Journal of lnsect Behavior, Vol. 5, No. 4, 1992
Behavior of Period-Altered Circadian Rhythm
Mutants of Drosophila in Light:Dark Cycles
(Diptera: Drosophilidae)
Melanie J. Hamblen-Coyle, 1 David A. Wheeler, l'a
Joan E. Rutila, 1'2 Michael Rosbash, 1'2 and Jeffrey C. Hall 1'4
Accepted November 27, 1991; revised December 20, 1991
Adults of Drosophila rnelanogaster had their locomotor activity monitored under
conditions of cycling light and dark (12 h each per cycle). The elementary
behavior of wild-type flies under these " L D " conditions fluctuated between
levels of high and levels of low activity. Two high-activity peaks occurred within
a given cycle: one at about dawn; the other, at around dusk. Such accentuated
activity levels gradually subsided to troughs in the middle of the day and of the
night, after which the flies anticipated the next environmental transition by
gradually becoming more active. Descriptions of these activity profiles were
augmented by newly developed formal analyses of the "diel rhythm" phases
(based in part on digital filterings of the raw behavioral data). The applications
of these analyses led to objective, automated determination of when in the
morning and the evening the flies'activity peaks occur. This normal diel behavior was compared to the locomotor activity and phase determinations for a
series of rhythm variants. Most of these involved mutations at the period (per)
locus and germ-line transformants bearing normal or altered forms of DNA
cloned from this "clock gene. '" Such genetic variants have been shown previously to exhibit, in constant darkness, strain-specific circadian periods ranging
from about 19 to about 29 h. We now show that the phases of the evening peaks
of activity under LD conditions were correspondingly earlier than normal for
the short-period mutants and later than normal for those with long circadian
~Department of Biology, Brandeis University, Waltham, Massachusetts 02254.
2Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254.
3Current address: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
4To whom correspondence should be addressed at 235 Bassine Building, Brandeis University,
Waltham, Massachusetts 02254-9110.
417
0892-7553/92/0700-0417506.50/09 1992PlenumPublishingCorporation
418
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
cycle durations. The morning peaks, however, moved (in comparison to the
normal phase position) minimally under the influence of a given per variant.
KEY WORDS: locomotoractivity;per-short mutant;per-long mutants;per-transgenics; Clock
mutant; blind norpA mutant;phase analysis.
INTRODUCTION
Circadian rhythms in Drosophila are increasingly being analyzed by monitoring
the locomotor activity of adults as opposed to eclosion of pupae into adulthood.
These recent behavioral experiments have usually focused on the "free-running" behavior of Drosophila in constant darkness and, in turn, have compared
the effects of normal flies to those expressing mutations (reviewed by Hall and
Kyriacou, 1990). Many of these mutants are due to chemically induced, or
molecularly manufactured, changes involving the period (per) gene of D. melanogaster (reviewed by Rosbash and Hall, 1989; Young et al., 1989).
There are a few reports of Drosophila's diurnal (or "diel") behavior, that
is, locomotor activity monitored against a background of light: dark (LD) cycling
(reviewed by Saunders, 1982; more recently by Helfrich and Engelmann, 1987;
Petersen et al., 1988; Dushay et al., 1989, 1990; Hamblen-Coyle et al., 1989).
Many of these diel results have been reported anecdotally.
The current study was designed to describe and analyze systematically the
behavior, under LD conditions, of D. melanogaster adults whose genotypes
allow for rhythms to be expressed in DD. Thus, we have monitored the activity
of normal adults and those expressing a variety of per-related, and other, genetic
variants that cause free-running periods (r' s) to be shorter or longer than normal
(reviewed by Hall and Kyriacou, 1990; Jackson, 1991; Vosshall and Young,
1991). By displaying the behavior of individual wild-type and rhythm-mutant
adults, and by plotting the superimposed behavioral records for several flies of
a given genotype, clock functions that underlie the fluctuating patterns of activity
in conditions of environmental cycling were revealed. We also show that all
r-altered types entrained to the 24-h cycles used in the current experiments.
We have, moreover, developed new algorithms to determine the phase of
the activity cycles. Our methods are based on processing digital data (activity
events) through a series of analytical stages that result, ultimately, in precise
and objectively determined phase values. The findings generated by application
of our programs indicate that the phase of Drosophila's "evening activity peak,"
which describes one element of its behavior in LD, moves in a direction that
can be predicted from the shortening or lengthening of a fly's free-running
rhythm period caused by a given clock-altering genotype.
Behavior of Circadian Rhythm Mutants of Drosophila
419
MATERIALS AND METHODS
Strains and Behavioral Tests
The flies used in this study were grown on a molasses, cornmeal, agar,
and yeast medium (supplemented with the mold inhibitor Tegosept) in a
12 h: 12 h light:dark cycle (lights on at 0800), at 25~ and about 75 % relative
humidity. To generate males for behavioral analysis, single p e r + (from Canton-S and Oregon-R wild-type strains), p e r s, per L1 (Konopka and Benzer, 1971),
and per L2 (Konopka, 1986) males were crossed to attached-X [C(1)DX, yf]
virgin females; the F 1 males were behaviorally tested (see below). The two
"non-per" variants in this study expressed a blind no-receptor-potential-A
mutant [the null allele norpA P24 (see the review by Pak, 1991)] or the Clock
(Clk) rhythm variant (cf. Dushay et al., 1990); for these, flies were simply taken
from the stocks for the behavioral tests.
Germ-line transformants involved the previously reported DNA fragments
cloned from the wild-type p e r locus (Zehring et al., 1984; Hamblen et al.,
1986; Citri et al., 1987; Yu et al., 1987a): Two transgenics carried a 14.6-kb
genomic fragment (lines 2l and 63, whose per + DNA is marked with Adh+),
one carded a 7.2-kb fragment (line 9, whose p e r + DNA is marked with ry+),
and two others carried 13.2-kb fragments (lines 2 and 34; both ry+). Transformed flies were outcrossed to virgin females from the stocks per~ Adh f~23
p r cn or per~ ry 5~ and the "marker-plus" progeny were tested behaviorally.
Newly constructed 13.2-kb lines, with novel nucleotide changes at the p e r s
mutation site [see Rutila et al. (1992) for the molecular methods], were also
crossed to per~ ry5~ flies. These in vitro-mutagenized lines [whose free-running periods are reported by Rutila et al. (1992); see also Table I] were
"13.2Gly," lines 5 and 45; "13.2Thr," lines 3, 5, 6, and 8; "13.2Asp," lines
7, 18, 19, and 34; "13.2Glu," line 39; and " 1 3 . 2 T y r , " lines 1 and 7. The
amino acids just noted replace the Set residue--at amino acid 598 in the perencoded protein--that had been mutated (by Konopka and Benzer, 1971) to Asn
on induction of per s (Yu et al., 1987a; Baylies et al., 1987).
Young (0- to 12-h) adult progeny from the crosses--mutant vs normal
X-chromosome males from those involving attached-X females and "single
insert-bearing" males from those involving transformants (see above)--were
collected under ether or CO2 anesthesia, placed in food vials, and entrained by
three cycles of LD 12 : 12 (lights on at noon). After this entrainment the flies
were placed in glass tubes that sit between infrared emitters and detectors (cf.
Hamblen et al., 1986) to have their activity monitored during the LD cycles,
in a 25~ incubator. The lights were switched on and off by a Hunter programmable timer (Model 41001). The light intensities varied from about 200 to 2500
420
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
Table I. Types and Numbers of Flies Monitored for Locomotor Activity~'
Number of flies
Genotype
~
Monitored
Not
included
Did
not entrain
Canton-S
Oregon-R
24
24
124
30
11
1
3
0
pe&
p e r LI
p e r L~
19
29
29
52
43
43
8
0
0
1
6
0
13.2:2
13.2:34
14.6:21
14.6:63
7.2:9
24.5-25
24.5-25
27-28
25-27
25.5-26
20
13
10
10
19
2
0
0
1
0
1
1
0
0
0
13.2Gly:5
13.2Gly:45
19
19
17
15
1
1
0
0
13.2Glu:39
22
12
1
1
13.2Asp: 7
13.2Asp:18
13.2Asp:19
13.2Asp:34
22.5
22.5
22
22.5-23
11
13
10
10
0
1
0
1
1
0
1
0
13.2Tyr:l
13.2Tyr:7
23
23.5
16
15
3
0
0
0
13.2Thr:3
13.2Thr:5
13.2Thr:6
13.2Thr:8
25
25.5-26
25.5-26
26
19
17
11
13
5
5
0
1
0
0
1
1
Clk
norpA P24
22.5
23
40
43
9
0
0
1
i
aThe top two rows designate outcrossed wild-type males (see Materials and Methods). The next
group is p e r mutants. The remaining groups (except for the bottom two rows) are germ-line
transformants involving cloned, possibly in vitro-mutated, p e r DNA (see text); note that the italicized numbers following the colons designate independently derived transgenic strains involving
a given transduced p e r DNA insert type; the boldface abbreviations indicate the amino acids
substituted for the Ser residue which was replaced by Asn in the p e r s mutant and has been
substituted by Gly, etc., in the in vitro-mutated forms of the " 1 3 . 2 " construct (see text). ~ is the
characteristic free-running period that has been determined in previous studies (see text) by monitoring the DD activity of flies expressing a given genotype. The free-running periods for the
" 1 4 . 6 " transformant types have been established in several reports (see text); that for the " 7 . 2 "
strain was noted as 25.7 + 0.1 h (N = 17) by Hamblen et aL (1986); this value was confirmed
(25.7 + 0.1 h; N = 14) in a DD test done contemporaneously with the current LD tests. The
number of the latter kind of data records analyzed for a given genotype is the third column in each
row, minus the total from the second two. Certain flies were not analyzed (e.g., for contribution
to the plots in Figs. 5-7 or the listings in Table II) because of irregularities in their behavioral
records "not included" or because they failed to "entrain" (see Materials and Methods).
Behavior of Circadian Rhythm Mutants of
Drosophila
421
lux at different positions within a given incubator (these intensities were measurements of filtered light; see below). The majority of the flies were tested
within a range of 300-1000 lux. One " D D run" was performed (on the Z 2 : 9
transformant type; see Table I, footnote a), wherein the flies were entrained by
exposure to 3 days of 12 : 12 LD; then they were transferred to DD, under which
conditions activity was monitored for 1 week (as described by Hamblen et al.,
1986).
Data Collection, Preparation, and Preliminary Analysis
Data were collected using an Apple IIe computer and the software described
by Sulzman (1982) and Hamblen et al. (1986). Activity events (each one of
which equals an interruption of the infrared beam) were stored in the memory
of the Apple IIe and written to disc every half-hour, throughout the 7-12 days
of monitoring. The activity files of the flies tested were transported to a VAX/
VMS system using an Apple IIe that is networked to the VAX/VMS system
through an Apple Super Serial card, employing Kermit v.384 software; thus,
the analysis and display programs (see below) were executed on the VAX.
Before analyzing the data from a given fly, its behavioral data (collected
as numbers of activity events for a series of 30-min bins) were examined for
irregularities. These were of three types.
(1) Out-of-Range Data. In some of our initial experiments, the AC-powered fluorescent lights used for entrainment caused certain detectors to add thousands of spurious bouts to a given bin during the L phase--compared to the
"typical" range of activity events/0.5-h bin in our system (Hamblen et al.,
1986), which, for peak activity was 50-500; for midpeak, 25-250; and for
trough, 0-50. Out-of-range records were discarded; the spurious-bout problem
was alleviated further by moving monitor boards that were relatively near the
light sources farther away and by placing neutral-density filters (Glare-Stop,
Visual Pursuits, Chicago, IL; percentage transmission, 35%) between all light
banks and the monitors beneath them; the result was that none of the detectors
registered pseudoevents when the lights were on and the glass tubes contained
no flies.
(2) Feeble Flies. An individual was considered to have behaved in too
desultory a manner if it failed to produce more than two 0.5-h bins with 10 or
more activity events in a given 24-h period; each day's activity was evaluated
consecutively in this manner, and when debilitation of this kind was encountered, that cycle's worth of data plus further ones were removed from the data
base; moreover, the entire record might have been expunged--based on the
following.
(3) Death. Some flies died before the end of a 7- to 12-day run; records
with fewer than 4 days of reasonably robust data (see point 2) were excluded.
422
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
The numbers o f irregular-fly records not included in the genotype-summarizing
histograms and further analyses are listed in Table I.
Flies whose behavior was monitored in these experiments showed transient
and relatively abrupt changes in their activity levels soon after the environmental
transitions. That is, the data bin following lights-on or lights-off usually included
a higher number of activity events than the one before or (less noticeably) the
one after. W e would argue that these D-to-L and L-to-D triggered peaks are not
related to an endogenous pacemaker function; nevertheless, these spike-like
peaks are displayed in the pictorial representations of the flies' diel behavior
(e.g., Fig. 5). Yet the activity spikes were removed before the low-pass filters
and other algorithms were applied for numerical analysis o f periodicity and phase
(see below). Such removal meant that, within a given LD cycle, the posttransition bin was emptied and replaced by a value that was an average o f that in
the just-pretransition bin and that which followed the posttransition bin.
The spike-purged files remaining after removal of cases 1, 2, and 3, (above)
were first analyzed by chi-square periodogram to obtain a value for the period
o f each behavioral record (cf. Sokolove and Bushnell, 1978). [The data from
the one DD run (see above) were also analyzed by this method.]
A double-plotted, continuous-line actogram (exemplified in Fig. 1) was
printed for every LD-monitored fly and examined to make sure that the numerical and analytical outputs at least roughly jibed with the pictorial records. If a
~
A
<
Day (n)
Day (n+ 1 )
Fig. 1. Double-plotted "continuous-line" actogram. This is an example
of a normal male's activity plot (the fly was obtained by outcrossing a
Canton-S wild-type strain; see Materials and Methods). It is in X - Y
coordinates, as opposed to the commonly used "raster"-type actograms, which are analog plots of event recorder-like activity markings
(e.g., Hamblen et al., 1986; Hamblen-Coyle et al., 1989; Newby et
al., 1991). These raster actograms do not readily reveal the times and
amplitudes of activity peaks (because of "saturation" of marking densities), whereas the behavioral record shown here, which displays the
digitized numbers of activity events (0.5-h X-axis bins) on the Y axis,
does (for each line of data, that portion of the Y-axisranges from 0 to
300 activity events). The double-plotted feature of this record means
that days 1 and 2 of the fly's activity are displayed on the first line,
days 2 and 3 on the second, etc. The data were collected during 12 h
light: 12 h dark (12:12 LD) cycles. Open boxes indicate the light portion of the cycle; black boxes indicate night.
Behavior of Circadian Rhythm Mutants of Drosophila
423
fly did not entrain (Table I)--meaning that the periodogram analysis of the
activity determined no significant rhythmicity (and the actogram usually looked
aperiodic as well)--its record was removed from the group to be analyzed by
application of the various pieces of software (see below).
Average Activity Plotting
The activity files of the flies' raw data, starting from the first activity bin
of the first full day (in all cases, a full day is from 4 h before lights-on to 4 h
before lights-on the next day), were processed by software that takes raw activity
data from a single fly and superimposes it, bin for bin, for several daily cycles
(usually about a week's worth). An average of the activity for each half-hour
bin was computed, and a histogram displaying this fly's behavior was prepared
by the computer (see, e.g., lower-fight panel in Fig. 5A). Then the data were
normalized for an entire set of flies to obtain group-average activity values for
each bin. Finally, histograms of this normalized activity were generated that
graphically superimpose not only the several cycles of activity for each fly, but
also the several flies tested for a given genotype [see Figs. 5-7 here and Fig. 4
of Hamblen-Coyle et al. (1989) for additional information about this method].
Phase Analysis
The purpose of this analysis was to locate the times of peak activity in
each day. To determine these values for a given fly whose data were deemed
analyzable (see Table I), we developed a new FORTRAN program, for the
VAX/VMS, that finds the daily peak locations of an individual fly's activity,
followed by averaging the daily peaks for that individual and then for a group
of animals (see Table II). This program is available on request.
A biological time series can contain many peaks (even within a given
cycle), only some of which would be likely to be linked to an underlying
circadian pacemaker. Thus, before the Phase program (per se) was run, a digital
filter was applied to the data sets, in order to screen out high-frequency noise
in the behavioral records, i.e., to remove what can be regarded as extraneous
activity peaks (Fig. 2A). This refers in the main to high-frequency components
of Drosophila's fluctuating locomotor activity, which could be unrelated to the
circadian phenomena of interest (though see Dowse and Ringo, 1991). This
procedure yielded a smoothed curve in which the main phases for wild-type
activity (Fig. 2B) or that of rhythm variants (Fig. 3). To remove noise, Phase
employs a two-pole Butterworth filter [see legend to Fig. 2; cf., e.g., Dowse
and Ringo (1989)]. The filter coefficients are such that peaks with periodicities
of less than 4 h were removed. The attenuation cutoff was chosen at 4 h to
ensure that there would be little or no effect on periodicities of interest, which
can range down to 10 or 12 h for certain rhythm mutants (cf. Hamblen-Coyle
Table II. Analytically Determined Phases of Peak Activity Levels for Drosophila Behaving in
Light : Dark Cyclesa
Evening phase
Genotype
Hour
Moming phase
Bin 4- SEM
Hour
Bin 4- SEM
Canton-S
Oregon-R
13.2:2
13.2:34
11:24
11:42
11:18
10:42
31.8
32.4
31.6
30.4
+
44+
0.1
0.1
0.1
0.4
23:39
23:18
23:48
0:30
8.3
7.6
8.6
9.5
+
_
4•
0.2
0.1
0.3
0.2
per LI
per L2
17:57
17:33
11:18
11:30
11:54
44.9
44.6
31.6
32.0
32.8
+
444•
0.1
0.1
0.3
0.1
0.1
0:39
0:18
0.48
0:36
23:48
10.3
9.6
10.6
10.2
8.6
44•
+
4-
0.4
0.3
0.2
0.1
0.2
14.6:21
14.6:63
7.2:9
13.2Gly:5
13.2Gly:45
8:51
8:27
8:45
26.7 4- 0.1
25.9 + 0.2
26.5 4- 0.2
23:54
23:30
0:06
8.8 4- 0.1
7.5 + 0.2
9.2 + 0.2
13.2Glu:39
10:03
29.1 • 0.2
23:39
8.3 • 0.3
13.2Asp:7
13.2Asp:18
13.2Asp:19
13.2Asp:34
10:06
10:00
10:21
10:42
29.2
29.0
29.7
30.4
0.2
0.1
0.2
0.3
0:30
0:27
0:09
23:45
9.5
9.9
9.3
8.5
13.2Tyr:l
13.2Tyr:7
10:36
10:30
30.2 4- 0.2
30.0 • 0.2
0:24
0:12
13.2Thr:3
13.2Thr:5
13.2Thr:6
13.2Thr:8
11:45
11:48
11:30
11:27
32.5
32.6
32.0
31.9
0.2
0.2
0.2
0.2
23:45
0:42
0:42
0:09
Clk
norpA e24
10:33
10:12
30.1 4- 0.1
29.4 + 0.1
23:24
23:45
per ~
•
+
4+
4444-
•
+
44-
0.4
0.2
0.3
0.3
9.8 4- 0.2
9.4 4- 0.2
8.8
10.4
10.4
9.3
4- 0.2
4- 0.2
4- 0.2
• 0.3
7.8 4- 0.2
8.5 4- 0.1
aThe phases are given in Zeitgeber time (ZT), whereby the beginning of a 12:12 LD cycle is called
0 h; lights-off is thus 12 h. The ZT values were derived from the bin numbers (No. 10, the first
0.5 h after lights-on; No. 34, the first 0.5 h after lights-off) in which the average evening or
morning peak fell; the standard errors for the bin values listed therefore represent fly-to-fly variabilities within a given genotype. In addition, there was intraindividual variability, e.g., in positions
of the evening peaks, from day to day (see upper right panels in Figs. 5-7). Thus, determination
of a given fly's evening peak involved computing a mean phase value (based on the several days
of monitoring its LD behavior) + SEM. The averages for these standard errors were usually in
the range of 0.5 to 1 bin, for a given series of adults monitored (i.e., one genotype's worth); some
individuals, however, had SEMs (referring to day-to-day fluctuations in their peak positions) of
more than 3 bins, whereas a few flies had no variation in this parameter. A proportion of the search
windows (cf. Fig. 4) applied to the behavioral record for a given fly, for a given day of its activity,
led to no morning peak extracted; that is, the animal expressing the genotype in question was
"inappropriately" inactive during that day, within that time span, or exhibited (during that time)
too broad a plateau of relatively high-level activity (hence no summit during the search window;
cf. Fig. 4). Such peak absences were dependent somewhat on genotype. Thus, for the nontransformed types, whether normal or rhythm mutant, the proportions of morning phase values included
analytically (vs being too low-amplitude to compute) were only 0-10% less than the number of
evening peaks. But some of the transformants had many fewer morning peaks in their records
compared to evening peaks: per +14.6:21, about 35% fewer; perSGly or perSThr transformants,
10-20% fewer (depending on the strain); perSGiu transformant, 45% fewer; perSAsp transformants, 20-40% fewer; perSTyr transformants, 20-35% fewer.
I I
i-0
-i
2
3
4
5
DAY
B
>,-
l->
m
e
i
0
1
s
2
i
t
!
3
4
5
DAY
Fig. 2. Raw vs filtered activity data collected from normal D r o sophila during LD cycles. (A) Raw numbers of locomotor events
monitored for 5 days in 0.5-h bins, events, from each of three
flies derived from outcrossing flies from a strain of Canton-S
wild type (see Materials and Methods). Unlike Fig. 1, each fly's
record here represents successive days of activity events plotted
left to right (one line's worth of plot per fly). The circadian
clock controls the timing of the major activity peak each evening
(activity encompassed by " a " ) . Another peak appears in the
morning (b); plus there are many small bursts of activity
throughout each day (c), which should be ignored in an analysis
of circadian rhythms (see text). Black boxes are 12-h segments
of dark. The ordinates range from 0 to 300 activity events. (B)
Smoothed plots of the activity data for the three individuals in
A were operated upon by a low-pass filter to remove periodicities
in the data of less than 4 h. Thus, high-frequency noise has been
removed, leaving smoother curves for which the time of a given
peak is almost always unambiguous (e.g., there are essentially
no cases of dual summits separated by a saddle). Peaks in the
first 2 days are labeled as follows: m, morning; e, evening; s,
secondary. The filter involved the two-pole Butterworth function: Y~ = (Xi + 2 " X i _ l + Xi 2 + A . Y~_~ + B . Y~_2)/C,
where Y~is the ith output value; X~ is the ith input value; i is a
specific data point; and A, B, and C are coefficients that vary
according to the desired frequency cutoff. For a 4-h cutoff, A =
16.944, B = 6.120, and C = 14.825.
426
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
A
'13.2' transformant
B
pers
C
perL 1
t•
Fig. 3. Actograms of raw and filtered data collected from per
variants during LD cycles. (A) A "13.2" transformed male,
whose actual activity record is on the left; this led (arrow), by
the filtering operation described in the legend to Fig. 2 (with
reference to wild-typeactograms), to the smoothed record on the
right. (B, C) Same kinds of raw, then filtered, actograms of a
per ~ and a per LI male's behavior, respectively.
et a l . , 1989; Dowse and Ringo, 1991). An undesirable effect of these filters is
to phase-delay the output, causing peaks in the data to be shifted one or more
bins later in the cycle. This problem was corrected by running the filtered output
back through the filter in the reverse direction. The examples shown in Figs.
2B and 3 were filtered in this way.
The metric of primary interest in performing a phase analysis is the time
during which the active portion of the cycles occurs. In our study, activity phase
peaks were sought o n e d a y a t a t i m e , as opposed to determining, by placing a
ruler onto a raster-plotted actogram, one global phase value for several days'
worth of evening activity offsets (cf. Fig. 4 of Dushay et a l . , 1990).
Behavior of Circadian Rhythm Mutants of
Drosophila
427
Peak searching was further confined to a specified window of bins within
a given 48-bin day. This is because inspection of the plotted locomotor behavior
indicated that two main peaks of activity, at about lights-on and lights-off,
describe the diel behavior of D. melanogaster. To choose a "search w i n d o w "
the investigator first viewed the actograms and average activity plots to find an
approximate point to start the search. This was performed separately for the
morning and the evening peaks. A search window's midpoint varied for each
genotype (see legend to Fig. 4 for details and examples), because the behavioral
plots indicated obvious differences in peak times, especially in the evening. The
number of 0.5-h bins in all search windows, that is, for all individual morning
and evening peaks in each day of an animal's record, for all genotypes, was
_+8 from the midpoint (see legend to Fig. 4 for further details). Had we not
" w i n d o w e d " our search for a given (morning or evening) peak, the separate
peaks frequently would have been blended together, resulting in one weightedaverage phase value. That is, the distinction between morning and evening peaks
of activity, which are genuine for this organism, would be lost.
All peaks within the window specified were located using the following
criterion (see Fig. 4 for further details): A given bin, N, was said to contain a
peak if the activity therein is greater than that of bin N - 1 (the previous bin)
and greater than or equal to the activity value of bin N + 1 (the next bin). If
window
I
I
2
i
./L2'J
I
I
0
12
24
36
48
BIN
Fig. 4. Depiction of the "search window" used to find an activity peak. All peaks within such a
window (hatched rectangle) and above the "d" threshold--a set fraction of the highest peak--are
tallied; the fraction depicted is 0.25, which was employed throughout this study (i.e., within all
morning and evening search windows, for all animals and genotypes). To exemplify how the search
windows were chosen, average activity plots for flies with (in this example) normal, short-period,
and long-period r's (see Figs. 5A, 6A, and 7A,B, respectively) were inspected, which indicated
that the search-window midpoints should be bins 33 (per+), 25 (per'), and 45 (perCt'~e); all
windows were then fixed as 8 bins to either side of the midpoints. The phase point of the activity
is tabulated as the weighted-averageposition of all peaks above d (in this case, peaks 2 and 3; see
text); that average position is indicated by the diamond.
428
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
the main filtered peak has a shoulder, or there is a double peak within a window
(as in Fig. 4), then the weighted-average location of all peaks within that window
was found as follows. "Weighted-average" bin, BINwa, was calculated as
BINwa
A i * BINi
=
i=l
Ai
"=
where Ai is the amplitude of the ith peak, BINi is the bin in which the ith peak
was found, and n is the number of valid peaks. (If N = 1, as is the usual case,
the weighted average position is just the maximum-amplitude peak position.)
RESULTS
Displays of Normal and Mutant Diel Activity
A fruit fly's locomotor activity changes in a cyclical manner over the course
of a 24-h period either under light: dark cycling conditions (LD) or in constant
darkness ("dark:dark"; DD). A genetically normal fly exhibits 24-h periodicities of its behavioral cycles in LD and, usually, in DD as well (for examples
of the latter, see Konopka and Benzer, 1971; Smith and Konopka, 1981, 1982;
Zehring et al., 1984; Hamblen et al., 1986; Hamblen-Coyle et al., 1989; Dushay
et al., 1990; Konopka et al., 1991; Liu et al., 1991; Newby et al., 1991).
In other Drosophila species locomotor activity has been described as shifting from bimodal segments in LD, to a single component (per day) of subjectiveevening activity in DD (see Saunders, 1982, particularly Fig. 2.10). That kind
of behavioral pattern has also been depicted for D. melanogaster, whereby
evening activity persists in DD as a broad segment, with moming activity being
much diminished [see Fig. la of Hall (1990) for examples]; however, more
prominent bimodality has been observed for this species, under constant conditions as well as in LD [e.g., Fig. 1 of Petersen et al. (1988); also J. M. Ringo
and H. B. Dowse (personal communication)].
The two relatively tight daily clusters of activity in LD are exemplified in
the average activity plot in Fig. 5A, which superimposes the behavior of several
separately monitored normal adults; these had been obtained by outcrossing
males from an Oregon-R wild-type strain [see Materials and Methods; cf. Hamblen-Coyle et al. (1989) and Dushay et al. (1990) for similar plots of CantonS wild-type behavior]. Another average activity plot is displayed for a p e r
transformant in which the effects o f p e r ~ (i.e., arrhythmicity in DD) are almost
completely rescued (by a 13.2-kb p e r + DNA fragment); these transgenic flies
behaved similarly to the wild type in LD (Fig. 5B; cf. Fig. 2B vs 3).
One of the relatively high-activity portions of these normal cycles commences a few hours before lights-on and peaks about 0.5 h before that environ-
O~
0
Time
wild-type
12
i[zZZ:::]lllmlR~
B
o
Fime
12
'13.2' transformant
Fig. 5. Diurnal behavior of normal flies. (A) Outcrossed wild type (T = 24 h). Locomotor activity data were collected, then "accepted" (cf. Table 1),
for a series of 29 males (of the 30 flies tested); these had been derived from an Oregon-R strain (males of which were crossed to attached-X females, to
yield the F I subjects). A typical actogram (cf. Fig. 1) fbr a given individual's behavior under these 12:12 LD conditions is shown in the upper right
corner; it is "double-plotted"--as implied by the nighttime segments, represented by filled bars, and the daytime-indicating open bars. In the lower right
corner, the several days of monitored behavior for this fly are superimposed with respect to a single 24-h time span; a given ordinate from this kind of
plot typically runs from 0 to 300 activity events (referring to this graph and those in Figs. 6 and ? as well; cf. Fig. 2). An open vertical bar represents
the average activity level (per day) during a 0.5-h bin of daytime behavior, with the filled bars indicating nighttime bins. The day-to-day variation in
activity levels for corresponding time bins (on successive days) is indicated by the small points; the distance between the top of a vertical bar and the
point represents the SEM. The large plot superimposes all 29 flies' worth of activity data. The same plotting conventions apply as are used in the individualfly record at the lower right--with the additional indication of lights-on being defined as Time 0 h, and lights-off as 12 h, though this standard manner of
quoting "Zeitgeber Times" [cf. Zerr et al. (1990); and see Table II, footnote a] did not correspond to literal local time during these monitoring experiments
(see Materials and Methods). To superimpose the separate animals' records, the levels of activity (e.g., peak numbers of events, which of course vary
from fly to fly) had to be normalized; this was done as described by Hamblen-Coyle et al. (1989), and the ordinate represents activity level proportions
(not numbers of events, as in the lower right panel). Each vertical bar in the large plot depicts a " m e a n of a mean," i.e., the average amount of activity
per fly per day _+SEM (indicated by the point; see above). The data leading to all three of these plots--and those in Figs. 6 and 7--were unfiltered (cf.
Figs. 2 and 3). (B) A germ-line transformant (z = 24.5-25 h), carrying a 13.2-kb DNA fragment that includes the normal per gene (genetic background:
hemizygosity forper~
In the large average activity plot, the behaviors of 17 males from strain " 2 " of this transformant type (cf. Table I) are superimposed,
as described above.
>~
'7
A
&
&
430
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
mental change; we call this the morning peak. The other begins a few hours
before lights-off, peaks about 0.5 h before that time, and is called the evening
peak. During the remainder of the cycle, in most cases, there is relatively little
activity. Note that a clock function would appear to underlie these changes in
behavior, in that the flies "anticipate" the environmental transitions by becoming more active well before lights-on or lights-off. Activity then subsides gradually, reaching troughs in the middle of the day and of the night. Such nonabrupt,
time-dependent changes in behavior suggest that the flies are not merely being
startled by, say, the dark-to-light transition (see below), followed by a quick
recovery from the effects of such an environmental change.
The diel behavior of the different mutants and transformants was essentially
the same as that of the wild type in two ways: All gave bimodal activity records
and 24-h periods (examples in Fig. 3). In fact, every wild-type or mutant fly
that entrained to the 12:12 LD cycles (which represented the vast majority of
flies monitored for a given genotype; Table I) had a 24-h best estimate of its
diurnal rhythmicity extracted by periodogram analysis.
The times of the z-altered mutants' activity peaks, however, differed from
normal behavior. Any type with a shorter than wild-type period (in D D ) - - p e r ~,
two of the in vitro-mutated transformants, and norpA--exhibited shifts of the
evening peak into the daytime. [See Dushay et al. (1990) for a similar kind of
plot for the short-z Clock mutant, numerical data for which are given in Table
II.] These shifts ranged from approximately 3 h earlier than the wild type, for
per s and the "Gly-substituted" short transformant (Figs. 6A and B), to less
than 1 h, for the blind norpA mutant (Fig. 6D).
In contrast, the per L1 and per L2 mutants exhibited shifts of their major
blocks of evening activity to later times than those of the wild type (or the pernormal transformants). The onset of such activity blocks for these long-period
mutants' evening peaks commenced after lights-off, and the summits (albeit not
sharp ones) were well into the night, i.e., about 5-6 h after lights-off for these
two particular mutants (Figs. 7A and B). In the per cl and per c2 average activity
plots there are additional components to the post-lights-off behavior. First, there
were rather abrupt rises in activity levels just after the on-off transition (observed
in other genotypes as well; see below); forper L1, the flies remained at this level
briefly, then exhibited an activity "spike" three bins into the night (Fig. 7A);
after the transition-following increase for per c2, the flies became relatively
quiescent during the next bin, which was followed by a sharp increase in activity
(Fig. 7B). Flies of the other genotypes exhibited environmental-transition peaks
that were milder (ca. 10-40% higher than the pretransition activity values) and
tended to occur for only one bin's worth of activity after lights-off or lights-on
(Figs. 5 and 6). Whereas these abrupt changes could be some kind of startle
response, we have no explanation for delayed spikes, following lights-off, shown
by the perL's. Perhaps it is the case that the slow clock in these long-period
Behavior of Circadian Rhythm Mutants of Drosophila
A
pers
-~
oo.
"
.
"
9
c,4
o
0
12
Time
B
pers Gly transf0rmant
>~
o
o
0
12
Time
Fig. 6. Diurnal behavior of short-period rhythm variants. (A) Locomotor
activity in LD of an individual per '~ male (two right-hand panels) and the
superimposed records of 43 such males' behavior. The plotting conventions,
and other methodological details, are described in the legend to Fig. 5; ~- for
this genotype is 19 h. (B) LD behavior of a transformant (r = 19 h) in which
a certain serine residue (substituted by asparagine in the original per" mutant)
has been replaced by glycine, by in vitro mutagenesis of the pertinent nucleotide in the 13.2-kb "construct" (cf. Rutila et al., 1991). Strain " 4 5 " of this
transformant type (Table I) has its behavior displayed here (in the large plot,
for 14 flies' worth of LD activity). (C) pert-defined Ser, in vitro-mutated to
aspartate (r = 22.5 h); in the large plot, 12 activity records for flies of line
" 1 8 " of this transformant type are superimposed. (D) Diurnal behavior of
the no-receptor-potential-A mutant. This short-period mutant [r = 23 h
(Dushay et al., 1989)] responds to cyclical changes between periods of light
and darkness, as implied by the actogram (upper right), in spite of having
nonresponsive external photoreceptors (reviewed by Pak, 1991). The LD
activity records for 42 males hemizygous for the norpA e24 "null" (and thoroughly blinding) allele are superimposed in the large plot.
431
432
Hamblen-Coyle,
Wheeler, Rutila, Rosbash, and
Hall
persAsp transformant
C
c3
~
c5
c5
12
o
Time
D
norpA
c5
>,
U.
CJ
12
Time
F i g . 6. Continued
mutants causes them sluggishly to respond in an anomalously late manner to
the environmental transition.
Another long-period type--a per transformant called 14.6:21, which
exhibits free-running periods 3-4 h longer than normal (e.g., Zehring et al.,
1984; Zerr et al., 1990)--was anomalous in its evening peak phase: This did
not occur at a time appreciably later than (or otherwise different from) normal
(Fig. 7C). The transduced per + DNA in this strain lacks the "5'-flanking"
region of the gene (Hamblen et al., 1986), but that may not be the reason for
the odd diel behavior of these 14. 6 flies (see below).
Morning activity seen in the various rhythm variants' behavioral records
did not correlate very well with free-running period values. Several of the short-
perL 1
A
o
o
0
12
Time
B
perL2
(5
O
0
12
Time
C
'14.6' transformant
.... i .... i ....
i ~ , , , i ....
o_
c5
i~ c5
o.
o
_.,,I
'
'
'
p
. . . .
i
.
.
.
0
.
.
.
.
i
. . . .
12
Time
Fig. 7. Diurnal behavior of long-period rhythm variants. (A) p e r u ( r =
29 h), for which the large average activity plot summarizes the behavior
of 37 males (cf. Table I) that were monitored in LD (see the legend to
Fig. 5 for plotting conventions). (B) p e r L2 (r = 29 h). Large plot: 43
males' worth of behavior (cf. Table I). (C) p e r transformant (7 = 2728 h), in which flies with a p e r ~ carded a 14.6-kb DNA fragment that
extends well beyond the 3' end of this gene, but whose 5' end is truncated
[missing "5'-flanking" sequences, the gene's first noncoding exon, and
part of the first intron (cf. Hamblen et a l . , 1986; Yu et a l . , 1987a,b)].
The LD behavior of 10 males from strain " 2 1 " of this transformant type
(cf. Table I) is summarized in the large average activity plot.
434
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
period types exhibited shifts of their morning activity peak to a phase slightly
later than that of the wild type (Figs. 6A-C; see Table II). Yet the slightly
short-r norpA flies showed a slightly earlier than normal morning peak, paralleling the direction in which this mutant's evening peak moved (Fig. 6D). Also,
Drosophila expressing three long-period genotypes--per L1, per Le, and the
14. 6:21 per transformant--exhibited relatively late morning peaks (Fig. 7), but
the magnitude of these shifts was much lower than for evening activity (also
see below, Table II).
Phase Analysis of Diel Activity
To find time bins in which peaks for the flies' activity fall, the numerical
versions of the data plotted in the foregoing figures were analyzed with the
Phase program, as described under Materials and Methods (see also Figs.
2-4). The average phase points of the morning and evening peaks were found
for each genotype; these are listed in Table II, which indicates the average
morning and evening peak phases for all the flies of a given genotype. Such
values are means of means, because there is some variability in the time of a
given individual's evening peak (for examlple) from day to day (see the upper
right panels in Figs. 5-7 and Table II, footnote a). To show the individual phase
values for the key short-period, long-period, and normal types, plots displaying
the numerical results of applying the filtering and peak-determining programs
were prepared: Each and every (daily) peak time--for morning plus evening
activity--was entered for all flies monitored (Figs. 8-10). These histograms
show that the automated analysis of Drosophila's did behavior does find characteristic phases for flies whose genotype is associated with a given free-running
,/-.
Thus, these numerical results are congruent with those that can be visually
extracted from the average activity plots discussed above. Notably, the phases
of the evening peaks are 3-7 h earlier or later than normal (Table II) for per
mutants with the most dramatic changes in their free-running periods. [The late
evening phases for the perL's were extracted using a "search window" (Fig.
4) that excluded the "delayed spikes" of activity seen rather early in the night
(see above).]
The morning phase values (Table II) were not very different from those
associated with the wild type ( < 2-h difference between the extremes) and, for
some of the rhythm variants, moved (slightly) in a direction opposite that of the
evening peak's change. Nevertheless, simple regression analysis revealed a significant correlation between r and morning phase (slope, 0.08, whereby earlier
phases tended to have shorter periods, and vice versa; P = 0.02, from ANOVA;
this correlation was much stronger for the evening phase values: slope, 0.68; P
< 10-3). Some of the morning peaks--for a given fly, on a given day of
Behavior of Circadian Rhythm Mutants of Drosophila
A
Fig. 8. Phase analysis of normal activity in
LD. Wild type-derived (A) or per+-trans formed (B) flies (cf. Fig. 5) had their phases
determinedformally (cf. Figs. 2 and 3) for each
morning and evening "search window" (cf.
Fig. 4) applied to a series of successive
light:dark Cycles. At the top of each panel, the
black bars designate nighttime portions of the
LD cycles. Each individual phase value
(excluding search windows that led to peaks
which were too feeble to tabulate; cf. Table
II)--for each day's activity, for each fly of a
given genotype that was monitored--entered
into the morning and evening parts of these
histograms; thus, all individual (daily) phase
values were lumped together for that genotype
(as opposed to computing per-fly mean phases,
as in Table II). The mean morning and evening
phases (again, as amassed from all wild typeor transformant-associated values, irrespective
of which individual flies they were derived
from) are designated by diamonds.
435
wild-type
II
............
Illl
Q
. . . . . . .
6
B
r
12
3,2' transformant
Ill
...........
Ill
~
o
6
12
Time
monitoring its LD activity--were essentially absent or undefinable; this was
especially true o f the amino acid-substituted p e r transformants (see Table II,
footnote a).
Certain o f the evening peak times that were extracted analytically (Table
II) in a sense validated some previous free-running period determinations, in
which r ' s are only a bit shorter or longer than normal (Table I). Thus, the Clock
mutant has a circadian pacemaker that, at most, runs 1.5 h fast in DD (Dushay
et al., 1990); correspondingly, this mutant's evening peak was determined to
be earlier than normal (Table II). Also consider the case o f blind norpA mutants,
which have been reported to free-run with short periods (23.0-23.3 h) that are
even closer to wild-type values (Dushay et al., 1989). Whereas there is still no
explanation for why a lack o f visual input through the fly's external photoreceptors (cf. Pak, 1991) would alter r, norpA seems to be a bona fide fast-clock
mutant, because its evening phase in LD is about l h earlier than that determined
for the wild type (Table II). The same can be said o f the in vitro-mutated p e r
mutants, some of whose r ' s in DD are not far from normal values [Table I (cf.
Rutila et al., 1991)]. Yet the movements o f their evening peaks in LD were in
the same directions (i.e., earlier or later than normal) as for the mutants with
more dramatically abnormal r ' s . Even the slightly long-period p e r transformants
with Thr substituted for Ser (at the amino acid position where p e r ~ is mutated
436
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
A
B
per s
pers GIV transformant
I
cn
~
6
= I
......
0
r2
C
per s
12
6
Time
Time
D
AS~ transformant
norpA
.
.
.
.
Illl
fi
.
tn
L~ _l~~.t ........
6
Time
12
18
0
6
12
r8
T~me
Fig. 9. Phase analysis of short-period mutant activity in LD. The strategies and conventions for
plotting these data are as described in the legend to Fig. 8. The genotypes leading to the analyzed
data in A through D correspond to those noted in Figs. 6A-D.
to Asn) had an evening phase (weighted average for the four strains grouped
near the bottom of Table II) that was about a quarter-hour later than the average
for the normal types (first group in Table II).
In two per transgenics whose 14.6 kb of transformed DNA is devoid of
the gene's 5'-flanking region, the evening phases corresponded to a bit before
lights-off, with values indistinguishable from normal ones (see first two sections
in Table II; cf. Figs. 7C and 5A). This anomaly is neither explainable nor
generalizable: Another long-period, 5'-flanking-minus transformant, carrying
7.2 kb of inserted per + DNA, yielded an evening phase that was a half-hour
later than the average for the two 14.6 types [Table II (second section); the
somewhat late evening peak was also apparent in the average activity plot for
7.2:9 (not shown)], even though free-running r's for the 14. 6 transgenics have
consistently been about an hour longer than in the case of 7.2:9's activity in
DD (Table I). Thus, the diel behavior of the 7.2 flies conforms to what one
would expect from the other long-period genetic variants (though it must be
noted that this transgenic's morning phase was earlier than in the case of other
Behavior of Circadian Rhythm Mutants of Drosophila
per
A
Ill
I
I
I
I
I
I
LI
i
~
I
437
per
B
I
i
Illl
I
L2
III ....................
cn
6
12
18
6
Time
12
18
Time
C
'14.6' transformant
Ill[
...................
ro
0
6
12
18
Time
Fig, 10. Phase analysis of long-period mutant activity in LD, The strategies and conventions for
plotting these data are as described in the legend to Fig. 8. The genotypes leading to the analyzed
data in A through C correspond to those noted in Figs. 7A-C,
long-period strains; Table II). Arguments in favor of such expectations can also
be made on a priori grounds, as taken up in the next section.
DISCUSSION
Basic Features of
Drosophila's Diel
Behavior
Under laboratory conditions of alternating light and dark entrainment cycles,
D. melanogaster adults display two peaks of locomotor activity: one near dawn
and the other near dusk. It could be argued that this kind of diel behavior is
adaptive, in that the animals are active when they can see (and hence locate
things such as food sources and potential mates, in part by visual cues), but
they are quiescent during the middle of the day, when they could be baked if
they were out and about. However, Saunders (1982) mentions that bimodal
activity rhythms, common in Drosophila, are observed in species from nonarid
438
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
as well as arid locales. He also notes (Saunders, 1982, Fig. 2.10) that the
morning peak of activity seems to be an exogenous effect of lights-on in one
species (D. pseudoobscura), in part because there appears to be no anticipation
of that environmental transition (also see Engelmann and Mack, 1978). In coptrast, our results indicate that D. melanogaster begins to increase its activity
1-2 h before lights-on (Fig. 5), in addition to showing an even more pronounced
anticipation of lights-off (the latter "phase lead" occurs in D. pseudoobscura
as well).
Another reason for believing that the morning peak of activity in D. melanogaster is under the control of an endogenous pacemaker is that it remains
present (and can be prominent in some behavioral records) on release of some
of the flies into free-run (see citations under Results). Moreover, this dawn peak
can be phase-shifted by endogenous r-altering mutations in a manner that is, in
general, consistent with the effects of these genetic variations on the evening
peak (see below).
The positions of these peaks, and other elements of D. melanogaster's diel
behavior, were, in the main, as would be predicted (see Aschoff, 1981) from
knowledge of (1) the period of the entraining stimulus (24 h in all cases), (2)
the pace at which the internal clock is running in flies of a given genotype, and
(3) the fundamental nature of phase response curves (PRCs) that contribute
heavily to descriptions (and understandings) of circadian pacemaker function
[see Winfree (1987) for discussion, and refer to Fig. 11].
Thus, a mutant with a short free-running period ( < 24 h) will advance the
onset of its DD activity every day, whereas a long-r mutant will exhibit daily
delays in such onsets [see Fig. 1 of Hall and Rosbash (1987) for examples].
One would surmise, therefore, that the first evening peak displayed during monitoring in LD will be early for the short-period mutant and late for the longperiod mutant. The subsequent evening peaks could, however, be at the same
time each day--though still relatively early or late in comparison to normal diel
behavior--if these mutants are reset every day by the Zeitgeber. Thus, the
period-altered mutants would, as in the wild type, entrain (be driven into 24
periodicities), though the phases of the formers' activity peaks would not be
normal. The current behavioral results are consistent with immunohistochemical
and molecular findings on per RNA and protein cycling: The periodicities of
such cycles, in LD, had normal 24-h values for the per r mutants, though the
phases for these biochemical rhythms were different from those for the wild
type (Hardin et al., 1990; Zerr et al., 1990).
The daily phase shifts need to be of rather high magnitudes to allow dramatically r-altered mutants to entrain; thus, per s, for example, must phase-delay
itself 5 h each day. Other mutants of this type may not be able to effect such
extensive shifts. Consider the tau mutant of the golden hamster, which was
discovered as an "early-onset" behavioral mutant in an LD activity-monitoring
Behavior of Circadian Rhythm Mutants of Drosophila
439
experiment (Ralph and Menaker, 1988): Free-running periods for mutant homozygotes are about 20 h, and that periodicity (i.e., no entrainment) is also observed
in many tau/tau individuals when their wheel-running activity is monitored
under LD 12 : 12 (Ralph and Menaker, 1988). Thus, 24-h cycles for this mutant
are beyond its "limit of entrainment;" such limits seem narrower, in general,
for mammals than insects (Aschoff and Pohl, 1978).
Details of Mutationally Induced Phase Resettings and Novel Phase Angles
To understand how a circadian period mutant entrains, consider the exemplary case of perS; imagine that a fly expressing that short-T mutation is
approaching the end of its (extrinsic) daytime. The endogenous pacemaker in
this mutant, running with a 19-h periodicity, will bring the animal to the end
of its "internal" day when it is still light (during LD 12:12). One would,
furthermore, infer that p e r s will arrive at the end of its day fully 5 h before the
lights go off, owing to the fact that the "subjective day" is only 7 h in duration,
with the subjective night having a normal 12-h time span (Konopka, 1979;
Konopka and Orr, 1980). In any event, exposure to light during internal-early
night for this mutant will effect a phase delay, by analogy to the effects of light
pulses delivered to free-running organisms (in general) early in the subjective
night [see Fig. 2 of Hall and Rosbash (1987) and Fig. 4 of Dushay et al. (1990)
for examples of these PRCs as determined for Drosophila activity rhythms].
The light-induced delay in p e / w i l l occur during each LD cycle and hence--if
of large enough magnitude (see above)--can bring this short-r mutant into register with the environmental cycles.
The question suggests itself as to why the position of the evening-peak
phase for p e r ~ is only 3 h earlier than that of the wild type (in LD), instead of
5 h as would be predicted by the known PRCs (Fig. 11). The problem is that
the phase shifts in question (Hall and Rosbash, 1987; Dushay et al., 1990) were
based on 10-min light pulses, whereas the current experiments have used 12-h
segments of light. It is possible that a different equilibrium phase (i.e., its new
position, postpulse) would be predicted by PRC based on the phase-shifting
effects of 12-h light exposures. A more interesting possibility is that the phase
angle between the endogenous pacemaker and the activity peak has been altered
in the mutant. In other words, p e r s might be a phase-angle variant as well as a
period mutant. The effect on phase angle could be to delay the expected onset
of evening locomotor activity, relative to the endogenous pacemaker (the delay
being, empirically, 2 h; see beginning of this paragraph). This argument could
also explain the approximately half-hour phase delay of perS's morning peak,
relative to the wild type (Table II). In this regard, recall that the PRC for this
mutant has a 12-h subjective night (e.g., Konopka and Orr, 1980); p e r s should
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
440
A
+ ~per +
|
-~
~
Cm
g
<
+ per
I~
-~
<
C
perL1
>
<
12
18
I
6
0
Time
12
(h)
Fig. 11.
therefore traverse the night phase of the LD cycle normally and, hence, anticipate lights-on in a manner similar to that of the wild type. But if p e r s is indeed
a phase-angle (i.e., delayed) mutant, then the later-than-normal morning peak
becomes comprehensible. Whereas these morning delays are not pronounced,
they were consistently observed (Table II) for essentially all the "per'-related"
types (involving the original in vivo mutation at this amino acid within the period
gene and the in vitro-effected substitutions at that position well).
That t h e p e r L mutants also entrain to the environmental cycles we employed
is readily understandable in general terms: Knowing the shapes of PRCs (Fig.
11), one assumes that these long-period mutants receive anomalous light exposure late in their long-duration subjective night (see below); this will cause phase
advances, which these mutants must accomplish if they are to entrain to 12 : 12
Behavior of Circadian Rhythm Mutants of Drosophila
441
Fig, 11. A model for the effects of period gene alleles, including ~"alterations and phase-delaying
effects caused by per mutations. The PRCs (solid lines) for the three per genotypes indicated are
taken from Konopka and Orr (1980), Orr (1982), and Hall and Rosbash (1987; in which Fig. 2
displays curves of this sort, determined by R. J. Konopka and D. Orr). The left-hand ordinates
indicate changes in phase (AO), whereby - means phase delay, and + phase advance. The PRC
amplitudes for the three genotypes are indicated to be the same; this is indeed the case for these
curves (Orr, 1982; Hall and Rosbash, 1987), as determined from phase shifts of locomotor activity
rhythms [whereas the "eclosion PRC" for per s has more substantial advances and delays than in
per + (Konopka, 1979)]. Locations of peak locomotor activity times (shaded humps) are taken from
the data in Figs. 8A, 9A, and 10A. The right-hand ordinates designate the relative activity levels.
Each abscissa represents 24 h, divided equally into nighttime (filled bar) and daytime (open bar).
(A) For this wild-type (per +) PRC, ~m and ~be are the morning and evening phases, which are
empirically correlated with the morning and evening activity peaks; the former is just a bit before
lights-on, and the latter about 0.5 h before lights-off. (B) Forper ~, t~ m and ~be are marked in positions
(shaded vertical bars) where they would fall if they occurred at the analogous positions on the PRC
as in the wild type. In this regard, note that--since the night portion of the PRC is of the same
duration as in the wild type in this mutant (Konopka and Orr, 1980)--~bm is marked at the same
position (relative to the Zeitgeber) as in A. The moming activity peak would thus be expected (left
bar) at the same position as in the wild type but, instead, was observed to be slightly later (left
hump). In the day portion of the PRC, the--the expected evening phase for this particular mutant
(see citations above)--is advanced by 5 h. Note, in this regard, that the dashed line indicates such
a premature resumption of the subjective night and the corresponding "delay portion" of the PRC;
this ~be should be correlated with an activity peak (fight bar), but the observed peak (right hump) is
quite a bit later. (C) ForperLl's PRC, ~br is shown (left bar) to be shifted about 3-4 h into the night
(cf. Orr, 1982), yet the observed activity peak (left hump) occurred appreciably later than this. In
the morning, what is hypothesized (see text) to be the clock's (standard) resetting (hence, ~bm is
fight at lights-on, as in the wild type) leads to the expectation (right bar) of a normally timed
morning peak, but this actually occurred somewhat later (right hump).
LD. The p e r L mutations also caused e v e n i n g activity peaks to be delayed 6 - 7
h relative to the wild type (Table II; if one discounts, for the sake o f the current
argument, the activity spikes seen, in Figs. 7 A and B, relatively early in the
night). U n l i k e p e r S ' s P R C , that for p e r L1 appears to be lengthened uniformly
over the entire cycle, so the subjective day is ca. 14 h (Orr, 1982). I n v o k i n g ,
once more, the supposition that the phases for D r o s o p h i l a ' s diel activity peaks
are determined by coupling to the e n d o g e n o u s p a c e m a k e r (and the phase
responses a s s o o a t e d with e n v i r o n m e n t a l inputs to it), one would predict e v e n i n g
phases for the p e r L ' s to be about 3 - 4 h after lights off, instead o f 5 - 6 h, as
observed (Fig. 11). Thus, the p e r L types are not only phase-delayed relative to
the wild type but also are later than w o u l d be predicted by these simple expectations. A g a i n (as for p e r S ) , it seems that these long-period mutants could also
be phase-angle variants.
H o w can one account for the differences in m a g n i t u d e b e t w e e n m o r n i n g
and e v e n i n g peak phase d e l a y s - - a s implied (in Table II) by the relatively small
differences b e t w e e n n o r m a l and m u t a n t phase values in the m o r n i n g , compared
to the more substantial intergenotypic variations in e v e n i n g peak times? O n e
possibility is to invoke, in a rather general way, the " d u a l oscillator" formalisms
442
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
of Pittendrigh and Daan (1976) and Illnerov~i (1982), which were based on their
analysis of phase-shifting behavioral or biochemical rhythms in rodents (see also
Vafopoulou and Steel, 1991). In Drosophila, one oscillator, which would be
more tightly under the control of the genetic factors we have applied, could
control the evening activity, with the other hypothetical oscillator, loosely influenced by the actions of these genes, underlying morning behavior. An alternative
explanation stems from a further feature of the model implied by Fig. 11 and
the idea that per mutants are phase-angle variants. Consider, first, that the
morning (or subjective morning) is the phase-reference point from which all
other times are measured, i.e., this would be the "clock-start" time in these
fruit flies. If the phase delay (i.e., phase-angle difference, relative to the wild
type) accumulates progressively during the day from the initial starting point,
then the peaks that occur later in a given cycle (here, high activity in the evening)
would be more greatly affected then the early ones. Another implication of this
idea is connected to the fact that the morning peaks are just barely later than
normal in the (hypothetically delayed) per s mutant: Just after the clock restarts
in the morning (in this mutant or any other type), its period-shortening effect
would begin to counteract the phase-angle delay, which could result in the everso-slightly delayed morning peaks that are observed (Table II). In contrast, the
slow clock properties of per L act in concert with the phase-angle effect (which
is again a delay), causing the morning peaks in this mutant to be appreciably
later than normal (Figs. 7A and B, Table II).
This model provides a unifying explanation for the phase properties of the
original period mutants (Konopka and Benzer, 1971), the recently generated per
transgenic strains (Rutila et al., 1991), and two additional short-period mutants
(Clk and norpA). The one exception involves the "14. 6"per transgenics (cf.
Zehring et al., 1984). The evening peaks for these two strains were about the
same as for the wild type, contrary to expectation for a long-period variant; the
14. 6 morning peaks, in contrast, exhibited phase delays not unlike those observed
for the perC's (Table II). We thus imagine that the 14. 6per variants are phaseangle mutants that are advanced relative to what would be predicted from simple
PRC assumptions. We cannot, however, explain how changes at this genetic
locus could (hypothetically) cause phase advances in some mutants and phase
delays in others, let alone the matter of phase effects being in opposite directions
for the morning vs evening peaks exhibited by the 14. 6 transgenics. But it should
be kept in mind that this molecularly engineered variant is of a different type
from (almost) all others used in this study, which contain amino acid substitutions. In contrast, this transgenic contains a normal coding region of the gene
and a "5'-flanking" deletion (see citations under Results); this could alter per
regulation, against a background of normal inherent activity of the gene product.
That altered regulation is not enough to eliminate the ability of the 14. 6 strains
to phase-shift themselves by a few hours per day, i.e., to entrain (as do all flies
Behavior of Circadian Rhythm Mutants of Drosophila
443
in this study). Instead, the way that time information is transduced to the fly's
behavior has somehow become different in this transgenic type from how it is
in the other mutants (and from the " 7 . 2 " transgenic, with its "properly" late
evening peak), in terms of the particular phase position at which high levels of
activity occur.
The proviso that must accompany the model implied by this discussion
(and Fig. 11) is that no "12-h light-pulse" PRC is available for any Drosophila
genotype, let alone the rhythm mutants (for which the curves lurk mainly in
theses and review articles). If such PRCs were different from those determined
using short pulses (Orr, 1982; Hall and Rosbash, 1987), they might predict
equilibrium positions (i.e., the new phases that eventually stabilize after the
light treatments) which are different from the simple expectations (Fig. 11). The
use of "skeleton photoperiods" may be most helpful in assigning accurate activity phases--as has been the case for eclosion rhythms in Drosophila (e.g.,
Pittendrigh and Minis, 1964)--and for determining the true equilibrium position
of the PRC associated with a given rhythm-related genotype.
In addition to the anomalies found for some of those genotypes (notably
the incomplete per-gene transformants just discussed), certain other rhythm variants in Drosophila are counterintuitive in terms of how their periodicities relate
to phase angles. These were obtained by directional selection (Pittendrigh, 1981)
or after chemical mutagenesis (Jackson, 1983) for early phases of eclosion rhythmicity in LD. Regarding their (subsequently tested) free-running periods, these
strains did not conform to expectation (cf. Aschoff, 1981) in that they paradoxically had longer-than-normal z values (Pittendrigh, 1981; Jackson, 1983). We
imagine that these strains also displayed phase shifts of the overt rhythms,
relative to the endogenous pacemaker. In these cases, more dramatically than
in those we have studied, the phases are not just a bit later than would be
expected from knowing ~-(as inper s, per L, etc.) but are actually in the opposite
direction. It will be interesting to determine whether any period-altered mutants
recovered in fruit flies (see Newby et al., 1991) will be apparently unaltered in
their phase angles as well.
ACKNOWLEDGMENTS
We thank Sherry Rails for testing one of the transgenic strains for its
rhythmicity in free-run. We appreciate comments on the manuscript from Harold
B. Dowse, Brigitte Frisch, Jay C. Dunlap, Mitchell S. Dushay, Paul E. Hardin,
and Carl H. Johnson. This work was supported by NIH Grant GM-33205 to
M.R. and J.C.H.
444
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
REFERENCES
Aschoff, J. (1981). Freerunning and entrained circadian rhythms. In Aschoff, J. (ed.), Handbook
of Behavioral Neurobiology, Vol. 4: Biological Rhythms, Plenum Press, New York, pp.
81-93.
Aschoff, J., and Pohl, H. (1978). Phase relations between a circadian rhythm and its Zeitgeber
within the range of entrainment. Naturwissenchaften 65: 80-84.
Baylies, M. K., Bargiello, T. A., Jackson, F. R., and Young, M. W. (1987). Changes in abundance
and structure of the per gene product can alter periodicity of the Drosophila clock. Nature
326: 390-392.
Citri, Y., Colot, H. V., Jacquier, A. C., Yu, Q., Hall, J. C., Baltimore, D., and Rosbash, M.
(1987). A family of unusually spliced and biologically active transcripts encoded by a Drosophila clock gene. Nature 326: 42-47.
Dowse, H. B., and Ringo, J. M. (1989). The search for hidden periodicities in biological time
series revisited. J. Theoret. Biol. 139: 487-515.
Dowse, H. B., and Ringo, J. M. (1992). Is the circadian clock a "metaoscillator"? Evidence from
studies of ultradian rhythms in Drosophila. In Young, M. W. (ed.), Molecular Genetics of
Biological Rhythms, Marcel Dekker, New York (in press).
Dushay, M. S., Rosbash, M., and Hall, J. C. (1989). The disconnected visual system mutations
in Drosophila melanogaster drastically disrupt circadian rhythms. J. Biol. Rhythms 4: 1-27.
Dushay, M. S., Konopka, R. J., Orr, D., Greenacre, M. L., Kyriacou, C. P., Rosbash, M., and
Hall, J. C. (1990). Phenotypic and genetic analysis of Clock, a new circadian rhythm mutant
in Drosophila melanogaster. Genetics 125: 557-578.
Engelmann, W., and Mack, J. (1978). Different oscillators control the circadian rhythm of eclosion
and activity in Drosophila. J. Comp. Physiol. 127: 229-237.
Hall, J. C. (1990). Genetics of circadian rhythms. Annu. Rev. Genet. 24: 659-697.
Hall, J. C., and Kyriacou, C. P. (1990). Genetics of biological rhythms in Drosophila. Adv. Insect
Physiol. 22: 221-298.
Hall, J. C., and Rosbash, M. (1987). Genes and biological rhythms. Trends Genet. 3: 185-191.
Hamblen, M., Zehring, W. A., Kyriacou, C. P., Reddy, P., Yu, Q., Wheeler, D. A., Zwiebel,
L. J., Konopka, R. J., Rosbash, M., and Hall, J. C. (1986). Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: Overlapping genomic fragments
that restore circadian and ultradian rhythmicity to per ~ and per- mutants. J. Neurogenet. 3:
249-291.
Hamblen-Coyle, M., Konopka, R. J., Zwiebel, L. J., Colot, H. V., Dowse, H. B., Rosbash, M.,
and Hall, J. C. (1989). A new mutation at the period locus with some novel effects on circadian
rhythms. J. Neurogenet. 5: 229-256.
Hardin, P. E., Hall, J. C., and Rosbash, M. (1990). Feedback of the Drosophila period gene
product on circadian cycling of its messenger RNA. Nature 343: 536-540.
Helfrich, C., and Engelmann, W. (1987). Evidences for circadian rhythmicity in the per~ mutant
of Drosophila melanogaster. Z. Naturforsch. 42C: 1335-1338.
Illnerov~i, H., and Vanecek, J. (1982). Two-oscillator stmcture of the pacemaker controlling the
circadian rhythm of N-acetyltransferase in the rat pineal gland. J. Comp. Physiol. 145: 539548.
Jackson, F. R. (1983). The isolation of biological rhythm mutations on the autosomes of Drosophila
melanogaster. J. Neurogenet. 1: 3-15.
Jackson, F. R. (1992). Circadian rhythm mutants of Drosophila. In Young, M. W. (ed.), Molecular
Genetics of Biological Rhythms, Marcel Dekker, New York (in press).
Konopka, R. J. (1979). Genetic dissection of the Drosophila circadian system. Fed. Proc. 38:
2602-2605.
Konopka, R. J. (1986). Neurogenetics of Drosophila circadian rhythms. In Huettel, M. D. (ed.),
Evolutionary Genetics oflnsect Behavior, Plenum Press, New York, pp. 215-221.
Konopka, R. J., and Benzer, S. (1971). Clock mutants of Drosophila melanogaster. Proc. Natl.
Acad. Sci. USA 68: 2112-2116.
Konopka, R. J., and Orr, D. (1980). Effects of clock mutations on the subjective day--Implications
Behavior of Circadian Rhythm Mutants of Drosophila
445
for a membrane model of the Drosophila circadian clock. In Siddiqi, O., Babu, P., Hall, L.
M., and Hall, J. C. (eds.), Genetics and Neurobiology of Drosophila, Plenum Press, New
York, pp. 409-416.
Konopka, R. J., Pittendrigh, C., and Orr, D. (1989). Reciprocal behavior associated with altered
homoeostasis and photosensitivity of Drosophila clock mutants. J. Neurogenet. 6: 1-10.
Konopka, R. J., Smith, R. F., and Orr, D. (1991). Characterization of Andante, a new Drosophila
clock mutant, and its interaction with other clock mutants. J. Neurogenet. 7:103-114.
Liu, X., Yu, Q., Huang, J., Zwiebel, L. J., Hall, J. C., and Rosbash, M. (1991). The strength
and periodicity of D. melanogaster circadian rhythms are differentially affected by alterations
in period gene expression. Neuron 6: 753-766.
Newby, L. M., White, L., DiBartolomeis, S. M., Walker, B. J., Dowse, H. B., Ringo, J. M.,
Khuda, N., and Jackson, F. R. (1991). Mutational effects of the Drosophila miniature-dusky
(m-dy) locus: Effects on cell size and circadian rhythms. Genetics 128: 571-582.
Orr, D. P.-Y. (1982). Behavioral Neurogenetic Studies of a Circadian Clock in Drosophila melanogaster, Ph.D. thesis, California Institute of Technology, Pasadena.
Pak (1991). Molecular genetic studies of photoreceptor function using Drosophila mutants. Prog.
Clin. Biol. Res. 362: 1-32.
Petersen, G., Hall, J. C., and Rosbash, M. (1988). Theperiod gene of Drosophila carries speciesspecific behavioral instructions. EMBO J. 7: 3939-3947.
Pittendrigh, C. S. (1981). Circadian organization and the photoperiodic phenomena. In Follett, B.
K. (ed.), Biological Clocks and Reproductive Cycles, John Wright, Bristol, UK, pp. 1-35.
Pittendrigh, C. S., and Daan, S. (1976). A functional analysis of circadian pacemakers in nocturnal
rodents. V. Pacemaker structure: A clock for all seasons. J. Comp. Physiol. 106: 333-355.
Pittendrigh, C. S., and Minis, D. H. (1964). The entrainment of circadian oscillations by light and
their role as photoperiodic clocks. Am. Nat. 98: 261-294.
Ralph, M. R., and Menaker, M. (1988). A mutation of the circadian system in golden hamsters.
Science 241: 1225-1227.
Rosbash, M., and Hall, J. C. (1989). The molecular biology of circadian rhythms. Neuron 3" 387398.
Rutila, J. E., Edery, I., Hall, J. C., and Rosbash, M. (1992). The generation and analysis of shortperiod behavioral rhythm mutants in D. melanogaster suggests potential mechanisms of period
gene function. J. Neurogenet. 8: 101-113.
Saunders, D. S. (1982). Insect Clocks, 2nd ed., Pergamon Press, Oxford, UK.
Smith, R. F., and Konopka, R. J. (1981). Circadian clock phenotypes of chromosome aberrations
with a breakpoint at the per locus. Mol. Gen. Genet. 183: 243-251.
Smith, R. F., and Konopka, R. J. (1982). Effects of dosage alterations at the per locus on the
period of the circadian clock of Drosophila. Mol. Gen. Genet. 185: 30-36.
Sokolove, P. G., and Bushnell, W. N. (1978). The chi square periodogram: Its utility for analysis
of circadian rhythms. J. Theoret. Biol. 72: 131-160.
Sulzman, F. M. (1982). Microcomputer monitoring of circadian rhythms. Comput. Biol. Med. 12:
253-261.
Vafopoulou, X., and Steel, C. G. H. (1991). Circadian regulation of synthesis of ecdysteroids by
prothoracic glands of the insect Rhodnius prolixus: Evidence of a dual oscillator system. Gen.
Comp. Endocrinol. 83: 27-34.
Vosshall, L. B., and Young, M. W. (1992). Circadian behavior defects of eye, optic lobe, and
brain mutants of Drosophila melanogaster. In Young, M. W. (ed.), Molecular Genetics of
Biological Rhythms, Marcel Dekker, New York.
Winfree, A. T. (1987). The Timing of Biological Clocks, Scientific American Library, New York.
Winfree, A. T., and Gordon, H. (1977). The photosensitivity of a mutant circadian clock. J. Comp.
Physiol. 122: 87-109.
Young, M. W., Bargiello, T. A., Baylies, M. L., Saez, L., and Spray, D. C. (1989). Molecular
biology of the Drosophila clock. In Jacklet, J. W. (ed.), Neuronal and Cellular Oscillators,
Marcel Dekker, New York, pp. 529-542.
Yu, Q., Colot, H. V., Kyriacou, C. P., Hall, J. C., and Rosbash, M. (1987a). Behaviour modification by in vitro mutagenesis of a variable region within the period gene of Drosophila.
Nature 326: 765-769.
446
Hamblen-Coyle, Wheeler, Rutila, Rosbash, and Hall
Yu, Q., Jacquier, A. C., Citri, Y., Hamblen, M., Hall, J. C., and Rosbash, M. (1987b). Molecular
mapping of point mutations in the period gene that stop or speed up biological clocks in
Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 84: 784-788.
Zehring, W. A., Wheeler, D. A., Reddy, P., Konopka, R. J., Kyriacou, C. P., Rosbash, M., and
Hall, J. C. (1984). P-element transformation with period locus DNA restores rhythmicity to
mutant, arrhythmic Drosophila melanogaster. Cell 39" 369-376.
Zerr, D. M., Hall, J. C., Rosbash, M., and Siwicki, K. K. (1990). Circadian fluctuations of period
protein immunoreactivity in the CNS and the visual system of Drosophila. J. Neurosci. 10:
2749-2762.