The photoperiodic clock in blackheaded buntings (Emberiza melanocephala) is mediated by a self-sustaining circadian system

Three experimental protocols were employed to clarify whether the circadian system is involved in photoperiodic time-measurement in the blackheaded bunting, Emberiza melanocephala. In a single-pulse paradigm, one 8-h light pulse was delivered at different times to groups of birds across three days of constant darkness (DD). Photoperiodic induction, as measured by a rise in plasma luteinizing hormone (LH), showed clear circadian rhythmicity. The second experiment examined the LH responses in birds exposed to lighting cycles using a Nanda-Hamner type of protocol and confirmed full photostimulation under 6L:30D. The third experiment measured the time of the first photo-induced rise in LH in birds subjected to 30 h of continuous light following entrainment under short days (6L:18D). This experiment aimed to identify the position of the photoinducible phase ( _i). LH first rose at hour 18 following dawn indicating that _i lies in the middle of the day. Plasma concentrations of melatonin were also measured under 6L:18D and 6L:30D light cycles as another physiological marker of the circadian system in buntings. The pattern of melatonin secretion under these LD cycles showed properties consistent with the driving oscillator being circadian in nature. It is concluded that the circadian pacemaker driving the photoinducible rhythm in blackheaded bunting is strongly self-sustaining and free-runs under constant conditions.     

Circadian rhythms of corneal mitotic rate,retinal melatonin and immunoreactive visual pigments,and the effects of melatonin on the rhythms in the Japanese quail

We investigated circadian ocular rhythms in the Japanese quail, Coturnix coturnix japonica. The birds were placed under light-dark cycles (LD 1212), constant light (LL) and constant darkness (DD), and the retinas were dissected out at four-hour intervals throughout 24 h. Following measurements were performed. (1) Melatonin content in the retina was measured by radioimmunoassay. It was low in light and several folds higher in darkness under LD 1212. The rhythm continued in DD, but disappeared in LL. (2) Mitotic figures in the corneal epithelium were counted. Similar rhythms to the melatonin content were observed in the corneal mitotic rate with a slight phase delay. (3) The retinas were fixed at 4-h intervals and immunostained with anti-bovine rhodopsin serum and anti-chicken iodopsin monoclonal antibodies. The outer segments of photoreceptor cells were stained intensively throughout 24 h in LD 1212, LL and DD. In contrast, the stainability of the locus close to the outer limiting membrane where the Golgi apparatus exists changed diurnally. Scores showing the ratio of cells with positive staining indicated high values from 4 h after the onset of light to the beginning of dark phase under LD 1212. The values were high throughout 24 h in LL and intermediate or low in DD. (4) To investigate the effect of melatonin on the corneal mitotic rate and visual pigments at the Golgi region, melatonin was injected into one eye and saline into the contralateral eye. Melatonin induced a phase advance in the corneal mitotic rate under LD 1212, but did not induce a rhythm under LL. The ratio of photoreceptor cells with positive staining to anti-visual pigment antibodies at the Golgi region was not affected by melatonin injection.Abbreviations ANOVA analysis of variance - BSA bovine serum albumin - DD constant darkness - Io-mAb monoclonal antibodies against chicken iodospin - LD light-dark - LL constant light - mRNA messenger ribonucleic acid - PBS phosphate buffer solution - Rh-As antiserum against bovine rhodopsin - SCN suprachiasmatic nucleus - T transducin - T transducin      

The circadian rhythm of thermoregulation in Japanese quail

Japanese quail exhibit a robust circadian rhythm in body temperature. This rhythm is readily entrainable by 24 h light-dark (LD) cycles and persists under constant conditions. Because both the pineal organ and the eyes have been implicated as major components of the circadian system of birds, the role of these organs in generating the rhythm of body temperature was investigated. Pinealectomy, when performed alone, had little effect on the body temperature rhythm of quail either under LD or under constant darkness (DD). Most birds subjected to optic nerve section alone remained rhythmic in DD although the robustness of the rhythm was decreased, and 25% became arrhythmic. Birds subjected to both pinealectomy and optic nerve section behaved similarly to birds subjected to optic nerve section alone. However, complete eye removal, when performed alone or in combination with pinealectomy, caused all birds to become arrhythmic in DD. The data support the hypothesis that the eyes are the loci of circadian pacemakers in quail that act, via both neural and hormonal outputs, to preserve the integrity of (self-sustaining or damped) circadian oscillators located elsewhere.     

Photoperiodic control of reproduction in the male lizardAnolis carolinensis

Summary In contrast to the higher vertebrates the photoperiodic time measuring system in the male lizardAnolis carolinensis seems to rely on an hourglass timer which lacks endogenous rhythmicity. This timer appears to measure the absolute length of the light portion of light-dark (LD) cycles. The present study further characterized the nature of theAnolis photoperiodic timer and demonstrated: (1) The gonadal response is quite sensitive to photostimulation. Exposure to as few as three 16 h photoperiods (over a 3 week period) can maintain testicular function in summer anoles whereas exposure to as few as six 16 h photoperiods (over a 3 week period) can elicit maximal testicular development in the fall. (2) The photoperiodic timer does not have to be reset daily by a dark interruption. (3) The dark portion of LD cycles may be involved in a complex fashion in reversing a light-initiated reaction and (4) Comparisons of entrained circadian activity rhythms with testicular responses to various light cycles argue against the participation of a circadian clock in photoperiodic time measurement.Abbreviation CRPP circadian rhythm of photoperiodic photo-sensitivity     

Complex circadian regulation of pineal melatonin and wheel-running in Syrian hamsters

Circadian regulation of pineal melatonin content was studied in Syrian hamsters (Mesocricetus auratus), especially melatonin peak width and the temporal correlation to wheel-running activity. Melatonin was measured by radioimmunoassay in glands removed at different circadian times with respect to activity onset (= CT 12). Pineal melatonin peak width (h; for mean 125 pg/gland) and activity duration () were both 4–5 h longer after 12 or 27 weeks than after 5 or 6 days in continuous darkness (DD). Increased peak width was associated with a delay in the morning decline (M) of melatonin to baseline, correlated with a similar delay in wheel-running offset. In contrast, the evening rise (E) in melatonin occurred at approximately the same circadian phase regardless of the length of DD. Fifteen min light pulses produced similar phase-shifts in melatonin and activity. In a phase advance shift, M advanced at once, while E advanced only after several days of adjustment. Independent timing of shifts in the E and M components of the melatonin rhythm suggest that these events are controlled separately by at least two circadian oscillators whose mutual phase relationship determines melatonin peak width. This two-oscillator control of melatonin peak width is integral to the circadian mechanism of hamster photoperiodic time measurement.Abbreviations CT circadian time - DD continuous dark - L: D light: dark cycle - PMEL pineal melatonin - PRC phase response curve - RIA radioimmunoassay; , duration (h) of the active phase of the circadian wheel-running rhythm; , free-running period     

Daily and circadian variation in the electroretinogram of the domestic fowl: effects of melatonin

Visual and circadian function are integrally related in birds, but the precise nature of their interaction is unknown. The present study determined whether visual sensitivity measured electroretinographically (ERG) in 7-week-old cockerels varies over the time of day, whether this rhythm persists in constant darkness (DD) and whether exogenous melatonin affects this ERG rhythmicity. ERG b-wave amplitude was rhythmic in LD and persisted in DD with peak amplitude during mid- to late afternoon in LD and mid-subjective day in DD, indicating that the ERG rhythm is endogenously generated. No daily or circadian variation in a-wave amplitude was observed, and ERG component latency and durations were not rhythmic. Intramuscular injection of 10 g/kg melatonin at ZT10 in LD significantly decreased b-wave amplitude but had no effect on a-wave. Intraocular injection of 600 pg melatonin, however, had no effect on any aspect of the ERG. These data indicate that a circadian clock regulates ocular sensitivity to light and that melatonin may mediate some or all of this effect. The level at which melatonin modulates retinal sensitivity is not known, but the present data suggest a central site rather than a direct effect of the hormone in the eye.Abbreviations DD constant darkness - ERG electroretinography - EW Edinger-Westphal nuclei - IMEL iodomelatonin - IO isthmooptic nucleus - LD light-dark cycle - SCG superior cervical ganglion - SCN suprachiasmatic nuclei - vSCN visual suprachiasmatic nucleus     

Evidence for a daily rhythmicity in the acute release of luteinizing hormone in response to electrical stimulation in the Japanese quail

Summary This study was undertaken to examine the effect of electrical stimulation of the hypothalamus at different times of day on luteinizing hormone (LH) secretion in male castrated quail on short days (8L16D). The posterior hypothalamus was stimulated with square-wave pulses of 80 A for 2 min through chronically-implanted platinum microelectrodes. Stimulation was carried out on each quail at 4 (treatment A), 10 (B), or 14 h (C) after dawn. Plasma LH levels were increased markedly within 2 min of ending the stimulation but reached basal levels again over the next 20 min or so. The absolute increase was significantly greater in treatment B (10 h after lights on) than at the other times tested. This is consistent with a rhythm in hypothalamic responsivity. The results are discussed in the context of the rhythm of photoinducibility which occurs early in the night and which is used by quail as a photoperiodic clock to regulate seasonal reproduction.Abbreviations LH luteinizing hormone - LH-RH luteinizing hormone-releasing hormone     

A coupled pacemaker-slave model for the insect photoperiodic clock: interpretation of ovarian diapause data in Drosophila melanogaster

A coupled circadian oscillator model for the insect photoperiodic clock is described which consists of a hierarchically arranged pacemaker and slave. The pacemaker is self-sustained, temperature compensated, and entrainable by the light cycle; the slave is a damping oscillation receiving entrainment from two sources, from the pacemaker via a coupling factor, and also directly from the light. The damping slave oscillation is seen as the photoperiodic oscillator, equivalent to that proposed earlier by Lewis and Saunders (1987). The present simulations describe the effect of the strength of the coupling factor between hypothetical short- and long-period pacemaker oscillations (modelled on the clock mutants per ^sand per ^L2in Drosophila melanogaster) and a slave oscillation with a period of about 24 hours. The output is presented in terms of photoperiodic response curves and Nanda-Hamner, or resonance, plots. With a high coupling strength, the pacemakers strongly entrain the slave, but with a low coupling strength the slave's properties are more evident. The model is presented as a possible explanation for recent ovarian diapause data in D. melanogaster clock mutants (Saunders 1990), but also as a more general model for the role of the insect circadian system in seasonal time measurement.     

Photoperiodic activation of Fos-like immunoreactive protein in neurones within the tuberal hypothalamus of Japanese quail

Photoperiodic stimulation of quail (Coturnix coturnix japonica) resulted in the appearance of a nuclear fos-like protein within neurones of the basal tuberal hypothalamus. On transfer to long days the number of neurones containing this fos-like immunoreactivity increased from about 150 to 700, the neurones being scattered throughout the length of the tubero-infundibular complex. This activation had occurred by early in the second long day and was maintained for at least three long days. Over this period circulating levels of LH increased seven-fold, indicating that photoperiodic induction had taken place in the birds. A similar time-course of fos-like induction occurred in castrated quail exposed to a single long day and then returned to short days. Activation mirrored the long-term changes in LH secretion found in this paradigm and fos-like immunoreactivity showed the same carry-over characteristics of photoperiodic induction, being maximal two days after the quail had been exposed to the single long day (and were again on short days) and when LH secretion was at its maximum. Activation of fos-like immunoreactive cells did not take place when long-day quail were transferred to short photoperiods. The evidence supports the view that the neurones being activated are involved in a specific fashion in the avian photoperiodic response.     

Rhythmicity of swimming activity in an axenic population of Paramecium multimicronucleatum

A unicellular organism, Paramecium, exhibits circadian rhythm activities in many physiological phenomena, i.e., mating reactivity, photoaccumulation in Paramecium bursaria and mating type reversals in Paramecium multimicronucleatum. In this study, we used an image-processing system to analyze swimming activity in a population of Paramecium multimicronucleatum cultured axenically under 12 h-light/12 h-dark cycles (LD 1212). Swimming behavior was recorded both under LD 1212 and constant darkness and images tracing the tracks of Paramecium were produced every 4 min. Swimming activity was represented by the occupied area by the tracks relative to the total observed area. It is high during daytime and low at night and exhibits a freerunning rhythm in constant darkness. Furthermore, criteria for two major components of swimming behavior, straight and circle swimming, were established and analyzed. The results indicate that swimming behavior alters depending on the time of day: straight swimming increased during the day and circling was dominant around dusk both under LD 1212 and constant darkness.Abbreviations ZT Zeitgeber Time - LD 1212 12h- light/12h-dark cycles - TF transversing frequency     

The effects of pinealectomy and blinding on the circadian locomotor activity rhythm in the Japanese newt,Cynops pyrrhogaster

We examined the effects of pinealectomy and blinding (bilateral ocular enucleation) on the circadian locomotor activity rhythm in the Japanese newt, Cynops pyrrhogaster. The pinealectomized newts were entrained to a light-dark cycle of 12 h light and 12 h darkness. After transfer to constant darkness they showed residual rhythmicity for at least several days which was gradually disrupted in prolonged constant darkness. Blinded newts were also entrained to a 12 h light/12 h dark cycle. In subsequent constant darkness they showed free-running rhythms of locomotor activity. However, the freerunning periods noticeably increased compared with those observed in the previous period of constant darkness before blinding. In blinded newts entrained to the light/dark cycle the activity rhythms were gradually disrupted after pinealectomy even in the presence of the light/dark cycle. These results suggest that both the pineal and the eyes are involved in the newt's circadian system, and also suggest that the pineal of the newt acts as an extraretinal photoreceptor which mediates the entrainment of the locomotor activity rhythm.Abbreviations circadian period - DD constant darkness - LD cycle, light-dark cycle - LD 12:12 light-dark cycle of 12 h light and 12 h darkness     

Photoperiodism and thermoperiodism in the predatory mite Amblyseius potentillae are probably based on the same mechanism

Summary A comparative study was made of the photoperiodic and thermoperiodic induction of diapause in the phytoseiid mite Amblyseius potentillae. Sensitivity to thermoperiod was found to be highest during the protonymphal and deutonymphal stages, with some sensitivity still being present in the young adult. Summation of both photoperiodic and thermoperiodic cycles was shown to take place, which demonstrated the presence of a photoperiodic counter as well as a thermoperiodic counter in these mites. Vitamin A appeared to be necessary for some early step in the physiological mechanism of diapause induction and not just for the expression of the diapause response. The light sensitivity threshold for photoperiodic induction of diapause was found to be extremely low, viz. less than 0.02 W/cm². Moreover, the light sensitivity threshold appeared to be strongly temperature dependent in A. potentillae. Experiments in which the mites experienced various sequences of short-day photoperiods and short-day thermoperiods, applied either concurrently or in succession, showed that the information collected by the photoperiodic counter and the thermoperiodic counter is integrated into one induction sum. These results strongly suggest that photoperiodic and thermoperiodic induction of diapause in these mites is based on the same physiological mechanism.Abbreviations DD continuous darkness - LL continuous light - LD light-dark cycle (e.g. LD 16:8 is a cycle of 16 h of light and 8 h of darkness) - TC thermoperiodic cycle (e.g. TC 16:8 (27°: 15°) is a thermoperiod with a 16 h thermophase of 27 °C and an 18 h cryophase of 15°C)     

Changes in gene expression in the leaf of Lolium temulentum L. Ceres during the photoperiodic induction of flowering

Unifoliated plants of Lolium temulentum L. Ceres were induced to flower by a unique 24-h long day (LD) consisting of the extension of the regular 8-h short day (SD) (400 mol photons·m^–2·s^–1, fluorescence + incandescence) with incandescence at 10–15 mol photonsm ^–2·s^–1. The polyadenylated-RNA complement of leaf blade tissues was analysed at 4-h intervals during the photoperiod extension in LD vs. SD, by using two-dimensional polyacrylamide gel electrophoresis to resolve in-vitro-translated products. Of the 991 spots that were analysed, none appeared or disappeared during the inductive cycle, i.e. no qualitative effect of floral induction was detected, at any time. Sixty-eight spots were found whose intensity was influenced by lengthening of the photoperiod; 50 of them, i.e. ca. 5% of the population analysed, were affected before the end of the extension period and were thus potentially related to floral induction. Many of these RNAs were not quantitatively constant during a 24-h cycle in SD. Seven of them oscillated according to the light-on and the light-off signals, among which three seemed to be controlled by phytochrome since their relative amount increased under the standard light conditions but decreased under incandescence even faster than in darkness. The large majority of other RNAs varied with a timing that was not clearly driven by the alternation of light and darkness, indicating that genes related to the biological clock may be especially sensitive to the lengthening of the photoperiod. Furthermore, seven spots were observed that underwent a phase-shift in LD, which consisted, for six of them, of a phase advance of 4–8 h. The steady-state level of CAB mRNA was analysed because the CAB gene family (encoding the chlorophyll a/b-binding proteins of the light-harvesting complexes) is known to be controlled both by the biological clock and phytochrome. In SD, the level was high in the light and low in darkness; the fluctuation was conducted by a circadian rhythm. When plants were exposed to the inductive LD, the peak of mRNA accumulation that was expected according to the endogenous rhythmicity was abolished, possibly because of the change in light quality during the LD extension.Abbreviations CAB chlorophyll a/b-binding proteins of the light-harvesting complexes - 2D two-dimensional - LD(s) longday(s) - LDP(s) long day plant(s) - SD(s) short day(s) - SDP(s) short day plant(s) This work was supported by the University of Liège through the Action de Recherche Concertée (# 88/93-129). Some analyses were performed with the collaboration of Dr. H. Ougham, Institute of Grassland and Environmental Research, Aberystwyth, UK. The authors also want to thank Dr. F. Cremer (Max Planck Institute for Plant Breeding, Köln, Germany) for critical discussion of the results.     

Entrainment and phase shifting of the circadian rhythm of cell division by light in cultures of the achlorophyllous ZC mutant ofEuglena gracilis

The photosynthesis-deficient ZC mutant ofEuglena gracilis Klebs (strain Z) was cultured at 16°C on an aerated, magnetically stirred, mineral medium containing 0.1% ethanol (pH 7.0). Cell division could be entrained by a 12: 12 light: dark cycle (LD: 12, 12) or even by a one-pulse skeleton photoperiod (LD: 1,23) The rhythm free-ran in DD for at least 8 days with a circadian period (=25.5 h) in populations that had been previously entrained by LD. The freerunning rhythm could be phase-shifted by a single 1-h light pulse (3000 lx). The strong (Type 0) phase-response curve derived from the resetting effects of such signals given at different circadian times was similar to that for the photosynthetic wild-type strain. These results demonstrate that the presence of a functional chloroplast compartment is not necessary for the circadian clock to function inEuglena and suggest that phase resetting of the circadian clock by light occurs via a similar pathway in both photosynthetic and non-photosynthetic cell types.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Photoperiodic induction in quail as a function of the period of the light-dark cycle: implications for models of time measurement.

The earliest detectable event in the photoperiodic response of quail is a rise in luteinizing hormone (LH) secretion beginning at about hour 20 on the first long day. The timing of this rise was measured in castrated quail after entrainment to short daylengths which cause significant phase angle differences in the circadian system: (1) LD 2:22 and LD 10:14, and (2) LD 3:21 (T = 24 hr) and LD 3:24 (T = 27 hr). The quail were then exposed to 24 hr of light (by delaying lights-off), and the time of the first LH rise was measured; it was similar in all schedules. Quail were also entrained to LD 3:21 or LD 3:24 and then given a single 6-hr nightbreak 6-12, 7-13, or 13-19 hr after dawn. The earlier pulse was marginally more inductive in the 27-hr cycle. Thus the entrainment characteristics of the photoinducible rhythm (phi i) in quail appear very different from those of the locomotor circadian rhythm, and raise doubts as to whether phi i is a primary circadian oscillator.     

Photoperiodic time measurement in the male lizardAnolis carolinensis

Summary Photoperiod plays an important role in controlling the annual reproductive cycle of the male lizardAnolis carolinensis. Groups of anoles were exposed to various experimental lighting regimens to determine how the lizards were measuring the length of the day. The experimental regimens were designed to discriminate between the following two general classes of hypotheses: (1) Photoperiodic time measurement is based on an hourglass or interval timer which measures the length of the light or dark or (2) Photoperiodic time measurement is based on an endogenous circadian rhythm of photoperiodic photosensitivity. The experiments demonstrated thatAnolis uses an hourglass mechanism which measures the absolute length of the light period. This is in contrast to the higher vertebrates (birds and mammals) which measure photoperiodic time by means of a circadian oscillation of responsiveness to light.     

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol

The circadian rhythm of CO₂ output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (10^-6 mol) and 2,4-dinitrophenol (10^-5 mol) applied via the transpiration stream. After having been suppressed by 10^-6 M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using ¹⁴CO₂ show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO₂. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO₂-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (K_i=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO₂ output.Abbreviations CAM crassulacean acid metabolism - PEP-C phosphoenol-pyruvate carboxylase - MDH malate dehydrogenase - CHM cycloheximide - DNP 2,4-dinitrophenol - LD light-dark-cycle - DD continuous darkness     

Abscisic acid in shoots and roots of Scots pine (Pinus sylvestris L.) seedlings grown in controlled long-day and short-day environments

Scots pine (Pinus sylvestris L.) seedlings grown in nutrient solution in controlled-environment chambers were used. The effects of a shortday (SD, early autumn) treatment on growth and the content of free and alkaline hydrolysable abscisic acid (ABA) in shoots and roots were investigated. The weekly relative growth rates of seedlings grown continuously under long-day (LD, summer) conditions were stable at approx. 0.08 g g^–1 d^–1 between weeks four and eight from germination. Weekly relative growth rates of seedlings transferred to SD conditions decreased rapidly to a then stable level of approx. 0.04 g g^–1 d⁰¹. Shoot elongation ceased within two weeks of SD treatment. The content of both free and alkaline hydrolysable ABA was approx. 40–50% higher in shoots of seedlings grown for five weeks in LD plus one week in SD than in shoots of seedlings grown for five or six weeks in LD. Two additional weeks of SD did not change the free ABA content. Three weeks in simulated late autumn (SD but decreased temperatures) and three weeks in simulated winter (lower light intensity and temperature) further increased the content of free ABA in the shoots. A transfer back to LD conditions reduced the ABA content to a level equal to the level found during the first LD period. The recovery of radioactive ABA at certain times after application ofr[³H] ABA was the same in shoots and roots of LD-grown and SD-treated seedlings.Abbreviations ABA abscisic acid - LD long day(s) - RGR₇ weekly relative growth rates - SD short day(s)     

Formal properties of the circadian rhythm of locomotor activity in Japanese quail

Summary Japanese quail have a circadian rhythm of locomotor activity whose free-run period () in constant darkness (DD) was 22.5±0.1 h (45). A phase response curve of typical form was obtained by illuminating the free-running rhythm with single 1 h light pulses. Using entrainment theory a derived phase response curve was calculated from the phase relationships between the locomotor rhythm and 1 h light periods in light-dark cycles of various lengths (T). Although the limits of entrainment to theseT cycles differed slightly from those predicted, there was a close correlation between the two phase response curves. The phase relationships between the locomotor rhythm and 1–9 h photoperiods in 24 h cycles were in general accord with a prediction based on the short free-run period and the relative sizes of the delay and advance portions of the phase response curve for 1 h light pulses.