the relationships between phase and period responses to light pulses
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The Relationships between Phaseand Period Responses to LightPulsesMark A. Stokes , Stephen Kent & Stuart MaxwellArmstrongPublished online: 09 Aug 2010.
To cite this article: Mark A. Stokes , Stephen Kent & Stuart Maxwell Armstrong (2002)The Relationships between Phase and Period Responses to Light Pulses, Biological RhythmResearch, 33:3, 303-317
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The Relationships between Phase and Period Responsesto Light Pulses
Mark A. Stokes1, Stephen Kent2 and Stuart Maxwell Armstrong3
1School of Psychology, Deakin University, Victoria 3125, Australia; 2School ofPsychological Sciences, La Trobe University, Bundoora, Victoria, 3083, Australia;3Brain Sciences Institute, Swinburne University of Technology, Hawthorn, Victoria,3122, Australia
Abstract
Current theories of stable circadian entrainment postulate phase delays should beassociated with period lengthening, while phase advances should be associated withperiod shortening. While characterising features of the rat PRC to light, we noted sub-stantial numbers of responses that displayed the opposite pattern. Forty-eight rats pro-vided data for 192 phase responses. Limiting our analysis to phase shifts greater than1 hour, we found 44 displayed the expected predicted relationship, and 33 displayedthe contrary paradoxical relationship. Paradoxical responders possessed significantlyshorter initial activity periods, compared to predicted responders. Activity was sig-nificantly lengthened by paradoxical responders and shortened by predicted respon-ders following light pulse exposure. These results suggest a second mode of stableentrainment. Additionally, these results indicate entrainment mode, predicted or para-doxical, is based upon activity period duration. Short activity period durations willbe associated with paradoxical responses, long durations will be associated with predicted responses. We argue that, given the dynamic changes in photoperiod, bothmodes of entrainment are necessary to provide stable entrainment across the year.
Keywords: Circadian rhythms, entrainment, phase response curves, period responses,multiple light pulses, light pulses, rat.
Introduction
The difference between the daily light-dark cycle duration (T) and the intrinsic dura-tion of circadian clocks (t) necessitates that all animals must reset their circadianrhythm daily, or they will slip out of phase with their environmental temporal niche.This is further complicated by annual variation in photoperiod, exacerbated by
Address correspondence to: Mark A. Stokes, School of Psychology, Deakin University, 221 BurwoodHighway, Burwood VIC 3125, Austrialia. [email protected]
Biological Rhythm Research 0165-0424/3303-303$16.002002, Vol. 33 No. 3. pp. 303–317 © Swets & Zeitlinger
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increasing latitude. Consequently, an animal must reset its circadian pacemaker tomaintain the appropriate phase relationship between their internal clock and the envi-ronmental cycle on a regular basis. Two adjustments are required. One to circadianpacemaker phase, and the second to pacemaker velocity. Since Pittendrigh and Daan’s(1976a–c; Daan & Pittendrigh, 1976a, b) seminal work, it has been believed that phaseadvanced animals would tend to shorten period, while phase delayed animals wouldtend to lengthen period. However, this interaction was not formalised as a model untilthe work of Beersma, Daan and Hut (1999).
Under Pittendrigh and Daan’s model species that possessed a t < 24 obtain a dailyinternally mediated phase advance, in turn, necessitating an environmentally inducedphase delay, conserving the phase-relationship to dusk. The converse was proposedfor species with t > 24. By inversely adjusting period at the same time, the need forfurther phase adjustments would be reduced. Pittendrigh and Daan suggested sim-ultaneous adjustment of period and phase would keep animals entrained regardlessof day length fluctuation across seasons. For instance, as days shorten, a nocturnalanimal missing dusk would not receive a phase delay, but would run into dawn andreceive a phase advance. Period would shorten, lessening the need for a further phaseadvance. As days lengthened, animals would receive a net phase delay from duskintruding into the night, period would lengthen, reducing the need for additional phasedelays (Pittendrigh & Daan, 1976b).
Under Beersma et al.’s (1999) model, simulations revealed that phase and periodchanges were unstable unless period behaved inversely to phase. Beersma et al.’smodel (1999) indicates that modification of pacemaker velocity sets internal dawn or dusk to the environment. This is achieved with fast pacemakers lengthening (prolongation of internal period) and phase delaying, while slow pacemakers needperiod shortening (reducing internal period) and phase advances. The opposite rela-tionship (i.e., fast pacemakers lengthening and phase advancing, and vice versa forslow pacemakers) was found to be highly unstable. Beersma et al.’s model was usedto successfully predicted the behaviour of the diurnal ground squirrel Spermophilluscitellus (Hut, 2001).
Pittendrigh and Daan’s theory necessitates several consequences, of which, many have been assessed. For example, animals with t < 24 should display largerphase delays and smaller phase advances than animals with t > 24 from light administered at the same subjective or circadian time (CT). Additionally, phaseadvances should be associated with period shortening, and phase delays with periodlengthening. These predictions have been supported (cf. Daan & Pittendrigh, 1976a;Pohl, 1984; Honma et al., 1985; Kennedy et al., 1989; Puchalski & Lynch, 1992).Beersma et al.’s model specifically indicates that pacemaker instabilities arise wherephase advances are associated with period lengthening or phase delays with periodshortening.
Nonetheless, while examining period related bias in the rat phase response curve(PRC), it was noted that although the majority of rats displayed the predicted phaseperiod relationship, large numbers of rats did not (Stokes, 1997; Stokes et al., 2001).Many rats were phase advanced with period lengthening, others with the expectedperiod shortening, contrary to Beersma et al.’s model. On the other hand, many ratswere phase delayed with period shortening, and others with the expected period
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lengthening. These unexpected relationships imply another mechanism by whichstable entrainment might be achieved, we present these results here.
Method
Animal maintenance
Seventy male Long Evans hooded rats (Rattus norvegicus) aged between 80 and 90days were obtained from Monash University, Australia. Following delivery, rats were housed in pairs for 3 weeks in constant temperature (21 ± 2°007C), on a 12 :12Light :Dark (LD) cycle. Thereafter, rats were placed into individual cages for 37 days(2 by 2 overhead 40-watt fluorescent lights; room temperature: 21 ± 2°C) before beingplaced into constant dark (DD; 21 ± 2°C). Sixty rats were used as experimentalanimals, and ten rats were kept as a reserve pool. All rats were assessed for rhythmclarity after 30 days, and six were replaced from the reserve pool.
Individual rat cages were mounted in racks, each containing 30 wire mesh cages(H = 20cm, W = 23cm, L = 33cm) and fitted with running wheels (W = 12cm, D =33cm) equipped with a magnetic reed switch that detected wheel revolutions (cf.Redman et al., 1983). Rats were maintained on an ad libitum diet of rat pellet food(GR2+, Clark King Co.) dispensed from an external hopper, and tap water from agravity fed reservoir. When rat cages were moved to the light pulse apparatus waterwas supplied by bottles with dispensing nipples. Cages were cleaned, food wasreplaced, and water refreshed weekly at random times between 06:00 and 20:00.
Phase shift and period change assessment
Data from the running wheels was collected into 5 minute bins, and were doubleplotted, presenting 48 hours of data per line, with a new line per day of data. Eachline consisted of a histogram where each pixel represented 10% of the maximumactivity data bin for the day. Analysis of the data was primarily by eyefit of activityonset and activity offset. Plots presented to the independent raters consisted of 10days data. This type of analysis has been reported to be highly reliable (Pittendrigh& Daan, 1976a; Klemfuss & Clopton, 1993). Reliability between raters herein wasconsistently found to be high (r = 0.986).
All estimates of phase shifts, calculated in circadian hours, were derived from estimates of activity onset before and after the light pulse. The initial phase estimatewas based upon the immediate ten days preceding the light pulse, while final phaseestimate was based upon days 11 to 20 following the phase shift. Experience over thelast twenty years in our laboratory with this species suggests transients would last forup to ten days. The difference between initial and final period gave change in period.Both initial period and final period were used to estimate phase shift for 2 reasons.First, this method makes fewer assumptions about period. Methods based upon initialperiod alone assume period is stable following the pulse. Second, methods based uponprojections of initial period from the first non-transient day following the phase shiftare biased by the number of days allowed for transients.
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Experimental protocol
Each of the 60 rats was randomly assigned to one of 12 circadian time (CT) groups(n = 4 each CT; CTs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24/0), or two control groups.The first control group (n = 6) was exposed to the pulsing apparatus without light, no pulse controls (NP). The second control group (n = 6) was never moved from the rat rack (Con) and, providing a control for non-experimental changes inperiod.
Light pulses (60min, 1,000 lux, two 40-watt flourescent tubelights) were adminis-tered by transporting the cage, with rat in situ, to an adjacent sound attenuated, tem-perature controlled room (21 ± 2°C), minimising the effects of handling stress. Thisprocedure was repeated four times, ensuring each rat received the same light dose atthe same CT on four different occasions. Pulse time was assessed on the day prior tothe pulse on the basis of the data for the ten preceding days, and then extrapolated tothe pulse day. The first light pulse was administered following 46 days constant dark(DD). The rats were returned to DD for 33 days, before the second light pulse. Thisprocedure was repeated with the third and fourth light pulses being administered aftera further 26 days DD each.
Analyses
To reduce the contribution of spurious data, the data analyses reported herein focusesonly on those responses where a phase shift (Df) greater than one hour was obtained.Data from 44 predictable and 33 paradoxical phase responses conformed to this criterion. These data were separated into four groups, advance and delay for both the predictable and paradoxical responders.
To avoid a violation of assumptions the response profile (period change (Dt) &Df) variables were excluded from all analyses. It was decided to analyse all data in2-way (Predicted vs Paradoxical by Advance vs Delay) general linear models. Vari-ables selected for the analyses included initial period (ti), initial activity period (ai),and change in initial activity period (Dai). Activity period was analysed as a propor-tion of circadian period (t) to obtain a measure independent of t length. As none of these variables (ti, ai, & t) are circular, standard linear statistics were employed(Batschelet, 1981).
Results
Of the 192 administered phase shifts, data was collected from 181 phase shifts. Equip-ment failures prevented data collection for the remaining 11 phase shifts. Phase andperiod changes were plotted as vectors from the initial phase and period, terminat-ing at the obtained phase and period (Fig. 1A). It was apparent that there were tworesponse types. Those where phase advances and t reductions were associated, orphase delays with t increases (n = 44, Fig. 1B), predicted responders. And those with associations between phase advances and t lengthening, or phase delaying witht shortening (n = 33, Figs. 1C), or paradoxical responders.
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The relationships between Dt and Df were examined for predicted and paradoxi-cal responders possessing Df > 1 hour. As no Df exceeded 7 hours, Df was able tobe treated as linear, and as Dt is not circular by its nature, standard Pearson’s regres-sions were used (Batschelet, 1981). When all data were analysed, no significant
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Figure 1. Raw data for period change and phase shift. The abscissa plots initial phase andfinal phase in circadian hours as a vector. The ordinate plots initial and final period as a vector.For an increase in period the vector points up, while for a phase delay, the vector points left.A: all data. B: results with absolute phase shift greater than one hour that conformed to Pittendrigh and Daan’s (1976b) theory. C: presents results with absolute phase shifts greaterthan one hour that do not conform with theory.
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Figure 2. Phase response patterns for predicted phase responses (A) and paradoxical phaseresponses (B) where the phase shift exceeded one hour. The abscissa plots time of pulse, whilethe left ordinate plots phase shift in circadian hours. The right ordinate plots period change inhours and change in the ratio of activity/period.
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relationship between Dt and Df was found (r = 0.06, ns, n = 77). However, predictedresponders possessed a significant negative relationship between Dt and Df (r =-0.56, p < 0.001, n = 44), while paradoxical responders displayed the opposite trend (r = 0.76, p < 0.001, n = 33).
Paradoxical responders possessed stronger phase delays at CT14 than did predictedresponders (Fig. 2). When extrapolated over the ten day measurement period, Dtswere similar in size to Dfs from CT12 to CT22 for both predicted and paradoxicalresponders. For predicted responders the effect of the Dt achieved was equal to 93%of the measured Df, while for paradoxical responders Dt was approximately equal to73% of the measured Df. The estimation methods we used do not confound Dt withinDf, therefore, measured Df cannot be attributed to Dt. Phase shift, period change,and rest/activity ratio changes for both predicted and paradoxical responders werethen examined. It was apparent from the data that predicted and paradoxical groups:1) displayed typical PRCs; 2) displayed different period responses (by definition); and3) differed in their activity period changes. Generally, predicted responders decreasedactivity, while paradoxical responders mostly increased activity.
The variables initial period, initial activity period, and change in activity period(Table 1) were analysed by 2-way general linear model (Predicted vs Paradoxical byAdvance vs Delay). There were no significant differences between groups on initial
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Table 1. Mean data (± SEM) for predicted type and paradoxical type phase responses.
ADVANCES DELAYS
PREDICTED RESPONSESAbbreviation n per group 9 35Dt Change in period (hours) -0.05 (± 0.01) 0.10 (± 0.01)Df Phase shift (circadian hours) 1.56 (± 0.11) -2.11 (± 0.17)
ti * Initial period (hours)† 24.26 (± 0.06) 24.29 (± 0.03)ai * Activity period (hours)† 11.61 (± 0.35) 11.29 (± 0.21)ai% * Activity period (%)† 47.89 (± 1.51) 46.47 (± 0.88)Da% * Change in activity period -14.14 (± 4.92) -2.50 (± 1.12)
(%)†
PARADOXICAL RESPONSESAbbreviation n per group 14 19Dt Change in period (hours) 0.12 (± 0.03) -0.10 (± 0.02)Df Phase shift (circadian hours) 1.54 (± 0.10) -2.34 (± 0.24)
ti * Initial period (hours)† 24.21 (± 0.06) 24.24 (± 0.04)ai * Activity period (hours)† 10.29 (± 0.48) 11.03 (± 0.23)ai% * Activity period (%)† 42.49 (± 1.93) 45.32 (± 0.96)Da% * Change in activity period 4.49 (± 6.28) 0.43 (± 2.70)
(%)†
*Variables included in the analyses.†Significant at p < 0.05.
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period duration. Initial activity period was longer among predicted responders thanamong paradoxical responders both in absolute hours (F(1,73) = 5.64, p = 0.02) andproportion of circadian period (F(1,73) = 5.87, p = 0.02). Moreover, predicted respon-ders significantly reduced activity period, while paradoxical responders significantlylengthened activity period (F(1,73) = 9.96, p = 0.02). Length of activity period wasfound to weakly, but significantly predict group membership, paradoxical or predicted,in a multiple regression (R2 = 0.06, p = 0.04, b = 0.24).
Activity period results must be understood in relation to changes in circadianperiod, as activity period is one component of circadian period. In order to facilitatethis, we modelled the group mean activity onset and offset results as a schematicactogram, measured against circadian time measured before the light pulse (Fig. 3).This revealed that when taken together, advances and delays, rats which respondedas predicted behaved as though photoperiod were expanding, and paradoxical respon-ders behaved as though photoperiod were contracting.
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Figure 3. Group mean activity onsets (�) and offsets (�) displayed by group: predictedadvances and delays, paradoxical advances and delays. By definition, activity onset is set atCT12 prior to the light pulse. The figure is divided by the datum which indicates the day ofthe light pulse. Elements above the datum indicate 10 days of data preceding the light pulse,while elements below the datum indicate 10 days of data following the transient period (10days). All measures are presented in circadian time based upon period prior the light pulse,thus showing the extent of changes. The general phase region where light pulses were pre-sented is shown by the open arrow (➩). Following the light pulse, the new activity onset is shown displaced from CT12 by the amount of phase shift and period change following the lightpulse.
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Discussion
The circadian literature indicates that in the absence of morning and evening lightpulses, the relationship between Dt and Df is phase dependent (Pittendrigh and Daan, 1976a–c; Moore-Ede et al., 1982). Pittendrigh and Daan (1976b) suggest phaseadvances should be associated with period shortening, while phase delays should beassociated with period lengthening. Furthermore, changes to activity period are pre-dicted to follow changes in period. The present results do not support these asser-tions, they suggest a more complex relationship between the durations of activityperiod and initial circadian period, and the magnitude of phase shift.
The most interesting result was that both delayed and advanced paradoxicalresponders significantly lengthened their activity period, or subjective night, follow-ing a light pulse, even though circadian period itself shortened among delayed para-doxical responders and lengthened among advanced paradoxical responders. While,on the other hand, predicted responders significantly shortened activity period fol-lowing a light pulse, even though circadian period lengthened among delayed predicted responders and shortened among advanced predicted responders. How-ever, paradoxical responders possessed shorter initial activity periods than did ratsthat behaved as predicted, indicating that activity period in both groups may havebeen returning to the mean; lengthening for paradoxical responders and shorteningfor predicted responders. Nonetheless, this indicates that change in t does not dictatechange in activity period.
It may be argued that the obtained paradoxical responses are spurious, or that theyare derived from spontaneous changes in t. The shape of the obtained PRC for para-doxical and predicted responders, conforming to a typical type 1 PRC, argues thatthese were not spontaneous or spurious results. Moreover, the estimation method usedmeasures Dt separately from Df, relying upon vectors from both initial and finalperiod. Because final period follows a vector to its origin on the pulse day, if its originhas not moved from that of initial period, the 2 vectors will show no net phase shift,even if t has itself changed. This method precludes the attribution of paradoxical Dfsto simple Dt. Even were these results due to Dt, this would not exclude their impor-tance, as an effective phase response would have arisen, requiring understanding.Hence, these paradoxical results need to be explained in a manner consistent withstable entrainment. The possible explanations include 1) a result generated by theexperimental conditions that are never replicated in nature and indicating period andphase responses are independent, 2) a result typical of the species or strain of rat used,or 3) a result revealing a further mode of stable entrainment.
Actographic data has been published for several species showing phase delaysassociated with period shortening. These include Sprague-Dawley rats (Bauer, 1992),the golden hamster (Mesocricetus auratus; Pittendrigh, 1981; Meijer & De Vries,1995; Reebs & Doucet, 1997), the Australian marsupial the kowari (Dasyuroidesbyrnei, Kennedy et al., 1989), the tree shrew (Tupaia belangeri; Meijer et al., 1990),the house sparrow (Passer domesticus; Binkley & Mosher, 1987), and the microchi-ropteran bat (Hipposideros speoris; Joshi & Chandrashekaran, 1984). This effect isalso apparent among insects. Page and Barrett (1989) report an actograph for the
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cockroach Leucophaea maderae with phase delays and period shortening. Similarly,actographic data has been published for several species showing phase advances asso-ciated with period lengthening. These include Sprague-Dawley rats (Bauer, 1992),hamsters (M. auratus; Reebs & Doucet, 1997), sparrows (P. domesticus; Binkley & Mosher, 1987), the Australian marsupial the dunnart (Sminthopsis macroura;Cassone, 1987), the microchiropteran bat (H. speoris; Joshi & Chandrashekaran,1984), and the cockroach (L. maderae; Page & Barrett, 1989). Pittendrigh and Daan(1976a) and Daan and Pittendrigh (1976a) found phase delays were associated withperiod decreases in golden hamsters. Meijer et al. (1990) could not clarify why theyfound phase advances associated with increases in t in the tree shrew T. belangeri.Further, Reebs and Doucet (1997) reported findings hamsters with ti < 24 hours whereincreased phase advances were significantly associated with shorter ti. Additionally,in hamsters with ti > 24 hours increased phase delays were associated with longertis. Most of these experiments used differing protocols, suggesting paradoxicalresponding was not a product of the experimental conditions. The experiments were undertaken across a wide variety of species, including some taken from the wild, suggesting paradoxical responding is not the product of a few species, nor anartefact of laboratory bred animals. No apparent methodological or limited explana-tion of these effects is apparent. Consequently, it must be concluded that the present finding of paradoxical results is not unique, is widespread across species, is not the product of a single protocol, is a genuine effect, and has not yet beenexplained.
Photoperiod varies across the year. The model of entrainment proposed by Pittendrigh and Daan (1976a–c, Daan & Pittendrigh, 1976a–b), where Dt and Dfbehave oppositely, t increasing for phase delays, t decreasing for phase advances,provides a mechanism where stable entrainment to dawn or dusk is possible. This isachieved by obtaining daily Dfs greater than Dt. In paradoxical responders the Dfexceeds and compensates for Dt and is able to retain a stable relationship to dusk ordawn (Fig. 4). However, under the standard paradigm an animal that is exposed to aphase delay as days shorten would obtain a longer period and need further re-adjustment, possibly increasing its exposure to environmental ‘threats’. During latesummer and autumn, as photoperiod decreases, and scotoperiod increases phasedelays associated with period lengthening will result in a requirement for much largerphase delays or loss of entrainment would ensue. Interestingly, in the present resultsphase delays among paradoxical responders were considerably larger than among predicted responders. Another model is possible where the need for compensatoryphase shifts is not required.
The presence of a second entrainment mode would offer a distinct advantage toan animal. If phase advances may be associated with period lengthening, and delayswith period shortening, paradoxical responses, then as photoperiod commences short-ening, and scotoperiod is lengthening, animals acquiring a paradoxical phase delayor paradoxical phase advance, are more able to maintain a stable relationship to thevectors of dawn and dusk (Fig. 5).
For instance, as scotoperiod starts to lengthen (autumn), a nocturnal animal is lesslikely to be phase shifted. Animals find dawn later and dusk earlier. As dusk and dawn
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intrude into the prior photoperiod, an nocturnal animal receiving a delay pulse, para-doxically shortening period would anticipate next dusk earlier the next day. The sameanimal receiving an advancing pulse would paradoxically lengthen period and antici-pate dawn later the next day. Thus, a second mode of entrainment that became active
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Figure 4. Schematic representation of the phase relationship between morning light and phaseshifts at dawn (yPmØD) and phase relationship between evening light and phase shifts at dusk(yPEØN) vectors as specified by Pittendrigh and Daan (1976b, c), projected as though from a single Df. These vectors are exaggerated to render visible otherwise small, imperceptibleeffects. The diagrams to the right represent the Df and Dt relationship that the yPmØD andyPEØN vectors are derived from.
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Figure 5. Schematic representation of the phase relationship between morning light and phaseshifts at dawn (yPmØD) and phase relationship between evening light and phase shifts at dusk(yPEØN). yPmØD and yPEØN vectors displayed across seasonal night length based upon sea-sonal entrainment. Vectors are provided for both predicted (Spring) and paradoxical (Fall/Autumn) responses as though from a single Df. Phase relationship vector indicates the vectorobtained following a Df. These vectors are exaggerated in the figures to render visible other-wise small, imperceptible effects. The diagrams to the right represent the Df and Dt relation-ship that the yPmØD and yPEØN vectors are derived from.
as nights lengthen would maintain tighter phase relationships with dusk and dawnregardless of the phase of light exposure.
When mean group data were assessed (Fig. 3) it became apparent that predictedresponders altered f, t, and a in a manner that anticipated the next light pulse asoccurring earlier the next morning or later the next evening, lengthening photoperiod.On the other hand, paradoxical responders changed f, t, and a in a manner indicat-ing the circadian system anticipated the next light pulse to occur later in the morning,or earlier in the evening, shortening photoperiod, lengthening scotoperiod. Addition-ally, paradoxical responders in the present analysis possessed shorter activity periods(nights) than predicted responders, and lengthened their activity period regardless of
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changes to circadian period; precisely as would be expected if they had been respond-ing to short nights that were lengthening (i.e., Autumn).
Evidence is available to support the contention that phase and period responsesare independent. Page (2000) reported finding no relationship in phase and period ineye of the marine mollusk Bulla gouldiana. Blocking protein synthesis by adminis-tering Cyclohexamide to B. gouldiana eyes, reset the oscillator phase, but did notresult in period differences between control and experimental eyes. Period was con-served. Thus phase is dependent upon protein synthesis, while period would appearindependent of protein synthesis. Here, we are suggesting that activity duration, ratherthan circadian period duration or phase response modulates entrainment response.
It is all very well to suggest a second mode of entrainment, however, some mech-anism should be inferred if this hypothesis is to be given credence. The present resultsindicate the duration of activity as important. If activity period mediates entrainmentmode, then entrainment mode must be controlled by reference to t, suggesting his-torical dependence, a second pacemaker, or amplitude based control.
Historical dependence has been suggested before, most notably by Pittendrigh andDaan (1976a) with after-effects to long and short light cycles. Pittendrigh and Daan(1976a–c) found historical dependence in mice at 60 days. Elsewhere, we report his-torical dependence in these rats at approximately 30 days (Stokes et al., 2001). Thismay be achieved by reference to a second pacemaker, or pacemaker sub-units. Othercircadian pacemakers are known to exist, such as the meal entrained pacemaker whichholds a stable period (Coleman et al., 1982; Kennedy et al., 1989; Stephan, 1989).One further possibility, pacemaker amplitude may provide information about relativeperiod length, and the direction this should be adjusted. Further experimentation willbe required to clarify this issue.
Seasonal influences in the PRC may explain other data that is presently unex-plained. Reebs and Doucet (1997) reported findings in hamsters they could notexplain. They found short t hamsters (t = 23.33) and long t hamsters (t = 24.67) dis-playing t lengthening and t shortening following release from entrainment. Whenexposed to a light pulse some hamsters behaved paradoxically, others as predicted.Rather than circadian period, activity period may be the explanation needed to under-stand this behaviour.
The hypothesis presented herein to explain these results posits 2 modes of en-trainment or seasonally modulated entrainment. It may be argued that this wouldcountervail against homoeostasis. However, treating phase and period change asmechanisms to preserve phase relationships, and to then provide the best predictionof later events indicates that a second mode of entrainment is required to maintainhomoeostasis. Under Pittendrigh and Daan’s scheme where t > 24 phase advancesshould outweigh delays, where t < 24 the PRC will be biased toward delays. Phaseadvances tend to reduce t and propel the t vector to earlier in the cycle and awayfrom dawn, enlarging the phase relationship between dawn and subjective morning(yPMfD), whereas a phase delay increases t and propels the t vector to later in thecycle and away from dusk, enlarging phase relationship between dusk and subjectivenight (yPEfN). During spring, dusk and dawn are converging nocturnally, and theserelationships would not lead to vectors heavily displaced from dusk and dawn. During
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the autumn, dusk and dawn are diverging nocturnally, and these relationships wouldlead to greatly increased yPMfD and yPEfN (Fig. 4). During autumn both phaseadvances and delays with the predictable relationships are contrary to maintainingstable entrainment for either nocturnal or diurnal animals. Should period changerender t > 24 by phase delays, and t < 24 following phase advances, entrainmentduring autumn would become intractable. Therefore, for autumnal entrainment to bestable, animals must utilise some other mode of entrainment, one such as is postu-lated herein based upon activity period length.
Given these results, we conclude that seasonally modulated entrainment is not onlypossible, but likely. However, given the exploratory nature of this analysis furtherexperimentation is needed to evaluate this possible entrainment mechanism. Suchexperimentation could be undertaken by examining the PRC of animals exposed tolengthening or shortening light cycles immediately prior to establishing the PRC.
References
Batschelet E (1981): Circular statistics in biology. (Mathematics in Biology). New York,New York, USA: Academic Press.
Bauer MS (1992): Irradiance responsivity and unequivocal type-1 phase responsivity ofrat circadian activity rhythms. Am J Physiol 263: R1110–R1114.
Beersma DGM, Daan S, Hut RA (1999): Accuracy of circadian entrainment under fluc-tuating light conditions: Contributions of phase and period responses. J Biol Rhythms14: 320–329.
Binkley S, Mosher K (1987): Circadian rhythm resetting in sparrows: Early response todoublet light pulses. J Biol Rhythms 2: 1–11.
Cassone VM (1987): Circadian organization and photoreception in an Australian Dasyuridmarsupial (Sminthopsis macroura). J Biol Rhythms 2: 261–268.
Coleman GJ, Harper S, Clarke J, Armstrong SM (1982): Evidence for a separate mealassociated oscillator in the rat. Physiol Behav 29: 107–115.
Daan S, Pittendrigh CS (1976a): A functional analysis of circadian pacemakers in noc-turnal rodents. II. The variability of phase response curves. J Comp Physiol A 106:253–266.
Daan S, Pittendrigh CS (1976b): A functional analysis of circadian pacemakers in nocturnal rodents: III. Heavy water and constant light: Homeostasis of frequency? JComp Physiol A 106: 267–290.
Honma K, Honma S, Hiroshige T (1985): Response curve, free-running period, and activ-ity time in circadian locomotor rhythm of rats. Jpn J Physiol 35: 643–658.
Hut RA (2001): Natural entrainment of circadian systems: a study in the diurnal groundsquirrel Spermophilus citellus. Unpublished Ph.D. Dissertation. Rijksuniversiteit,Groningen, the Netherlands.
Joshi D, Chandrashekaran MK (1984): Bright light flashes of 0.5 milliseconds reset thecircadian clock of a microchiropteran bat. J Exp Zoo 230: 325–328.
Kennedy GA, Armstrong SM, Coleman GJ (1989): Phase-response curve to 1-hour lightpulses for the marsupial, Dasyuroides byrnei. Physiol Behav 46: 667–670.
316 M.A. Stokes et al.
Dow
nloa
ded
by [
Nor
thea
ster
n U
nive
rsity
] at
17:
39 1
5 O
ctob
er 2
014
Kirk RE (1982): Experimental design: Procedures for the behavioral sciences. PacificGrove, California, USA: Brooks/Cole.
Klemfuss H, Clopton PL (1993): Seeking tau: A comparison of six methods. J InterdiscCycle Res 24: 1–16.
Meijer JH, De Vries MJ (1995): Light-induced phase shifts in onset and offset of runningwheel activity in the Syrian hamster. J Biol Rhythms 10: 4–16.
Meijer JH, Daan S, Overkamp GJF, Hermann PM (1990): The two-oscillator circadiansystem of tree shrews (Tapaia belangeri) and its response to light and dark pulses. J Biol Rhythms 5: 1–16.
Moore-Ede MC, Sulzman FM, Fuller CA (1982): The clocks that time us: Physiology ofthe circadian timing system. Cambridge, Massachusetts, USA, Harvard UniversityPress.
Page T (2000): A novel mechanism for the control of circadian clock period by light. J Biol Rhythms 15: 155–162.
Page TL, Barrett RK (1989): Effects of light on circadian pacemaker development. II.Responses to light. J Comp Physiol A 165: 51–59.
Pittendrigh CS, Daan S (1976a): A functional analysis of circadian pacemakers in nocturnal rodents: I. The stability and lability of spontaneous frequency. J CompPhysiology A 106: 223–252.
Pittendrigh CS, Daan S (1976b): A functional analysis of circadian pacemakers in noc-turnal rodents. IV. Entrainment: Pacemaker as clock. J Comp Physiol A 106: 291–331.
Pittendrigh CS, Daan S (1976c): A functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: A clock for all seasons. J Comp PhysiolA 106: 333–355.
Pittendrigh CS (1981): Circadian systems: Entrainment. In: Aschoff J. ed., Handbook ofbehavioural neurobiology. Vol. 4 Biological rhythms. New York, USA, Plenum Press.
Pohl H (1984): Differences in responses of the circadian system to light in the Syrianhamster. Physiol Zool 57: 509–520.
Puchalski W, Lynch GR (1992): Relationship between phase resetting and the free-runningperiod in Djungarian hamsters. J Biol Rhythms 7: 75–83.
Redman J, Armstrong S, Ng K (1983): Free-running activity rhythms in the rat: Entrain-ment by melatonin. Science 219: 1089–1091.
Reebs SG, Doucet P (1997): Relationship between circadian period and size of phaseshifts in Syrian hamsters. Physiol Behav 61: 661–666.
Stephan FK (1989): Entrainment of activity to multiple feeding times in rats with suprachi-asmatic lesions. Physiol Behav 46: 489–487.
Stokes MA (1997): The effect of single and multiple pulses of light upon the circadianphase response of the rat. Unpublished Ph.D. Dissertation, La Trobe University, Melbourne, Victoria, Australia.
Stokes MA, Kent S, Armstrong SM (2001): The effect of repeated pulses of light at thesame time on period responses of the rat circadian pacemaker. Chron Int 18: 187–202.
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