment can involve a considerable time investment. However, recent studies indicate that a single pulse of bright light can produce significant phase shifts in the circadian pacemaker. If a single pulse of bright light can produce significant phase-shifting effects, multiple-pulse designs may be unnecessary. This study examined the phase-shifting effects of a single 4-h pulse of bright light (12,000 lux) in 14 male and one female subject aged between 19-45 years. With use of a “constant routine” to estimate circadian phase, a single 4-h pulse of light produced significant shifts in the phase of the core temperature rhythm. The timing of the exposure, relative to the core temperature rhythm, determined the degree and direction of the phase shift. Exposure immediately prior to habitual bedtime produced a mean phase delay in the core temperature of 2.39 h (SD = 1.37 h). In contrast, exposure immediately following habitual wake-up produced a mean phase advance of 1.49 h (SD = 2.06 h). In addition, the magnitude of the shift increased the closer the light pulse was to the individual's estimated endogenous core temperature minimum. There was, however, considerable interindividual variability in this relationship. Overall, these results confirm that a single pulse of bright light can produce significant phase shifts in the phase of the circadian pacemaker controlling core temperature. Key Words: Bright light—Circadian rhythm—Core body temperature—Sleep-wake disorders—Chronobiology.     

Growth in the area of the inferior dental foramen of rats

The object of the present investigation was to see if the bone around the inferior dental nerve remodelled during mandibular growth and development. The investigation was carried out by injecting 27 albino Lewis rats with three fluorescent bone seeking dyes--oxytetracycline HCl (OTC), alizarin red S (ARS), and 2,4 bis-[N,N'-di' (carbomethyl-aminomethyl)] fluorescein (DCAF)--and then studying the bone around the inferior dental foramen. The mandibles of the animals were studied both macroscopically and microscopically under ultraviolet light to investigate the growth processes occurring and to see if the inferior dental foramen was relocated during growth. A quantitative analysis utilizing two specimens was also carried out for the same purpose. The results of both the qualitative and the quantitative analyses showed that the bone around the inferior dental nerve remodeled during mandibular growth. The mandible grew in an upward and backward direction, and the inferior dental foramen was correspondingly relocated in an upward and backward direction to maintain exactly the same position relative to the condyle and the posterior border of the ramus. This study, then, supports Moss's concept of the "unloaded" nerve, and is in keeping with his view of mandibular growth based on the functional matrix theory.     

Phase Resetting of the Human Circadian Pacemaker with Use of a Single Pulse of Bright Light

Since the initial studies reporting that light can alter the phase position of the human circadian system, there has been increasing interest in the use of bright light as a tool for manipulating the phase position of the circadian pacemaker. Exposure protocols typically require subjects to receive 2–5 h of exposure over several circadian cycles. As a consequence, bright light treatment can involve a considerable time investment. However, recent studies indicate that a single pulse of bright light can produce significant phase shifts in the circadian pacemaker. If a single pulse of bright light can produce significant phase-shifting effects, multiple-pulse designs may be unnecessary. This study examined the phase-shifting effects of a single 4-h pulse of bright light (12,000 lux) in 14 male and one female subject aged between 19–45 years. With use of a “constant routine” to estimate circadian phase, a single 4-h pulse of light produced significant shifts in the phase of the core temperature rhythm. The timing of the exposure, relative to the core temperature rhythm, determined the degree and direction of the phase shift. Exposure immediately prior to habitual bedtime produced a mean phase delay in the core temperature of 2.39 h (SD = 1.37 h). In contrast, exposure immediately following habitual wake-up produced a mean phase advance of 1.49 h (SD = 2.06 h). In addition, the magnitude of the shift increased the closer the light pulse was to the individual's estimated endogenous core temperature minimum. There was, however, considerable interindividual variability in this relationship. Overall, these results confirm that a single pulse of bright light can produce significant phase shifts in the phase of the circadian pacemaker controlling core temperature.     

Phase behavior of a monoacylglycerol: (myverol 18-99K)/water system

The phase behavior of Myverol 18-99K, a food emulsifier rich in monoacylglycerols, in combination with water has been determined. X-ray diffraction and polarized light microscopy (PLM) were used for phase identification and structure characterization. Phase behavior was established in the temperature range from -15 to 50 degrees C and in the composition range from dry to full hydration. Phases identified include the solid lamellar crystal (Lc) phase, the liquid fluid isotropic phase and three liquid crystal phases, the lamellar liquid crystal, the cubic-Ia3d and the cubic-Pn3m phase. Phase information is reported in the form of temperature-composition phase diagrams. It was collected under equilibrium conditions where measurements were made in the heating direction beginning with the Lc phase at -15 degrees C. Phase metastability was also examined in which the natural tendency of the liquid crystal phases to undercool was facilitated. Under this condition, both cubic phases were found to remain free of the solid Lc phase over a relatively wide range of hydration values down to 0 degrees C. The microstructure of the different phases and its dependence on temperature and hydration has been determined. Compositional analysis using thin layer chromatography and gas chromatography/mass spectrometry shows that Myverol 18-99K consists of 82% monoacylglycerols (86.6% monoolein, 7. 0% monostearin, 3.5% monopalmitin, 0.9% monoarachidin, 2.0% unidentified). The equilibrium and metastable phase diagrams of the Myverol 18-99K/water system show remarkable similarity to those reported for the monoolein/water system (Qiu, H., Caffrey, M., 2000. The phase diagram of the monoolein/water system: metastability and equilibrium aspects Biomaterials 21, 223-594.).     

The circadian locomotor rhythm of Hemideina thoracica (Orthoptera; Stenopelmatidae): the circadian clock as a population of interacting oscillators

ABSTRACT. The behaviour of the circadian locomotor rhythm of the New Zealand weta, Hemideina thoracica (White), supports the model that the underlying pacemaker consists of a population of weakly coupled oscillators. Certain patterns of locomotor activity, previously demonstrated almost exclusively in vertebrates, are presented here as evidence for the above hypothesis. They include after-effects of various pre-treatments, rhythm-splitting and spontaneous changes in the rhythm. After-effects, which describe the unstable behaviour of free-running circadian rhythms following particular experimental perturbations, have been observed in Hemideina following single light pulses, constant dim light, and laboratory and natural entrainment. Period changes occurred in the activity rhythm after single light pulses of 8-h and 12-h duration (25 lx). Constant dim light (0.1 lx) increased the free-running period (τ) of the activity rhythm, but the after-effect of constant dim light was either an increase or a decrease in τ. After-effects upon both τ and the active phase length of the activity rhythm were found following non-24-h light entrainment cycles with 8-h and 12-h light phases of 25 lx. Qualitative measurements of these after-effects upon τ are presented which reveal a relationship between both the direction and amount of change in τ, and the difference between entrainment cycle length (T) and pre-entrainment free-running period. The after-effect of natural entrainment was an initial short-period free-run (τ < 24h) lasting 5–10 days, generally followed by a rapid period lengthening to τ= 25–26 h. Support for the population model was provided by spontaneous dampening, recovery, and period changes of the rhythm, together with the disruption of the active phase following critical light perturbations, and rhythm-splitting. These Hemideina results are compared with the simulations of the Coupled Stochastic System of Enright (1980).     

Imaging of single light-responsive clock cells reveals fluctuating free-running periods

Zebrafish tissues and cell lines contain circadian clocks that respond directly to light. Using fluorescence-activated cell sorting, we have isolated clonal cell lines that contain the reporter construct, zfperiod4-luciferase. Bioluminescent assays show that oscillations within cell populations are dampened in constant darkness. However, single-cell imaging reveals that individual cells continue to oscillate, but with widely distributed phases and marked stochastic fluctuations in free-running period. Because these cells are directly light responsive, we can easily follow phase shifts to single light pulses. Here we show that light acts to reset desynchronous cellular oscillations to a common phase, as well as stabilize the subsequent free-running period.     

Re-Entrainment Behavior of Djungarian Hamsters (phodopus sungorus) with Different Rhythmic Phenotype Following Light-Dark Shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts. (Author correspondence: weinert@zoologie.uni-halle.de)     

Re-entrainment behavior of Djungarian hamsters (Phodopus sungorus) with different rhythmic phenotype following light-dark shifts

Djungarian hamsters bred at the authors' institute reveal two distinct circadian phenotypes, the wild-type (WT) and DAO type. The latter is characterized by a delayed activity-onset, probably due to a deficient mechanism for photic entrainment. Experiments with zeitgeber shifts have been performed to gain further insight into the mechanisms underlying this phenomenon. Advancing and delaying phase shifts were produced by a single lengthening or shortening of the dark (D) or light (L) time by 6?h. Motor activity was recorded by passive infrared motion detectors. All WT hamsters re-entrained following various zeitgeber shifts and nearly always in the same direction as the zeitgeber shift. On the other hand, a considerable proportion of the DAO animals failed to re-entrain and showed, instead, diurnal, arrhythmic, or free-running activity patterns. All but one of those hamsters that re-entrained did so by delaying their activity rhythm independently of the direction of the LD shift. Resynchronization occurred faster following a delayed than an advanced shift and also after changes of D rather than L. WT animals tended to re-entrain faster, particularly following a zeitgeber advance (where DAO hamsters re-entrained by an 18-h phase delay instead of a 6-h phase advance). However, the difference between phenotypes was statistically significant only with a shortening of L. To better understand re-entrainment behavior, Type VI phase-response curves (PRCs) were constructed. To do this, both WT and DAO animals were kept under LD conditions, and light pulses (15 min, 100 lux) were applied at different times of the dark span. In WT animals, activity-offset always showed phase advances, whereas activity-onset was phase delayed by light pulses applied during the first half of the dark time and not affected by light pulses applied during the second half. When the light pulse was given at the beginning of D, activity-onset responded more strongly, but light pulses given later in D produced significant changes only in activity-offset. In accord with the delayed activity-onset in DAO hamsters, no or only very weak phase-responses were observed when light pulses were given during the first hours of D. However, the second part of the PRCs was similar to that of WT hamsters, even though it was compressed to an interval of only a few hours and the shifts were smaller. Due to these differences, the first light-on or light-off following an LD shift fell into different phases of the PRC and thus caused different re-entrainment behavior. The results show that it is not only steady-state entrainment that is compromised in DAO hamsters but also their re-entrainment behavior following zeitgeber shifts.     

Phase shift of the circadian rhythm of lemna caused by pulses of a leucine analog, trifluoroleucine

Pulses of a fluorinated analog of leucine, 5′,5′,5′-trifluoroleucine, reset the phase of the circadian rhythm of K⁺ uptake in Lemna gibba G3 under continuous light conditions. The trifluoroleucine pulse caused the largest delay phase-shifts during the early subjective phase but it caused only small phase advances. The action of trifluoroleucine was investigated and the following results were obtained. (a) The uptake of trifluoroleucine was essentially the same at all circadian phases, even though phase shifting was dramatically different at different phases. At effective phases, the magnitude of phase shifting was well correlated with the amount of trifluoroleucine taken up by the duckweed. (b) The trifluoroleucine pulse lowered the endogenous content of valine and leucine but these decreases did not correlate with phase shifting. (c) Protein synthesis was not affected by trifluoroleucine pulses which caused large phase shifts. (d) Pulses of 4-azaleucine, a different structural analog of leucine, also caused phase shifting. However, neither the direction nor the effective times of phase shifting were similar to those of trifluoroleucine. Taken together, these results negate the proposition that trifluoroleucine and azaleucine caused phase shift by disturbing amino acid metabolism and/or inhibiting protein synthesis, but they suggest instead that these analogs are incorporated into some protein(s) which are necessary for normal clock operation.     

Effect of behavioral history on subsequent responding: experiments using rotary fixed-ratio schedules

The present study proposes a novel experimental approach to examine the effect of previous schedule history on subsequent responses. College students used a three-button device to indicate their choice of response. We performed two experiments. Each experiment consisted of three phases. In experiment I, fixed-ratio 10 (FR 10) reinforcement schedule was employed in phases 1 and 3. Phase 2 employed "Rotary-FR 10" schedule: after obtaining a point produced by pressing one of three specified buttons in a triangular array 10 times, the effective button changed to another in a clockwise direction. Optimal response pattern for Rotary-FR 10 was analyzed. Two types of behavior, referred to as "consecutive" and "scatter" were observed in phase 1. The mean number of optimal responses in consecutive type was significantly higher than scatter type in phases 2 and 3. In experiment II, two schedules were used in phase 1, and the same protocol used in experiment I was followed. We found that the reinforcement schedule used in phase 1 affected acquisition of the optimal response in phase 2. These results suggest that the earlier behavioral tendencies play an important role in determining subsequent behavior and that such behavioral tendency can be modified by specifically designed protocols.