Light-pulse phase response curves for the locomotor activity rhythm in Period mutants of Drosophila melanogaster

J. Insect Physiol. Vol. 40, No. II, pp.951-968, 1994 Copyright 0 1994 Elsevier Science Ltd

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0022-1910(94)00057-3

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Light-pulse Phase Response Curves for the Locomotor Activity Rhythm in Period Mutants of Drosophila melanogaster D. S. SAUNDERS,*

S. W. GILLANDERS*,

R. D. LEWIS?

Received 4 March 1994; revised 3 May 1994

Parameters (z, a and p) of the adult locomotory rhythm, and phase response curves (PRCs) of this rhythm to light pulses, were examined in wild type and period mutants (per’ and per Lz) of Drosophila mefunogaster. When compared to wild type, perS was shown to have a shortened active phase (a) whereas per L2 was shown to have a lengthened rest phase ( p). PRCs for all three strains were shown to change from a ‘low amplitude’ Type 1 to a ‘high amplitude’ Type 0 with increased duration of the light pulse, and perS to have an apparently increased light ‘sensitivity’. These PRCs offered good predictions for steady state entrainment (ofpers) to 24 h light-dark cycles. The shortening of circadian period in perS and its lengthening in perL2 were shown to be consequences of changes in the ‘time course’ of the oscillation in the late subjective day. Since the late subjective day was lengthened in perL2 but a was not, these two parameters are considered to be independently regulated. Drosophila melanogaster

Adult

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rhythms

Period mutants

INTRODUCTION

University

curves

(Siwicki et al., 1988), and the levels of per gene product and its underlying mRNA have been shown to undergo circadian oscillations, appropriate for the particular mutation, suggesting that the per protein regulates its own production through negative feedback (Hardin et al., 1990, 1992). per-mutant flies have also been investigated at the behavioural level. For example, the shortening of T in perS from about 24 to about 19 h was shown to be due to a shortening of the active phase of the cycle (CI) from about 12 to 7 h; the duration of the inactive or rest phase (p) remained unaltered (Konopka and Orr, 1980). In addition, temperature compensation of z was altered in mutant flies, the period of perS slightly lengthening, and that of per L’ substantially shortening, as the temperature was lowered from 26 to 17 or 15°C Konopka et al., 1989; see also Ewer et al., 1990). Also associated with mutation at the period locus were differential changes in T with age, or with exposure to constant low intensity light (Konopka et al., 1989). Strangely, there has been relatively little attention given to phase response curves (PRCs) in D. melanogaster, mutant or wild type, despite the fact that such analyses provide considerable information about the ‘time course’ of the circadian pacemaker, its ‘structure’, and the nature of entrainment to environmental light cycles (Pittendrigh, 1965, 1966; Johnson, 1992). Early PRCs, for wild type and pus, concerned the eclosion rhythm (Winfree and Gordon,

Over 20 years ago, Konopka and Benzer (1971) isolated three behavioural mutants of Drosophila melanogaster (called period) with altered circadian clocks controlling both pupal eclosion and adult locomotor rhythmicity. Whereas wild type flies showed free-running circadian periods (in darkness) close to 24 h, a short-period mutant (pers) expressed rhythms with a period (z) close to 19 h, a long-period mutant (per’; subsequently called perL’) a period close to 29 h, and a third class of mutants (per’) which were apparently arrhythmic. All three mutations mapped to the same genetic locus in the zeste-white region of the X-chromosome (Konopka and Benzer, 1971; Young and Judd, 1978), and subsequent molecular analysis has shown the importance of a fragment from this region which is sufficient for P-element restoration of rhythmicity to arrhythmic flies (Bargiello et al., 1984; Zehring et al., 1984; Hamblen et al., 1986). Mutational changes in the genome and in the per gene product have also been elucidated (Baylies et al., 1987; Yu et al., 1987). In adult flies, which concern us here, immuno-histochemical studies using antibodies raised against the relevant per protein, have demonstrated expression in the brain

*Institute of Cell, Animal and Population Biology, Edinburgh, Edinburgh EH9 3JT, Scotland. tSchool of Biological Sciences, University of Auckland,

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D. S. SAUNDERS et al.

1977; Konopka, 1979); one later study involved adult locomotor rhythmicity (Dushay et al., 1990) but the most complete data set (Orr, 1982) has remained formally unpublished. For eclosion rhythmicity (Konopka, 1979; Konopka and Orr, 1980) it was shown that 40 min pulses of white light at 300 ft-c gave a ‘weak’ Type 1 PRC (Winfree, 1970) for wild type flies, but a ‘strong’ Type 0 PRC for pus, suggesting an increased ‘sensitivity’ in the mutant flies. These studies also showed that the light-insensitive

A

‘dead-zone’ of the PRC (i.e. the subjective day) was shortened in perS by about 5 h, thus matching the shortened r for this strain referred to above. For locomotor rhythmicity, published PRCs are even rarer, the only example being one for wild type flies (Dushay et al., 1990) which showed, for 10 min pulses of light at 2000 lx, a Type 1 PRC with maximum delays of about 4 h and maximum advances of about 2 h. No PRCs for a long period mutant have ever been published. However. in a comprehensive but formally unpublished

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Period (h) FIGURE 1. Representative actograms for adult locomotor activity rhythms in females of Drosophila meiunogusler in continuous darkness (DD) and at 20°C. (A) wild type (per+)); (B) theshort period mutant, pe?; (C) a long period mutant, perLZ. Free-running periods were calculated by two methods: at least-squares regression through the mid-points of each day’s activity band (a) (lines on left), and by periodogram analysis (right).

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study, Orr (1982) showed that pulse durations of 1, 10 and 80 min, all with 2000 lx at several temperatures, gave rise to almost identical low amplitude Type 1 PRCs, for all three mutants. These data confirmed that the subjective day was shortened in perS and, moreover, showed a lengthening of the subjective day in perL’. In the present paper, phase response curves are reported for 1, 2, 6 and 10 h pulses of dimmer white light (-300 lx) applied to wild type, perS and another long-period strain, per L2 (see below). These studies, which show significant differences to those reviewed above were initiated as part of an analysis of the induction of ovarian diapause in photoperiodic D. melanogaster (Saunders et al., 1989; Saunders, 1990); hence the use of longer duration pulses. This paper is restricted to reports of the PRCs and some other basic data; predictions using the PRCs for diapause induction studies-within a framework of the entrainment phenomenon-will be considered elsewhere. MATERIALS

AND METHODS

Flies

Wild type (Canton-S) and mutant D. melanogaster were maintained on a standard cornmeal medium at 25°C under a cycle of 12 h of light and 12 h of darkness (LD 12: 12). Mutant flies comprised two strains homozygous for alleles of the per locus (Konopka and

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Benzer, 1971). These were perS with a shorter than normal free-running period in darkness (about 19 h), and perL2 with a longer than normal period (about 29 h). [per L2 is considered to be a re-isolation of the original long-period mutant (Gailey et al., 1991) but the terminology, perL’, per L2, is preserved here because some differences between them have been observed.] These per mutants were derived from a Canton-S strain (R. J. Konopka, personal communication); a version of that strain was employed here as a wild type. Recording locomotor activity rhythms

The locomotor activity rhythms of adult flies were recorded by placing single virgin females of the various strains into small glass tubes (50 x 5 mm o.d.) in the path of an infrared light beam (using Radio Spares IR emitters, type 306-077, and detectors, type 306-083) at a constant temperature of 20 * 0.5”C. In early experiments, one end of the tube contained a wicked water supply, the other was blocked by a crystal of sugar. Since flies often became stuck on the sugar, the water was replaced by a sugar solution and the sugar crystal by a cotton wool plug in later experiments. For about 3 days before their introduction to the tubes, the flies were maintained at 20°C and LD 12 : 12 with an ample food supply to build up their reserves. They were then placed in the recording apparatus for 2 to 3 weeks, during which time they were allowed to free-run in DD or exposed to timed light pulses (see below).

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Period (h) FIGURE 2. Phase delay caused by a single 6 h pulse of white light (open box) delivered on day 7. The delay phase shift is calculated as the shift (in circadian hours) between successive mid-points of a (equivalent to Circadian time, Ct 6). Right: periodograms,

to a perS female of D. melunogaster the two lines extrapolated between before and after the light pulse.

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Interruptions of the infrared light beam by the movement of the flies were recorded using a BBC B plus microcomputer in IO-min bins. These data were then assembled into the conventional ‘double-plotted’ actogram format (Fig. 1) and subjected to periodogram analysis and determinations of phase shift and activity duration using an Acorn Archimedes 400 series computer and specially written software. Delivery of light pulses

Light pulses were presented to flies in their activity monitors which were housed in light-tight wooden boxes held in a constant temperature room at 20 + 05°C. Light was provided by 4 W fluorescent tubes, water jacketted to control any temperature rise when the light was on, and regulated by 24 h timers. Flies were allowed to free-run in DD for 6 days and then exposed to single pulses of white light (1.13 + 0.05 W m-‘) of 1, 2, 6 or 10 h duration, timed to come on at different circadian phases. After the light pulse the flies were allowed to free-run again for a further 7 days before the records were analysed.

ef al.

squares regression line was then computed through the daily midpoints. A similar calculation was performed for the free-running activity band following the light pulse, and the computer program was used to calculate the phase shifts caused by the pulse, in circadian time. Phase shift data are presented in the form of standard PRCs, plotting phase shifts (in circadian h) as a function of the circadian time at which the light pulse commenced. Type 1 curves were eye-fitted to the data or calculated by fitting polynomials; Type 0 were calculated by linear regression. RESULTS Parameters of the free-running period mutant flies

rhythm of wild type and

The period of the free-running rhythm (r) in DD, and the duration of the active phase of the cycle (c() were determined in females of wild type (per + ), perS and perL2. Circadian period was determined by two methods, periodogram analysis (based on Enright, 1965 and Determination of phase shifts Williams and Naylor, 1967) and by calculation of the Since the onsets and offsets of locomotor activity in slope of the line drawn through successive midpoints of CI.In groups of 30 females of each strain a comparison D. melanogaster were not always sharply defined, the of these two methods showed differences to be non-sigmidpoint of the activity band (a) was used as a phase reference point, and regarded as the middle of the nificant or of only marginal significance; therefore, for subjective day, or Ct 6 (see Fig. 2). The midpoint of CI ease of analysis, the second method was adopted in was determined with a computer program in which the further calculations. In separate samples of 50 flies of each strain, z and cursor was used to select the beginning and the end of each days activity by subjective judgement. A least CIwere determined for each fly (Fig. 3). The duration of

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+ 23.72

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FIGURE

3. Distributions

FL2

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Period,z h

of (A) free-running females, showing

Activity duration, Q h

periods (DD, 20°C) and (B) active phase durations means and standard deviations (horizontal bars).

(a) for pers, per + and per’-*

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p in each fly was calculated by subtracting c1 from z. Table 1 shows that mean r for per + was 23.72 + 0.9 h, whereas that for pus was 19.70) 0.57 and that for per L2 was 29.10 f 3.64 h. These are all significantly different (per + vs. pus, t = 26.451, P < 0.001; per+ vs. per L2, t = 19.434, P < 0.001; pe? vs. per’*, t = 36.469, P < 0.001). The shortening of z in perS was entirely due to a shortening of CI from 12.8 to 8.47 h (t = 6.613, P < 0.001) whereas the lengthening of t in perL2 was entirely due to a lengthening of p (t = 6.510, P < 0.001). Mean values of E in per + and perLZ were not significantly different (t = 1.311); neither were the differences between the values of p between per+ and perS (t = 0.574). Figure 3 also shows that between-fly variations of T and u (expressed as standard deviations) were least for perS and greatest for perL2 (see also Konopka and Benzer, 1971; Hamblen et al., 1986). PRO Figures 4-6 show PRCs for wild type (per + ), perS and perL2 females of D. melanogaster generated by exposure of flies free-running in DD to single light pulses of 1 and 6 h, or in the case of perS, to 1, 2, 6 and 10 h. In per+, 1 h pulses gave rise to a low amplitude Type 1 PRC (Winfree, 1970) [Fig. 4(A)]. In the eye-fitted curve maximum delays and advances were of com-

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parable magnitude (about 4 h); the ratio of the area under the delay part of the PRC to that under the advance part (D/A) is therefore close to 1. Pulses of light of the same intensity but extended to 6 h [Fig. 4(B)], however, gave rise to a Type 0 PRC with an average slope close to - 1 (Winfree, 1970). This suggests that 6 h light pulses reset the oscillation to a near constant phase regardless of the phase at which the pulse commenced. In the short period mutant, perS, 1 h pulses of light gave an eye-fitted PRC with delay phase shifts approaching the maximum (12 circadian hours) but advances only up to about 6 Ct h [Fig. 5(A)]. Two data points close to maximum (at Ct 15-16) could be plotted as delays or advances, but are here treated as delays. The 1 h perS PRC is therefore treated here as though it is of Type 1, although it is clearly very close to a Type 0. This PRC showed increased magnitudes for both delays and advances when compared to that for per+, and also a D/A ratio > 1. Pulses of light lasting 2, 6 and 10 h gave rise to strong Type 0 PRCs with an average slope close to - 1 [Fig. 5(B-D)]. The change from Type 1 to 0, therefore, must occur with pulse durations between 1 and 2 h, and possibly with pulses very close to 1 h. The eye-fitted 1 h light pulse PRC for perL2 [Fig. 6(A)] shows differences to those of per + and pers. Maximum

FIGURE 4. Phase response curves (PRCs) for wild type (per’) females of D. melunogaster. (A) 1 h pulses, (B) 6 h pulses of white light. In A, the ‘solid’ curve was ‘eye-fitted’, the dotted curve was computed from a 5th order polynomial, +A+, phase advances; -A+, phase delays; V, data points double plotted along the abscissa. The PRC for 1 h pulses is Type I; that for 6 h pulses is Type 0 (slope = -0.967, r = 0.935).

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phase delays and advances both approached 8 circadian hours and the PRC was almost symmetrical (D/A ratios close to I). However, the whole curve appears to be displaced along the abscissa, with the ‘dead zone’ occurring between Ct 8 and 12 and the abrupt change between delays and advances occurring between Ct 22 and 23. Increase of pulse duration to 6 h [Fig. 6(B)] converted this PRC to Type 0. The PRCs fitted by eye and by polynomials are very close for per L2 [Fig. 6(A)] but somewhat divergent for per + and pd. In per + [Fig. 4(A)], for example, phase advances are smaller in the polynomial curve, and in per’ [Fig. 5(A)] the advance section, although of the same magnitude in eye-fitted and polynomial fitted curves, is shifted along the abscissa. However, these differences

et al

were found to have no effect on interpretations arising from the application of these PRCs (see below); for this reason the eye-fitted curves were employed in further analyses. The eye-fitted 1 h PRCs [Figs 4-6(A)] and the calculated PRCs for 6 h pulses [Figs 4(B), 5(C) and 6(B)], for the wild type and the two mutant strains, are thus shown for comparative purposes as data-less curves in Fig. 7. Figure 7(A) highlights the altered amplitudes of delay and advance phase shifts and the slippage of per L2along the x-axis. Figure 7(B) shows the ‘break-point’ between delays and advances occur earlier than wild type in perS and later than wild type in perL2. The 1 h PRCs [Figs 46(A)] are notable for their lack of a distinct dead zone in the middle of the subjective

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Phase h,Ct FIGURE 5. PRCs for the short period mutant, pd. (A) 1 h pulses; (B) 2 h pulses (slope = - 1.019, r = -0.970); (C) 6 h pulses (slope = - 1.016, r = -0.954); (D) IO h pulses (slope = - 1.042, r = -0.973). In A, the ‘solid’curve was ‘eye-fitted’, the dotted curve was computed from a 4th order polynomial. Other details as in Fig. 4.

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963

FIGURE 6. PRCs for the long period mutant, perL2. (A) 1 h pulses; (B) 6 h pulses (slope = - 1.000, r = -0.872). In A, the ‘solid’ curve was ‘eye-fitted’, the dotted curve was computed from a 4th order polynomial. Other details as in Fig. 4.

day. Only in the 1 h PRC for perLZ is there any suggestion of a portion of the subjective day that is immune to the phase-shifting effects of light. Steady-state phase relationships to light-dark cycles

The PRCs in Fig. 7 were used to predict steady-state phase relationships to trains of 1 and 6 h pulses

(T = 24 h) according to entrainment theory (Pittendrigh and Minis, 1964; Pittendrigh, 1966; Johnson, 1992). Thus, for entrainment to be achieved, the light pulse must commence, in each cycle, on that part of the PRC which generates a phase shift (Acp) equal to the difference between the period of the free-running rhythm (7) and the period of the entraining light cycle (T)

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FIGURE 7. (A) 1 h (eye-fitted), and (B) 6 h PRCs for pus (curves a), per + (b) and perL2 (c) replotted to show relationships on the circadian time scale. For calculations of steady-state phase relationships to the light cycles LD 1:23 and LD 6: 18 (lower panels),

see text.

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D. S. SAUNDERS TABLE circadian

Genotype perS per+ per Lz

et al.

1. Values of circadian period (T), duration of activity (a) and duration of rest (p) in locomotor activity records of females of Drosophila melanogasrer wild type (per +), short period (pe?) and long period (perL2) mutants.

Number flies 50 50 50

of

Mean circadian period (z) h

Mean duration of activity (m) h

19.70 * 0.57 23.72 k 0.90 29.10 + 3.64

8.47 f 2.35* 12.18 k 2.84 12.99 k 3.64

Mean duration of rest (0) h Il.23 + 2.37 1I.54 k 2.87 16.11 + 3.99*

Mean values expressed in hours + deviation. *Values significantly different from per + (P < 0.001).

(i.e. (t - T( = Aq). Therefore, for 1 h light pulses [Fig. 7(A)] and wild type flies (curve b) a phase delay of about 0.28 h must be achieved in each cycle when the light comes on in order to reach steady state: this can only be realised when the light pulse commences at Ct 3.7. For perS (z = 19.7 h) (curve a), a phase delay of 4.3 h (-5.24 circadian hours) is required in each cycle; this is only realised for pulses commencing at Ct 10.8. For perL2 (z = 29.1 h) (curve c), however, a phase advance of 5.1 h (+4.21 circadian hours) is required; this is only achieved at Ct 4.8. Using this information together with the average data for the duration of a (Table l), predictions of steady-state entrainment to a cycle of LD 1:23 [Fig. 7(A), lower panel] could be determined, showing in particular, a strong phase lead to the pulse in pers. Similar calculations for entrainment to LD 6 : 18 were carried out for the 3 strains using the PRCs for 6 h pulses [Fig. 7(B)]. These also predicted a phase lead for perS and a relative phase lag for perL2. These predicted phase relationships were tested using perS flies exposed to LD 1: 23 and LD 6 : 18, representative actograms being shown in Fig. 8. In Fig. 8(A), the fly was exposed to LD 1: 23 from day 6 to 14, otherwise it was in DD. The fly illustrated in Fig. 8(B) was in DD for the first 5 days, then in LD 6: 18. In both cases (and others not shown) the activity rhythms were entrained by the light cycle. In LD 1: 23 entrainment occurred with a marked phase lead to the light as predicted from the 1 h PRC in Fig. 7. Thus, in steady state (day 8 to 14) the light pulse illuminated the late subjective day (N Ct l&l 1) to produce the phase delay required to alter z to T. The activity observed whilst the light pulse was on is considered to be exogenous activity due to the masking effect of light; it ceased immediately after release into DD on day 15. The fly exposed to LD 6: 18 [Fig. 8(B)] also achieved the predicted phase lead in steady state.

FIGURE 8. Steady-state phase relationships of perS females to LD 1: 23 (A) and LD 6: 18 (B) showing, after an initial free-run in DD, a period of entrainment to the LD cycle with a pronounced phase lead to the light, as predicted by the relevant PRCs (see text). In A, the locomotor activity occurring during the light is regarded as an exogenous ‘masking effect’.

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DISCUSSION

Mutations at the period locus in Drosophila melanogaster cause a number of alterations to parameters of the adult locomotor rhythm, the most obvious of which is a change in circadian period (Konopka and Benzer, 1971). Others noted here and elsewhere, however, include apparent changes in light sensitivity (Konopka, 1979), temperature compensation (Konopka et al., 1989; Ewer et al., 1990), relative durations of the active (c() and rest (p) phases of the cycle (Konopka and Orr, 1980), and the ratio of delay to advance phase shifts in the PRC. The values of z for per’ and per L2females free-running in DD at 20°C (Table 1) are similar to those given elsewhere (e.g. Hamblen et al., 1986), as is the substantial shortening of !X in perS (Konopka and Orr, 1980). However, the present investigation has shown that the lengthening of t in per L2 is almost entirely due to a lengthening of the rest phase (p) of the cycle, c( remaining unchanged from the wild type. Period lengthening in perL’, therefore, appears to be quite different to that in the original long-period mutant (perL’) (Konopka and Benzer. 1971; Orr, 1982) which showed a lengthened active phase, despite the fact that perL2 is probably only a re-isolation of the original perL’ (Gailey et al., 1991). How these changes in z, CLand p are brought about needs to be elucidated, and these changes must also have important consequences for any comprehensive model for the circadian clock (in D. melanogaster, and other species). The PRCs described in this paper illustrate a number of important features, some of which are novel. Unlike other PRCs measured for adult locomotor rhythmicity in D. melanogaster (Orr, 1982; Dushay et al., 1990), which were all of a ‘weak’ Type 1 (Winfree, 1970), showing relatively small phase shifts even with bright (2000 lx) pulses, those presented here show a change from Type 1 to ‘high-amplitude’ Type 0 as the stimulus strength (pulse duration) was increased from 1 to 6 h (or from 1 to 2 h in the case of pers). Such a change is well documented for ‘population rhythms’ such as eclosion rhythmicity (Winfree, 1970; Saunders, 1978), but less frequently for rhythms in individual insects. Notable cases of the latter are the cockroach Nauphoeta cinerea (pulses increased from 3 to 12 h; Saunders and Thomson, 1977), the weta Hemideina thoracica (pulse durations of 6 to 8 h; Christensen and Lewis, 1982) and the mosquito Culex pipiens quinquefasciatus (pulses increased from 7.5 min to 2 h; Peterson, 1980). Although Type 0 curves have not been observed previously for adult locomotor rhythmicity in D. melanogaster, a change from Type 1 to 0 with increased signal strength was observed for eclosion rhythmicity (Winfree and Gordon, 1977; Konopka, 1979; Konopka and Orr, 1980), as a consequence of the perS mutation. A second systematic change in PRC shape is the timing of the ‘data-less’ breakpoint between delay and

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advance phase shifts seen in Type 0 PRCs when they are plotted in a non-monotonic form (Johnson, 1992). When 6 h PRCs for the 3 strains are compared [Fig. 7(B)], this breakpoint occurred at an earlier circadian time in perS than in the wild type, and at a later circadian time in per L2. This observation has connotations when we consider apparent light sensitivity (below). Results of the present investigation confirm earlier suggestions, for eclosion rhythmicity (Konopka, 1979), that perS flies respond to short light pulses with larger phase shifts than wild type flies, thus suggesting that mutation to shorter period also carries increased light sensitivity. Taken together with the observation that the breakpoint in the 6 h PRCs occurs earlier in perS and later in per L2, this suggests that the short period mutant responds to light pulses as if they were brighter (Johnson, 1992) and the long period mutant responds as if the pulses were less so. Apparent light intensity, or ‘subjective light intensity’, therefore is greatest in perS and least in perL2. An apparent increase in light sensitivity has also been observed in the short-period tau mutant of the hamster (Menaker, 1992). Other mutation-induced changes involve PRC shape. In earlier PRCs for locomotor activity in D. meIanogaster (Orr, 1982; Dushay et al., 1990) delays and advances were of approximately the same magnitude in wild type and mutant strains. In this study, however, the 1 h pulse PRC for perS showed a much larger delay section than an advance section (i.e. D/A ratio was > 1). For several species of rodents, Daan and Pittendrigh (1976) showed that a short freerunning period was often associated with a larger delay section in the PRC, whereas longer values of z were associated with a larger advance section (D/A < 1). These differences were considered appropriate for the entrainment of shorter and longer period animals to natural (T = 24 h) light cycles, because the former would need delays rather than advances, and vice versa. A similar relationship between T and D/A ratio has also been observed for several strains of inbred mice (Schwartz and Zimmerman, 1990). It is therefore of interest that the 1 h PRC for perS shows D/A > 1 [Fig. 5(A)], which might be expected for a short period mutant, even though it is not a product of natural selection involving evolutionary pressures from entrainment to natural light-dark cycles. On the other hand, perL2 does not show an increased advanced section [Fig. 6(A)], which might be expected for a fly with a long period. The PRCs shown here (Figs 4-6) are also notable for their lack of a distinct ‘dead zone’ in the subjective day. Only in the case of the 1 h PRC for perL2 [Fig. 6(A)] is there any suggestion of such a region. This situation is quite unlike that described earlier for wild type flies (Dushay et al., 1990) and for wild type and mutant flies (Orr, 1982) exposed to 1, 10 or 80 min pulses of light at 2000 lx, which all showed substantial ‘dead zones’. In the

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latter study, for example, per’ was shown to have a ‘dead zone’ of about 4 to 6 h, per + about 10 h, and perL’ a ‘dead zone’ extended to about 14 h. Konopka and Orr (1980) proposed a simple model for the circadian clock in D. melanogaster based on the observation that a was shortened in perS from about 12 to 7 h whilst the duration of p was unaltered, indicating that a ‘segment’ was deleted from the subjective day. A similar shortening of the subjective day has been reported for some short period frq mutants in Neurospora (Nakashima, 1985) and in the tau mutant hamster (A. S. I. Loudon, personal communication). Konopka and Orr’s idea, interpreted in the context of hypothetical ion gradients, imagined that oscillations might occur because of the alternating establishment and depletion of a gradient across a membrane. The perS mutation was imagined to increase the activity of the ion pump resulting in a shorter subjective day since the maximum value of the gradient was attained more rapidly. It was predicted, therefore, that a long period mutant might have decreased pump activity and hence a lengthened CI.Lengthening of c1 (and of the subjective day) was subsequently observed for per L1 (Orr 1982), but the present results (Table 1) shows a lenithening of p in perL2 while tl remains the same as in wild type. This raises again the important question whether the circadian period in mutant flies is generated by an overall shortening or lengthening, or by the deletion or addition of a ‘segment of time’. This question is addressed in Fig. 9 where the 1 h PRCs for the three strains are replotted with ‘real’ time on the abscissa. Superimposing the phase advance sections of the three PRCs clearly shows that the late subjective night-early subjective day sec-

tions are practically identical in duration (but not in the magnitude of the phase shifts generated) whereas the late subjective day-early subjective night (delay) sections are markedly different, being shortened by 4-5 h in perS and lengthened by about 5 h in perL2. This suggests that mutation shortens or lengthens the period of the oscillation, particularly in the second half of the subjective day. Since tl was not lengthened in per L2 this further suggests that the durations of the subjective day and a are determined independently. A more recent per-as-feedback model of Hardin et al. (1990, 1992) was based on observations that headspecific per gene product and its messenger RNA fluctuated in abundance with a maximum at night, and on observations that the mRNA rhythm persisted in DD. Furthermore, it was shown that the period of the mRNA abundance rhythm was about 19 h in perS, as opposed to about 24 h in wild type, and in steady state entrainment to a light cycle (LD 12 : 12), the elevation in mRNA abundance in perS phase-led wild type, whereas that in perL’ phase-lagged it, exactly as one would predict from the generalized entrainment model (Pittendrigh, 1965). The Per protein oscillated with a peak 6 to 8 h after that for its mRNA, suggesting that it was involved in a feedback loop that influenced the cycling of its own mRNA, although it was not clear whether this loop involved intermediate steps in addition to transcription, or how the differences in z, a and p were generated. A feedback control systems model (considered in various forms by Gander and Lewis, 1979; Christensen and Lewis, 1983; Christensen et al., 1984; Lewis and Saunders, 1987) effectively describes the known properties of circadian oscillations and includes parameters

Phase advances

FIGURE 9. Eye-fitted PRCs for 1 h pulses replotted with ‘real’ time on the abscissa, and the phase advance portions of the curves superimposed. a, pers; b, per +; c, perL2. The phase advance portions of the three PRCs occupy the same number of real hours, but the delay portion (late subjective dayxarly subjective night) is curtailed in perS (1) and lengthened in perLZ (3).

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MELANOGASTER

PERIOD

which may be identified as simple and feasible biochemical components. This model may also be used to account for what is known about the regulation of circadian activity in D. mehogaster. For example, the model describes an oscillation in the abundance of activityrelated chemical (c) which could be identified as a given per gene product. The most important components of the model are the temperature dependent rate of synthesis of c, its temperature independent loss, and a time-delayed function in the feedback loop that allows rhythmicity to be generated. Light destroys c or holds it at a low level until the onset of darkness. In steady state entrainment to the light-dark cycle, therefore, the concentration of c is high at night and low during the day. In nocturnal organisms c is activity-promoting, activity occurring when the concentration of c is above an arbitrary threshold; in diurnal organisms such as D. melanogaster, activity occurs when c is below threshold. Synthesis rate (SR) determines whether the oscillation is self-sustained (if SR is high) or damping (if SR is low), and time delay (TD) determines the period of the oscillation, the smaller the value of TD the shorter the period. This model suggests that the period of the oscillation in D. melanogaster may depend on the time delay function in the feedback loop, perS having a low value of TD, whereas perL’ and per L2 have higher values (see also Hardin et al., 1992; Hall and Rosbash, 1992). Since changes between the mutants and wild type have been shown to occur during the late subjective day (Fig. 9) mutation-induced changes in the time-delay function must operate at this phase. Since regulation of a has been shown to occur independently of that for z, mutation at the period locus might also alter the model’s threshold value: lowering the threshold in perS to reduce a, lowering it in perL2 to maintain Mat about 12 h, but raising threshold in perL’ to increase its value. These considerations suggest that the period mutation has many subtle effects on the system that generates rhythmicity.

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Acknowledgements-This work was supported by a grant from the Science and Engineering Research Council. The authors would like to thank Drs S. Daan and J. C. Hall for comments on an earlier draft of the manuscript.