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Entrainment properties of the circadian system changing with reproductive state and molt in the canary
Physiology& Behavior,Vol. 55, No, 5, pp. 803-810, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0031-9384/94 $6.00 + .00
Pergamon 0031-9384(93)E0008-E
Entrainment Properties of the Circadian System Changing With Reproductive State and Molt in the Canary H. P O H L
Max-Planck-Institut flir Verhaltensphysiologie, D-82346 Andechs, Germany Received 20 July 1993 POHL, H. Entrainment properties of the circadian system changing with reproductive state and molt in the canary. PHYSIOL BEHAV 55(5) 803-810, 1994.--Changes in entrainmentproperties of the circadian pacemaking system with respect to changes in reproductive state and molt were investigated in the male domestic canary (Serinus canaria). In two experimentscanaries were subjected to three and two similar sequences of light regimes, respectively, during 1 year. Locomotor activity, feeding, and vocalization were simultaneouslyrecorded. Only birds showing complete postnnptial wing feather molt and related changes in behavior (vocalization)expressed differences in the ability of their circadian system to entrain to low-amplitudelight/dark (LD) cycles with periods (T) > 24 h. It is suggested that changes in the organization of the circadian system, including differences in entrainmentproperties, are associated with the state of photorefractorinessand related functions (testicnlar regression,postnuptial molt). Regardlessof the molt status, over 90% of the birds that were arrhythmic in constant light (LL) entrained to low-amplitude LD cycles with T 25.5 h (Experiment 1) or T 25.0 h (Experiment2). Birds that did not entrain or were relatively entrained with the respective light regimes were (with one exception) rhythmic in LL. It is therefore likely that the occurrence of arrhythrniain LL facilitates entrainmentto low-amplitudelight cycles. Circadian rhythms Domesticcanary Locomotoractivity Reproduction Photorefractoriness Molt Arrhythmia
PREVIOUS investigations on general properties of the avian circadian system and their variability (adaptability) in European starlings (Sturnus vulgaris) and Cardueline finches have shown that essential parameters, namely period, measured in continuous dim light, or phase, measured under constant artificial LD cycles, varied seasonally (10,24,26,36). Seasonal differences were also observed in activity time, precision of activity onset, and in the range of entrainment of the circadian rhythms with LD cycles deviating from 24 h (T-cycles) (10,24,26). Likewise, the patterns of circadian rhythms of activity or metabolic rate, measured under the same light and temperature conditions, changed with season (27). Comparable seasonal changes in properties and organization of the circadian system were found in mammals (1517,28,39). It has been suggested that changes in the organization of circadian systems are causally related to seasonal or circannual functions, such as reproduction, migration, or hibernation, and that they are, consequently, under the control of the endocrine system. Although gonadal hormones can affect (or modulate) circadian parameters (15,33,36,40), direct effects of androgens (i.e., testosterone) on entrainment properties of the circadian pacemaker system have not been found in the male domestic canary (Serinus canaria) (in preparation). Studies on the endocrine system controlling the annual reproductive cycle in the European starling (Sturnus vulgaris) revealed that secretion of thyroxine as well as prolactine coincides with
Feeding
Vocalization
Entrainment
the photorefractory state (6,7). In the domestic canary it was found that the state of refractoriness is characterized by a fall in circulating luteinizing hormone (LH) levels (22) as well as by testicular regression (32). Is is also noteworthy that in the canary the change from the state of photorefractoriness to the subsequent state of photosensitivity, which are both characterized by small testis size (i.e., low levels of testosterone), is accompanied by changes in the circadian system involved in the photoperiodic control of these functions (12,20,21,31). Phase shifts of the rhythm(s) of photosensitivity relative to each other and to the daily LD cycle may be necesSary for the temporal coincidence of the photoinducible phases responsible for secretion of various hormones (35). Concomitant changes in behavioral rhythms as well as changes in properties of the circadian pacemakers controlling these rhythms are likely when the system changes between the photorefractory and the photosensitive state. In the presented experiments on male domestic canaries, Serinus canaria, it was tested whether the transitions from the state of reproductive competence to the state of photorefractoriness correlates with changes in properties and in the organization of the circadian system, including overt circadian rhythmicity, period length (T), the ability to entrain to low-amplitude LD cycles, and the phase relations between various behavioral rhythms. Two experiments were conducted in which canaries were subjected to similar sequences of cyclic light regimes (LD) interrupted by constant light (LL), either three times during 1 year (Experiment 803
804
POHL
1) or twice, that is, during the reproductive and the nonreproductive phase of the annual gonadal cycle (Experiment 2). To study changes in phase relations between behavioral rhythms, locomotion, feeding, and vocalization were simultaneously recorded. METHOD
Animals and Maintenance The male canaries that were used in the following experiments had been hatched during summer and were obtained from the breeder (T. Veit, Singen, Germany) in late autumn or early winter. Nine birds were transferred to the test conditions on January 19 (Experiment 1), and another nine birds on February 24 of the following year (Experiment 2). The respective sequences of light conditions and illumination levels are listed in Table 1. Before testing (and between tests in Experiment 2), the birds were individually held under natural daylights. Ambient temperatures were between 20 ° and 25°C before and during the experiments. During tests the birds were housed individually in cages (42 x
23 x 26 cm) placed in sound-proof and air-ventilated cabinets. Each cabinet was made of three wooden boxes that were mounted into each other. Foam rubber and air between the boxes provided acoustic insulation. Fluorescent white light illuminated the cage from the top through a frosted glass window. Light intensity was controlled by a motor-driven dimmer and measured by a FLMX 007 luxmeter (OPTRONIK) with a silicon cosV(h) receptor. Locomotor activity was monitored by an infrared motion detector; feeding was detected by a photocell that was activated by an infrared light source in front of the feeder. Total vocalization (calling and singing) was recorded by an U H E R microphone that was connected to an amplifier and a transformer for digital processing on a data logging system (Kr~iussling) together with the data from locomotion and feeding. An integrated pulse generator allowed recording song separately from total vocalization. The birds were fed a commercial seed mixture for canaries, biscuit, salad, apples, and water. During molt they received additional vitamins and minerals mixed in food or sand. Feeding and cleaning of the cages was done in 3 - 4 - d a y intervals at different circadian times. The feeder was not directly attainable from the perch and the bird had to move under an opaque screen to obtain food.
TABLE 1 EXPERIMENTAL PROTOCOL OF EXPERIMENTS l AND 2
Date
Days
Light Regimen
T (h)
Illumination (Ix)
24.0 -24.0 24.5 25.0 25.5 24.0 -24.0 24.5 25.0 25.5 -24.0 24.5 25.0 25.5 --
67:21 21 67:21 67:21 67:21 67:21 67:21 21 67:21 67:21 67:21 67:21 21 67:21 67:21 67:21 67:21 21
Experiment 1 Jan 19-Mar 01 Mar 02-Mar 23 Mar 24-Apr 06 Apr 07-Apr 27 Apr 28-May 12 May 13-May 28 May 29-Jul 06 Jul 07-Jul 30 Jul 31-Aug 24 Aug 25-Sep 08 Sep 09-Sep 24 Sep 25-Oct 10 Oct l l - N o v 18 Nov 19-Dec 17 Dec 18-Jan 02 Jan 03-Jan 16 Jan 17-Jan 31 Feb 01-Feb 23
42 22 14 21 15 16 39 24 25 15 16 16 39 29 16 14 15 23
LD LL LD LD LD LD LD LL LD LD LD LD LL LD LD LD LD LL
12:12 12:12 12.5:12.0 12.5:12.5 13.0:12.5 12:12 12:12 12.5:12.0 12.5:12.5 13.0:12.5 12:12 12.5:12.0 12.5:12.5 13.0:12.5
Experiment 2 Test 1 (no. 1-9) Mar 01-Mar 15 Mar 16-Apr 05 Apr 06-Apr 27 Test 2 (no. 1-6) Aug 01-Aug 31 Sep 01-Sep 21 Sep 22-Oct 13 Oct 14-Nov 04 Test 2 (no. 7-9) Aug 01-Aug 18 Aug 19-Sep 08 Sep 09-Sep 30 Oct 01-Oct 26
15 21 22
LL LD 12.5:12.0 LD 12.5:12.5
-24.5 25.0
24 55:24 55:24
31 21 22 22
LL LD 12.5:12.0 LD 12.5:12.5 LL
-24.5 25.0 --
24 55:24 55:24 24
18 21 22 26
LL LD 12.5:12.0 LD 12.5:12.5 LL
-24.5 25.0 --
24 55:24 55:24 24
Periodogram Analysis Chi-square periodograms were obtained according to the method of Sokolove and Bushell (30). Q(p)-values are given on the ordinate, periods from 18 to 30 h on the abscissa (Figs. 3 and 4). All Q(p)-values above the sloping line were significant with p < 0.001. Entrainment was characterized by one significant mean period 0") that was similar within _+ 0.1 h to the period (T) of the zeitgeber. If a second significant mean period was present, the rhythm was considered as relatively entrained. Relative coordination between rhythm and zeitgeber was denoted when the predominent mean T's differed from Tby at least 0.5 h (examples in Figs. 3 and 4).
Experimental Protocols In the first experiment, nine male canaries were tested, seven of which were subjected to three series of similar light regimes beginning on January 19 and ending on February 23 of the following year (see Table 1). One canary (No. 8) died after the end of the second series; canary No. 3 was replaced at the end of the first series by a new bird that had been previously held under natural daylight. This bird (No. 3a) was tested only twice. Molt was checked by counting the number of growing wing feathers (primaries and secondaries) on June 2 and 17, July 6, August 5, November 18, and February 22. On March 2 and June 2, the cloacal protuberance (length x width) was determined. In the second experiment, nine male canaries were subjected to two series of similar light regimes (see Table 1) from March 1 to April 27 (first series) and from August 1 to November 4 (second series). Eight birds were tested in both series; one bird (No. 1) was substituted after the first series by a bird (No. la) that had previously been held in natural daylight. Individual birds were tested in the same cage (cabinet) in both test series. In between these tests the birds were held individually under natural daylight conditions. Molt was checked on February 24, April 27, June 1, June 29, and July 28 in all canaries, and on October 26 (birds Nos. 7 - 9 ) or November 4 (birds Nos. 1-6). On February 24 and April 27, the cloacal protuberance was also measured in all birds.
CIRCADIAN ENTRAINMENT AND REPRODUCTION
805
RESULTS
Locomot.
No 9
Feeding
Vocalizat.
1
Experiment I
Canaries normally breed during increasing photopefiods in spring and early summer and molt after the summer solstice when day length is already decreasing. Onset of photorefractoriness is associated with gonadal regression, onset of wing feather molt, and cessation of singing (9,13,22). Three parameters (wing feather molt, cloacal protuberance, and vocalization) were used for determining whether a bird was in the state of gonadal regression and photorefractoriness. In five of the six male canaries (Nos. 2, 3, 6, 7, 8, and 9) that molted at the end of the first or during the second test series, cloacal protuberance was smaller by 23%, on average, during molt than during the reproductive phase. Singing and calling recommenced after termination of molt in only two of the six birds (Nos. 2 and 6). The three birds (Nos. 1, 4, and 5) that did not molt completely showed no systematic changes in either cloacal protuberance or vocalization. Recordings of locomotion, feeding, and vocalization (calling and singing) of two canaries (Nos. 6 and 9) are presented in Figs. 1 and 2. The respective periodograms are shown in Figs. 3 and 4. The records exemplify prop-
2O 40 6O 80
100 120 0 "0 ~D
140 ! 60
E I.--
~!~ii~ii!i~,,~, !,i~!! ,i~ ¸
180 200 220 240
No 6
Locomot.
Feeding
Singing 1" 24.0
20
LL
40
T 24.0
60
T 24.5
80
T 25,0 T 25.5
I00
120
LL
160
Ill
E
I 0
I 24
I
I
'-8 0
I
2~
I
I
La 0
I
I
2~
~8
T i m e of d o y {h)
FIG. 2. Original double-plotted recordings of locomotion, feeding and total vocalization (calling and singing) of male canary No. 9. Vocalization ceased almost completely at the onset of molt. Further details as in Fig. 1.
T 24.0
1/,0
0 "10
260
T 24.0
180
T 24.5
200
T 25.0
220
T 2~5
240 LL
260 280'
T 24,0
300 T 24.5
320
T 25.0
340
T 25.5
360
LL
I
I
0
2~-
I
I
I
48 0
24
I
i
48 0
I
I
2~
~s
T i m e of d a y (h}
FIG. 1. Original double-plotted recordings of locomotion, feeding, and singing of male canary No. 6 under various light regimes of Experiment 1 (see right margin and Table 1). Lines represent onset or end of light time (L), respectively. Activity begins before onset of L. Mean illumination levels were 67:21 Ix in LD and 21 Ix in LL. Blank areas indicate recording failure.
erties of the circadian system that were analyzed for all birds tested during this experiment. First series. At the beginning of the experiment all three functions established relatively stable phase relations to the light regime (LD 12:12; 67:21 Ix). Subsequent free-running rhythms in continuous light (LL; mean intensity: 21 Ix) showed similar periods and phases (onsets) in all functions. After reentrainment to LD 12:12 the period of the zeitgeber (T) was changed from T 24.0 to 25.5 in three steps. In canary No. 9 (Fig. 2), that did not molt until the middle of June (ca. day 140), all three rhythms showed relative entrainment at T 25.5, which is characterized by a second peak in the periodograms (see Fig. 4 and the Method section). In canary No. 6 (Fig. 1), that started molt during the middle of May (ca. day 85), locomotion and feeding were entrained, and singing gradually ceased, associated with the onset of molt. Second series. Reentrainment to LD 12:12 was quickly achieved in both canaries, but the patterns of the three rhythms and their phase relations to the zeitgeber showed marked differences compared to the beginning of the experiment. In canary No. 9 locomotion became arrhythmic during LD 12:12 and LL, although feeding was still rhythmic. Vocalization was totally inhibited during molt. During the following stepwise lengthening of T from 24.0 to 25.5 h locomotor activity and feeding were both rhythmic and entrained to the LD regimes. At T 25.5 locomotion showed one significant peak at 25.6 h; feeding had two significant mean periods in the periodogram (23.3 and 25.4 h), indicating relative entrainment (see Fig. 4). In the subsequent LL
806
POHL
No 6
L
F
S
Q(P) 600 400 200 T 25.0 h
T 25.5 h
LL
T 25.0 h
23.4
ii
23.3
i T 25.5 h
locomotion and feeding had similar patterns and periodograms as during the previous LL. In canary No. 6, that molted between the end of May and the middle of August (from ca. day 85 to day 180), locomotion and feeding changed their phase relations to the zeitgeber (LD 12:12) compared to the beginning of the experiment, and tended to become arrhythmic in LL. All rhythms lost entrainment to the zeitgeber even at T 24.5 after molt was completed and singing recommenced at the end of August. Third series. After reentrainment to LD 12:12 the phase relations between all three rhythms and the LD cycle of bird No. 6 and between the feeding rhythm and the LD cycle of bird No. 9 were similar to those observed at the beginning of the experiment, when the two birds were in their reproductice phase and not molting. During the third stepwise lengthening of T the rhythms of locomotion and feeding (as well as singing of canary No. 6) lost entrainment at T24.5 (No. 6) or T25.0 (No. 9). During the subsequent LL all measured functions were free running, with similar circadian periods in both canaries (see Figs. 3 and 4). Table 2 shows the results of all canaries tested in this experiment. In three birds (Nos. 2, 6, and 9) that were rhythmic in LL in all three tests, entrainment of the locomotor and feeding rhythms to T 25.5 was only observed during molt. When not molting, the birds were either relatively entrained or not entrained. These three canaries went through a complete molt cycle, and two of them (Nos. 2 and 6) started singing again after termination of molt. Three birds (Nos. 3a, 7, and 8) that completely molted their wing feathers but did not sing after molt, and the three birds (Nos. 1, 4, and 5) showing incomplete or interrupted molt patterns entrained to the LD regimes at T 25.5, regardless of their molt status.
Experiment 2
LL
-1 22.7
25.3
T 25.0 h
T 25.5 h
.L Period
18
22
26
30
23.2
LL 18
22
26
30
18
22
26
30 h
FIG. 3. Chi-square periodograms of male canary No. 6 (cf. Fig. 1), analyzed over 14-day intervalsunder the respectivelight regimes indicated on the right margin. Only periodograms for T 25.0, T 25.5, and LL are shown in chronologicalorder. Significantpeak Q(p)-values (p < 0.001) are indicatedfor each periodogram.L, locomotion;F, feeding;S, singing.
The increase in cloacal protuberance (46%, on average) between February 24 and April 27 indicated that all canaries were in reproductive state during the first entrainment test (see Table 1). No bird molted during that time. Postnuptial molt began during July under natural daylight in all canaries but was terminated in three of the nine birds (Nos. 2, 3, and 5) before the end of the second test series. Locomotor activity recordings of three canaries are presented in Fig. 5. Canaries Nos. 2 and 6 showing free-running rhythms in LL did not entrain to the LD regimes at T 24.5 and 25.0 during the first test. Their rhythms were relatively coordinated with the zeitgeber (see the Method section). Canary No. 8, which was arrhythmic in LL, relatively entrained to the LD regimes at T 25.0 but desynchronized from the zeitgeber shortly before the end of the test. All three canaries were molting at the beginning of the second test, and their activity patterns and/or periods differed from those during the first test. Canary No. 8, that was previously arrhythmic, showed two significant periods in the periodogram (22.0 and 25.7 h). The previously rhythmic bird (No. 2) was arrhythmic during molt, and canary No. 6 shortened its ~from 23.7 to 23.2 h. Canaries Nos. 6 and 8 entrained to the LD regimes at T 24.5 and 25.0. Bird No. 2, that had already terminated molt, was relatively entrained at T 24.5 and 25.0. Table 3 presents the results of all birds tested during this experiment. In the first test, when all birds were in reproductive state, only three birds were not entrained (Nos. 2 and 6) or relatively entrained (No. 8) to the LD regime at T 25.0, whereas six canaries were entrained regardless of whether they were rhythmic or arrhythmic in LL. In the second test all molting birds as well as two of three birds that had already completed molt entrained at T 25.0. The amount of locomotor activity was greatly reduced during molt in six of eight birds measured in both tests (p <
CIRCADIAN ENTRAINMENT AND REPRODUCTION
No 9
L
V
F
Q(p) 4o0
2OO
LL
T 25.0 h
T 25.5 h
LL
T 25.0 h
T 25.5 h
LL
807
ness, molt) and in properties of the circadian pacemaking system, it was necessary to determine the physiological state of each individual bird during the test conditions. Normal seasonal changes in cloacal protuberance, molt, and vocalization were observed in about 30% of the birds tested. In the majority of birds seasonality of physiological and behavioral functions was interrupted. These birds might have interpreted the relatively high light intensities during the test periods (see Table 1) as constant long days and photorefractoriness might not have broken. W h e n male domestic canaries were kept under constant long days (LD 16:8) for 3 years, about 20% of the birds tested showed circannual rhythms in wing feather molt and associated changes in plasma L H (unpublished data). All 15 birds that were molting (i.e., in photorefractory state) during one or two entrainment tests of both experiments were entrained to either T 25.5 (Experiment 1) or T 25.0 (Experiment 2), including only one case of relative entrainment. However, it must be taken into account that a relatively large percentage (ca. 60%) of nonmolting canaries was also entrained to the respective light regimes. It has recently been found that the circadian system of the domestic canary is highly sensitive to small changes in light intensity (29). Thus, the experimental light conditions used in the present tests might not have been subtle enough to force more birds' circadian rhythms out of the range of entrainment (1). From the results of the two experiments, it is indicated that the ability of the circadian system of the canary to entrain to lowamplitude LD cycles with periods longer than 24.0 h changes in relation to the transition from the reproductive to photorefractory state and from photorefractoriness to a state in which the birds are again photosensitive. Changes in entrainment properties were not correlated with systematic changes in r or with changes between rhythmicity and arrhythmia, though nearly all birds that were arrhythmic in LL entrained to T 25.5 (Experiment 1) or T 25.0 (Experiment 2). Only one of 11 birds (involving seven individuals) that did not entrain to, or were relatively entrained with, the respective LD regimes was arrhythmic in LL (see Tables 2 and 3). Thus, it seems likely that the occurrence of ar-
TABLE 2
T 25.0 h
CIRCADIAN PERIOD (7") IN LL*, ENTRAINMENT TO T 25.5, A N D O C C U R R E N C E OF W I N G FEATHER M O L T IN MALE C A N A R I E S D U R I N G EXPERIMENT 1 Test No. T 25.5 h 1
Bird No. LL Period
18
22
26 30
10
22
26
30
18
22 26 30 h
FIG. 4. Chi-square periodograms of male canary No. 9 (cf. Fig. 2). Details as in Fig. 3. L, locomotion; F, feeding; V, total vocalization (calls and song).
0.01; Wilcoxon signed rank test; Fig. 6), but there were no systematic changes in either 7- or between rhythmicity and arrhythmia. DISCUSSION
To evaluate corresponding changes in the endocrine system controlling seasonal functions (reproduction, photorefractori-
1 2 3 3a 4 5 6 7 8 9
2
7" (h)
E
M
ar 23.5 23.3
+ + (+)
+ +
24.1 23.3 23.6 ar ar 23.4
+ + + + + (+)
+ + + -
3
7" (h)
E
M
7" (h)
E
M
ar 23.2
+ (+)
-
ar 23.8
+ (+)
(+) -
23.5 ar ar 23.5 23.3 ar 23.4
+ + + + + +
(+) +
23.5 ar ar 23.3 23.4
+ + + +
(+) (+) -
23.5
-
-
* Measured after T 25.5. ar = arrhythmia; E+ = entrained, E(+) = relatively entrained, E - = not entrained, M+ = complete molt, M(+) = partial molt, M = no molt.
808
POHL
No 6
No 2
No 8
1
LL
20
T 24.5
40
T 25.0
60 1
t,L
O "O ID
20 T 24.5
E I---
40 T 25.0 60
LL 80 I
i
0
2z
i
I
t
48 0
2~
I
I
I
48 0
2~
.J
~8
Time of doy (h) FIG. 5. Original double-plotted recordings of locomotion of three male canaries (Nos. 2, 6, and 8) under various light regimes of Experiment 2 from March 1 to April 27 (upper diagrams) and from A u g u s t 1 to Ocober 26 (lower diagrams). Lines indicate onsets and ends of light time. Mean illumination levels were 55:24 lx in L D and 24 lx in LL. Indices on the right margin refer to canary No. 8.
rhythmia in LL facilitated entrainment to low-amplitude LD cycles. It w a s f o u n d e a r l i e r i n f r i n g i l l i d s (Fringilla coelebs, Pyrrhula that temporal dissociation (splitting) of two circadian activity components after inversion of the LD cycle accelerates the rate of reentrainment (25). Comparable results were reported o f t h e d i u r n a l S i b e r i a n c h i p m u n k , Eutamias sibiricus ( 2 3 , 2 5 ) .
pyrrhula)
TABLE
More recently, differences in responses to daily melatonin injections were observed in nocturnal rats (5). Individuals that lengthen r after being transferred from total darkness to continuous light (LL) did not entrain to the melatonin zeitgeber, although individuals with disrupted patterns of activity or arrhythmia in LL did entrain. This remarkable congruence in responses to different zeitgeber modalities of birds and diurnal as well as
100 %
3
CIRCADIAN PERIOD (r) 1N LL, ENTRAINMENT AT T 25.0, AND OCCURRENCE OF WING FEATHER MOLT IN MALE CANARIES DURING EXPERIMENT 2 Test No.
g,
1
2
T
o
E 8
T
Bird No.
(h)
E
M
1 la 2 3 4 5 6 7 8 9
23.3
+
-
23.2 23.7 ar 23.5 23.7 23.3 ar ar
+ + + + (+) +
-
5 8
(h)
ar 23.2* ar 23.2 ar 23.2 23.6 ar 23.4
E
+ (+) + + + + + + +
M
+ + + + + +
ar = arrhythmia, E + = entrained, E ( + ) = relatively entrained, E = not entrained, M + = molt, M - = no molt. * Measured after T 25.0.
3
o
"6
50
~
9 6 7 4 2
0
[ March
I August
FIG. 6. Percentage change in the amount of locomotion per circadian period, measured in LL, between March and A u g u s t (Experiment 2). N u m b e r s refer to i n d i v i d u a l s (see Table 3).
CIRCADIAN ENTRAINMENT AND REPRODUCTION
nocturnal mammals with disrupted or arrhythmic activity patterns in LL strongly suggests that changes in the organization of the circadian system influence the ability and the range of entrainment of the rhythms to external and internal zeitgebers. Based on experiments on greenfinches (Carduelis chloris) and house sparrows (Passer domesticus), a hypothesis was proposed that explained photorefractoriness by seasonal differences in the circadian organization of the photoperiodic timing mechanism (18-20). Different states of the annual reproductive cycle are accompanied, or caused, by changes in phase relations between circadian rhythms of photosensitivity that trigger secretion of LH and the follicle stimulating hormone (FSH), both controlling testicular development (21). Although this specific hypothesis was not substantiated by experiments on domestic canaries (31), phase shifts between photoinducible phases of sensitivity rhythms regulating other hormones are still likely (14,32). The observed changes in the organization of the circadian system and related changes in entrainment properties may be associated with alterations of the phase relations between circadian rhythms within the endocrine and neuroendocrine systems as well as between behavioral rhythms. Such changes may derive from different causes: for example, from temporary desynchronization of subsidiary oscillators as a result of uncoupling from the main pacemaker(s), or from a reduction of the degree (or loss) of self-sustainment of the circadian pacemaker, that is, by a decrease (or inhibition) of periodic release of melatonin from the pineal (2,3). Both possibilties extend the range of entrainment of circadian oscillators with an LD zeitgeber (1,38). The role of hormones controlling these changes still remains to be clarified. In the majority of birds tested in the two experiments, the periods and phases of the rhythms of locomotor activity, feeding, and vocalization were similar in LL and under the LD regimes.
809
In less than 50% of all birds tested, activity time (a) of the locomotor rhythm was between 1 and 5 h longer than that of the feeding rhythm. Activity time of the rhythm of vocalization (song and calls) was shorter than that of locomotion or feeding, and its temporal pattern was more variable in most birds. Stable phase relationships between rhythms of locomotor activity and feeding have been reported from finches, sparrows, and pigeons exposed to a variety of experimental light conditions, suggesting that timing of both functions is controlled by the same pacemaker (4), but independent control by two pacemakers is more likely (8,27). Close synchrony between locomotor activity and vocalization (crowing) was also observed in Japanese quail, Coturnix coturnix japonica (37). Entrainment patterns of the rhythms of locomotion, feeding, and vocalization, estimated by their periodograms, were also similar in most birds of this study. Some exceptions, however, are instructive with respect to the organization of the circadian system. Significant differences in the periodograms between locomotion and feeding were found in birds Nos. 7, 8, and 9 of Experiment 1. In canaries Nos. 7 and 8 only one significant peak was present at ca. 25.0 h (T 25.0) or 25.5 h (T 25.5) in the periodograms for feeding, and three or two significant peaks, respectively, were shown in the periodograms for locomotion. In canary No. 9, locomotion was arrhythmic during molt and feeding was still rhythmic. After the low-amplitude LD cycle was reintroduced, the rhythmic pattern of locomotion immediately reappeared (see Figs. 2 and 4). These findings support previous hypotheses: (a) that behavioral functions (e.g., locomotion, feeding) are independently controlled by separate oscillators (or groups of ocscillators), and (b) that internal desynchronization of these subsidiary oscillators may occur as a result of uncoupling from the main pacemaker(s) (11,34).
REFERENCES 1. Aschoff, J.; Pohl, H. Phase relationsbetween a circadianrhythm and its zeitgeber within the range of entrainment. Naturwissenschaften 65:80-84; 1978. 2. Cassone,V. M. Effects of melatoninon vertebratecircadiansystems. Trends Neurosci. 13:457-464; 1990. 3. Cassone, V. M.; Menaker, M. Is the avian circadian system a neuroendocrine loop? J. Exp. Zool. 232:539-549; 1984. 4. Chabot, C. C.; Menaker, M. Circadian feeding and locomotor activity rhythms in pigeons and house sparrows. J. Biol. Rhythms 7:287299: 1992. 5. Chesworth,M. J.; Cassone, V. M.; Armstrong,S. M. Effects of daily melatonin injections on activity rhythms of rats in constant light. Am. J. Physiol. 22:R101-R107; 1987. 6. Dawson,A. Changesin plasma thyroxine concentrationsin male and female starlings (Sturnus vulgaris) during a photo-inducedgonadal cycle. Gen. Comp. Endocrinol. 56:193-197; 1984. 7. Dawson, A.; Goldsmith,A. R. Plasma prolactin and gonadotrophins during gonadal development and the onset of photorefractorinessin male and female starlings (Sturnus vulgaris) on artificial photoperiods. J. Endocrinol. 97:253-260; 1983. 8. Ebihara, S.; Gwinner, E. Different circadian pacemakers controlling feeding and locomotor activity rhythms in European starlings. J. Comp. Physiol. [A] 171:63-67; 1992. 9. Follett, B. K.; Hinde, R. A.; Steel, E.; Nicholls, T. J. The influence of photoperiod on nest building, ovarian development and luteinising hormone secretion in canaries (Serinus canarius). J. Endocrinol. 59:151-162; 1973. 10. Gwinner, E. Effects of season and external testosteroneon the freerunning circadian activity rhythm of European starlings (Sturnus vulgaris). J. Comp. Physiol. 103:315-328; 1975.
11. Gwinner, E. Melatonin in the circadian system of birds. Model of internalresonance.In: Hiroshige,T.; Honma,K., eds. Circadianclocks in ecology. Sapporo: Hokkaido UniversityPress; 1989:127-153. 12. Hamner,W. M. The photorefractory period of the house finch.Ecology 49:111-117; 1968. 13. Hinde, R. A. Interaction of internal and external factors in the integration of canary reproduction.In: Beach, F. A., ed. Sex and behavior. New York: Wiley & Sons; 1974:381-415. 14. Meier, A. H. Temporal synergism of prolactin and adrenal steroids. Gen. Comp. Endocrinol. Suppl. 3:499-508; 1972. 15. Morin, L. P.; Fitzgerald, K. M.; Zucker, I. Estradiol shortens the period of hamster circadian rhythms. Science 196:305-307; 1977. 16. Morin, L. P.; Cummings, L. A. Effect of surgical or photoperiodic castration, testosterone replacement or pinealectomyon male hamster running rhythmicity. Physiol. Behav. 26:825-838; 1981. 17. Mrosovsky,N.; Boshes, M.; Hallonquist,J. D.; Lang, K. Circannual cycle of circadian cycles in a golden-mantledground squirrel. Naturwissenschaften63:298-299; 1976. 18. Murton, R. K.; Lofts, B.; Westwood, N. J. The circadian basis of photoperiodically controlled spermatogenesis in the greenfinch, Chloris chloris. J. Zool. 161:125-136; 1970. 19. Mutton, R. K.; Lofts, B.; Westwood, N. J. Manipulationof photorefractoriness in the house sparrow, Passer domesticus, by circadian light regimes. Gen. Comp. Endocrinol. 14:107-113; 1970. 20. Murton, R. K.; Westwood, N. J. An investigationof photorefractodness in the house sparrow by artificialphotoperiods. Ibis 116:298313; 1974. 21. Murton, R. K. Ecological adaptation in avian reproductivephysiology. Symp. Zool. Soc. Lond. 35:149-175; 1975.
810
22. Nicholls, T. J. Changes in plasma LH levels during a photoperiodically controlled reproductive cycle in the canary (Serinus canarius). Gen. Comp. Endocrinol. 24:442-445; 1974. 23. Pohl, H. Die Aktivit~itsperiodik yon zwei tagaktiven Nagern, Funambulus palmarum und Eutamias sibiricus, unter Dauerlichtbedingnngen. J. Comp. Physiol. 78:60-74; 1972. 24. Pohl, H. Circadian rhythms of metabolism in Cardueline finches as function of light intensity and season. Comp. Biochem. Physiol. [A] 56:145-153; 1977. 25. Pohl, H. Comparative aspects of circadian rhythms in homeotherms: Re-entrainment after phase shifts of the zeitgeber. Int. J. Chronobiol. 5:493-517; 1978. 26. Pohl, H. Properties of the circadian clock of the redpoll Carduelis flammea. I. Stability of period and ranges of entrainment. Physiol. Zool. 53:186-198; 1980. 27. Pohl, H. Properties of the circadian clock of the redpoll Carduelis flammea. II. Overt functions in relation to the pacemaker. Physiol. Zool. 53:199-209; 1980. 28. Pohl, H. Circadian pacemaker does not arrest in deep hibernation: Evidence for desynchronization from the light cycle. Experientia 43:293-294; 1987. 29. Pohl, H. Optimal light sensitivity--its significance for measurement of daylength. Second Meeting of SRBR, Abstract; 1990. 30. Sokolove, P. G.; Bushell, W. N. The chi-square periodogram: Its utility for analysis of circadian rhythms. J. Theor. Biol. 72:131-160; 1978. 31. Storey, C. R. Circadian photosensitivity in the photoperiodic control of gonadotropin secretion in the canary, Serinus canarius. Gen. Comp. Endocrinol. 30:198-203; 1976.
POHL
32. Storey, C. R.; Nicholls, T. J. Some effects of manipulation of daily photoperiod on the rate of onset of a photorefractory state in canaries (Serinus canarius). Gen. Comp. Endocrinol. 30:204-208; 1976. 33. Subbaraj, R.; Gwinner, E. Differential effects of testosterone on circadian rhythms of locomotor activity and feeding in the European starling. Naturwissenschaflen 72:663; 1985. 34. Takahashi, J. S.; Menaker, M. Entrainment of the circadian system of the house sparrow: A population of oscillators in pinealectomized birds. J. Comp. Physiol. 146:245-253; 1982. 35. Turek, F. The termination of the avian photorefractory period and the subsequent gonadal response. Gen. Comp. Endocrinol. 26:562564; 1975. 36. Turek, F. W.; Gwinner, E. Role of hormones in the circadian organization of vertebrates. In: Aschoff, J.; Daan, S.; Groos, G. A.; eds. Vertebrate circadian systems. Heidelberg: Springer-Verlag; 1982:173-182. 37. Wada, M. Circadian rhythms of testosterone-dependent behaviors, crowing and locomotor activity in male Japanese quail. J. Comp. Physiol. [A] 158:17-25; 1986. 38. Wever, R. A. Circadian rhythms of finches under bright light: Is self-sustainment a precondition of circadian rhythmicity? J. Comp. Physiol. 139:49-58; 1980. 39. Wollnik, F.; Breit, A.; Reinke, D. Seasonal change in the temporal organization of wheel-running activity of the European hamster, Cricetus cricetus. Naturwissenschaften 78:419-422; 1991. 40. Zucker, I. Hormones and hamster circadian organization. In: Suda, M.; Hayaishi, O.; Nakagawa, H.; eds. Biological rhythms and their central mechanisms. Amsterdam: Elsevier/North Holland Biomedical Press, 1979:369-381.
Pergamon 0031-9384(93)E0008-E
Entrainment Properties of the Circadian System Changing With Reproductive State and Molt in the Canary H. P O H L
Max-Planck-Institut flir Verhaltensphysiologie, D-82346 Andechs, Germany Received 20 July 1993 POHL, H. Entrainment properties of the circadian system changing with reproductive state and molt in the canary. PHYSIOL BEHAV 55(5) 803-810, 1994.--Changes in entrainmentproperties of the circadian pacemaking system with respect to changes in reproductive state and molt were investigated in the male domestic canary (Serinus canaria). In two experimentscanaries were subjected to three and two similar sequences of light regimes, respectively, during 1 year. Locomotor activity, feeding, and vocalization were simultaneouslyrecorded. Only birds showing complete postnnptial wing feather molt and related changes in behavior (vocalization)expressed differences in the ability of their circadian system to entrain to low-amplitudelight/dark (LD) cycles with periods (T) > 24 h. It is suggested that changes in the organization of the circadian system, including differences in entrainmentproperties, are associated with the state of photorefractorinessand related functions (testicnlar regression,postnuptial molt). Regardlessof the molt status, over 90% of the birds that were arrhythmic in constant light (LL) entrained to low-amplitude LD cycles with T 25.5 h (Experiment 1) or T 25.0 h (Experiment2). Birds that did not entrain or were relatively entrained with the respective light regimes were (with one exception) rhythmic in LL. It is therefore likely that the occurrence of arrhythrniain LL facilitates entrainmentto low-amplitudelight cycles. Circadian rhythms Domesticcanary Locomotoractivity Reproduction Photorefractoriness Molt Arrhythmia
PREVIOUS investigations on general properties of the avian circadian system and their variability (adaptability) in European starlings (Sturnus vulgaris) and Cardueline finches have shown that essential parameters, namely period, measured in continuous dim light, or phase, measured under constant artificial LD cycles, varied seasonally (10,24,26,36). Seasonal differences were also observed in activity time, precision of activity onset, and in the range of entrainment of the circadian rhythms with LD cycles deviating from 24 h (T-cycles) (10,24,26). Likewise, the patterns of circadian rhythms of activity or metabolic rate, measured under the same light and temperature conditions, changed with season (27). Comparable seasonal changes in properties and organization of the circadian system were found in mammals (1517,28,39). It has been suggested that changes in the organization of circadian systems are causally related to seasonal or circannual functions, such as reproduction, migration, or hibernation, and that they are, consequently, under the control of the endocrine system. Although gonadal hormones can affect (or modulate) circadian parameters (15,33,36,40), direct effects of androgens (i.e., testosterone) on entrainment properties of the circadian pacemaker system have not been found in the male domestic canary (Serinus canaria) (in preparation). Studies on the endocrine system controlling the annual reproductive cycle in the European starling (Sturnus vulgaris) revealed that secretion of thyroxine as well as prolactine coincides with
Feeding
Vocalization
Entrainment
the photorefractory state (6,7). In the domestic canary it was found that the state of refractoriness is characterized by a fall in circulating luteinizing hormone (LH) levels (22) as well as by testicular regression (32). Is is also noteworthy that in the canary the change from the state of photorefractoriness to the subsequent state of photosensitivity, which are both characterized by small testis size (i.e., low levels of testosterone), is accompanied by changes in the circadian system involved in the photoperiodic control of these functions (12,20,21,31). Phase shifts of the rhythm(s) of photosensitivity relative to each other and to the daily LD cycle may be necesSary for the temporal coincidence of the photoinducible phases responsible for secretion of various hormones (35). Concomitant changes in behavioral rhythms as well as changes in properties of the circadian pacemakers controlling these rhythms are likely when the system changes between the photorefractory and the photosensitive state. In the presented experiments on male domestic canaries, Serinus canaria, it was tested whether the transitions from the state of reproductive competence to the state of photorefractoriness correlates with changes in properties and in the organization of the circadian system, including overt circadian rhythmicity, period length (T), the ability to entrain to low-amplitude LD cycles, and the phase relations between various behavioral rhythms. Two experiments were conducted in which canaries were subjected to similar sequences of cyclic light regimes (LD) interrupted by constant light (LL), either three times during 1 year (Experiment 803
804
POHL
1) or twice, that is, during the reproductive and the nonreproductive phase of the annual gonadal cycle (Experiment 2). To study changes in phase relations between behavioral rhythms, locomotion, feeding, and vocalization were simultaneously recorded. METHOD
Animals and Maintenance The male canaries that were used in the following experiments had been hatched during summer and were obtained from the breeder (T. Veit, Singen, Germany) in late autumn or early winter. Nine birds were transferred to the test conditions on January 19 (Experiment 1), and another nine birds on February 24 of the following year (Experiment 2). The respective sequences of light conditions and illumination levels are listed in Table 1. Before testing (and between tests in Experiment 2), the birds were individually held under natural daylights. Ambient temperatures were between 20 ° and 25°C before and during the experiments. During tests the birds were housed individually in cages (42 x
23 x 26 cm) placed in sound-proof and air-ventilated cabinets. Each cabinet was made of three wooden boxes that were mounted into each other. Foam rubber and air between the boxes provided acoustic insulation. Fluorescent white light illuminated the cage from the top through a frosted glass window. Light intensity was controlled by a motor-driven dimmer and measured by a FLMX 007 luxmeter (OPTRONIK) with a silicon cosV(h) receptor. Locomotor activity was monitored by an infrared motion detector; feeding was detected by a photocell that was activated by an infrared light source in front of the feeder. Total vocalization (calling and singing) was recorded by an U H E R microphone that was connected to an amplifier and a transformer for digital processing on a data logging system (Kr~iussling) together with the data from locomotion and feeding. An integrated pulse generator allowed recording song separately from total vocalization. The birds were fed a commercial seed mixture for canaries, biscuit, salad, apples, and water. During molt they received additional vitamins and minerals mixed in food or sand. Feeding and cleaning of the cages was done in 3 - 4 - d a y intervals at different circadian times. The feeder was not directly attainable from the perch and the bird had to move under an opaque screen to obtain food.
TABLE 1 EXPERIMENTAL PROTOCOL OF EXPERIMENTS l AND 2
Date
Days
Light Regimen
T (h)
Illumination (Ix)
24.0 -24.0 24.5 25.0 25.5 24.0 -24.0 24.5 25.0 25.5 -24.0 24.5 25.0 25.5 --
67:21 21 67:21 67:21 67:21 67:21 67:21 21 67:21 67:21 67:21 67:21 21 67:21 67:21 67:21 67:21 21
Experiment 1 Jan 19-Mar 01 Mar 02-Mar 23 Mar 24-Apr 06 Apr 07-Apr 27 Apr 28-May 12 May 13-May 28 May 29-Jul 06 Jul 07-Jul 30 Jul 31-Aug 24 Aug 25-Sep 08 Sep 09-Sep 24 Sep 25-Oct 10 Oct l l - N o v 18 Nov 19-Dec 17 Dec 18-Jan 02 Jan 03-Jan 16 Jan 17-Jan 31 Feb 01-Feb 23
42 22 14 21 15 16 39 24 25 15 16 16 39 29 16 14 15 23
LD LL LD LD LD LD LD LL LD LD LD LD LL LD LD LD LD LL
12:12 12:12 12.5:12.0 12.5:12.5 13.0:12.5 12:12 12:12 12.5:12.0 12.5:12.5 13.0:12.5 12:12 12.5:12.0 12.5:12.5 13.0:12.5
Experiment 2 Test 1 (no. 1-9) Mar 01-Mar 15 Mar 16-Apr 05 Apr 06-Apr 27 Test 2 (no. 1-6) Aug 01-Aug 31 Sep 01-Sep 21 Sep 22-Oct 13 Oct 14-Nov 04 Test 2 (no. 7-9) Aug 01-Aug 18 Aug 19-Sep 08 Sep 09-Sep 30 Oct 01-Oct 26
15 21 22
LL LD 12.5:12.0 LD 12.5:12.5
-24.5 25.0
24 55:24 55:24
31 21 22 22
LL LD 12.5:12.0 LD 12.5:12.5 LL
-24.5 25.0 --
24 55:24 55:24 24
18 21 22 26
LL LD 12.5:12.0 LD 12.5:12.5 LL
-24.5 25.0 --
24 55:24 55:24 24
Periodogram Analysis Chi-square periodograms were obtained according to the method of Sokolove and Bushell (30). Q(p)-values are given on the ordinate, periods from 18 to 30 h on the abscissa (Figs. 3 and 4). All Q(p)-values above the sloping line were significant with p < 0.001. Entrainment was characterized by one significant mean period 0") that was similar within _+ 0.1 h to the period (T) of the zeitgeber. If a second significant mean period was present, the rhythm was considered as relatively entrained. Relative coordination between rhythm and zeitgeber was denoted when the predominent mean T's differed from Tby at least 0.5 h (examples in Figs. 3 and 4).
Experimental Protocols In the first experiment, nine male canaries were tested, seven of which were subjected to three series of similar light regimes beginning on January 19 and ending on February 23 of the following year (see Table 1). One canary (No. 8) died after the end of the second series; canary No. 3 was replaced at the end of the first series by a new bird that had been previously held under natural daylight. This bird (No. 3a) was tested only twice. Molt was checked by counting the number of growing wing feathers (primaries and secondaries) on June 2 and 17, July 6, August 5, November 18, and February 22. On March 2 and June 2, the cloacal protuberance (length x width) was determined. In the second experiment, nine male canaries were subjected to two series of similar light regimes (see Table 1) from March 1 to April 27 (first series) and from August 1 to November 4 (second series). Eight birds were tested in both series; one bird (No. 1) was substituted after the first series by a bird (No. la) that had previously been held in natural daylight. Individual birds were tested in the same cage (cabinet) in both test series. In between these tests the birds were held individually under natural daylight conditions. Molt was checked on February 24, April 27, June 1, June 29, and July 28 in all canaries, and on October 26 (birds Nos. 7 - 9 ) or November 4 (birds Nos. 1-6). On February 24 and April 27, the cloacal protuberance was also measured in all birds.
CIRCADIAN ENTRAINMENT AND REPRODUCTION
805
RESULTS
Locomot.
No 9
Feeding
Vocalizat.
1
Experiment I
Canaries normally breed during increasing photopefiods in spring and early summer and molt after the summer solstice when day length is already decreasing. Onset of photorefractoriness is associated with gonadal regression, onset of wing feather molt, and cessation of singing (9,13,22). Three parameters (wing feather molt, cloacal protuberance, and vocalization) were used for determining whether a bird was in the state of gonadal regression and photorefractoriness. In five of the six male canaries (Nos. 2, 3, 6, 7, 8, and 9) that molted at the end of the first or during the second test series, cloacal protuberance was smaller by 23%, on average, during molt than during the reproductive phase. Singing and calling recommenced after termination of molt in only two of the six birds (Nos. 2 and 6). The three birds (Nos. 1, 4, and 5) that did not molt completely showed no systematic changes in either cloacal protuberance or vocalization. Recordings of locomotion, feeding, and vocalization (calling and singing) of two canaries (Nos. 6 and 9) are presented in Figs. 1 and 2. The respective periodograms are shown in Figs. 3 and 4. The records exemplify prop-
2O 40 6O 80
100 120 0 "0 ~D
140 ! 60
E I.--
~!~ii~ii!i~,,~, !,i~!! ,i~ ¸
180 200 220 240
No 6
Locomot.
Feeding
Singing 1" 24.0
20
LL
40
T 24.0
60
T 24.5
80
T 25,0 T 25.5
I00
120
LL
160
Ill
E
I 0
I 24
I
I
'-8 0
I
2~
I
I
La 0
I
I
2~
~8
T i m e of d o y {h)
FIG. 2. Original double-plotted recordings of locomotion, feeding and total vocalization (calling and singing) of male canary No. 9. Vocalization ceased almost completely at the onset of molt. Further details as in Fig. 1.
T 24.0
1/,0
0 "10
260
T 24.0
180
T 24.5
200
T 25.0
220
T 2~5
240 LL
260 280'
T 24,0
300 T 24.5
320
T 25.0
340
T 25.5
360
LL
I
I
0
2~-
I
I
I
48 0
24
I
i
48 0
I
I
2~
~s
T i m e of d a y (h}
FIG. 1. Original double-plotted recordings of locomotion, feeding, and singing of male canary No. 6 under various light regimes of Experiment 1 (see right margin and Table 1). Lines represent onset or end of light time (L), respectively. Activity begins before onset of L. Mean illumination levels were 67:21 Ix in LD and 21 Ix in LL. Blank areas indicate recording failure.
erties of the circadian system that were analyzed for all birds tested during this experiment. First series. At the beginning of the experiment all three functions established relatively stable phase relations to the light regime (LD 12:12; 67:21 Ix). Subsequent free-running rhythms in continuous light (LL; mean intensity: 21 Ix) showed similar periods and phases (onsets) in all functions. After reentrainment to LD 12:12 the period of the zeitgeber (T) was changed from T 24.0 to 25.5 in three steps. In canary No. 9 (Fig. 2), that did not molt until the middle of June (ca. day 140), all three rhythms showed relative entrainment at T 25.5, which is characterized by a second peak in the periodograms (see Fig. 4 and the Method section). In canary No. 6 (Fig. 1), that started molt during the middle of May (ca. day 85), locomotion and feeding were entrained, and singing gradually ceased, associated with the onset of molt. Second series. Reentrainment to LD 12:12 was quickly achieved in both canaries, but the patterns of the three rhythms and their phase relations to the zeitgeber showed marked differences compared to the beginning of the experiment. In canary No. 9 locomotion became arrhythmic during LD 12:12 and LL, although feeding was still rhythmic. Vocalization was totally inhibited during molt. During the following stepwise lengthening of T from 24.0 to 25.5 h locomotor activity and feeding were both rhythmic and entrained to the LD regimes. At T 25.5 locomotion showed one significant peak at 25.6 h; feeding had two significant mean periods in the periodogram (23.3 and 25.4 h), indicating relative entrainment (see Fig. 4). In the subsequent LL
806
POHL
No 6
L
F
S
Q(P) 600 400 200 T 25.0 h
T 25.5 h
LL
T 25.0 h
23.4
ii
23.3
i T 25.5 h
locomotion and feeding had similar patterns and periodograms as during the previous LL. In canary No. 6, that molted between the end of May and the middle of August (from ca. day 85 to day 180), locomotion and feeding changed their phase relations to the zeitgeber (LD 12:12) compared to the beginning of the experiment, and tended to become arrhythmic in LL. All rhythms lost entrainment to the zeitgeber even at T 24.5 after molt was completed and singing recommenced at the end of August. Third series. After reentrainment to LD 12:12 the phase relations between all three rhythms and the LD cycle of bird No. 6 and between the feeding rhythm and the LD cycle of bird No. 9 were similar to those observed at the beginning of the experiment, when the two birds were in their reproductice phase and not molting. During the third stepwise lengthening of T the rhythms of locomotion and feeding (as well as singing of canary No. 6) lost entrainment at T24.5 (No. 6) or T25.0 (No. 9). During the subsequent LL all measured functions were free running, with similar circadian periods in both canaries (see Figs. 3 and 4). Table 2 shows the results of all canaries tested in this experiment. In three birds (Nos. 2, 6, and 9) that were rhythmic in LL in all three tests, entrainment of the locomotor and feeding rhythms to T 25.5 was only observed during molt. When not molting, the birds were either relatively entrained or not entrained. These three canaries went through a complete molt cycle, and two of them (Nos. 2 and 6) started singing again after termination of molt. Three birds (Nos. 3a, 7, and 8) that completely molted their wing feathers but did not sing after molt, and the three birds (Nos. 1, 4, and 5) showing incomplete or interrupted molt patterns entrained to the LD regimes at T 25.5, regardless of their molt status.
Experiment 2
LL
-1 22.7
25.3
T 25.0 h
T 25.5 h
.L Period
18
22
26
30
23.2
LL 18
22
26
30
18
22
26
30 h
FIG. 3. Chi-square periodograms of male canary No. 6 (cf. Fig. 1), analyzed over 14-day intervalsunder the respectivelight regimes indicated on the right margin. Only periodograms for T 25.0, T 25.5, and LL are shown in chronologicalorder. Significantpeak Q(p)-values (p < 0.001) are indicatedfor each periodogram.L, locomotion;F, feeding;S, singing.
The increase in cloacal protuberance (46%, on average) between February 24 and April 27 indicated that all canaries were in reproductive state during the first entrainment test (see Table 1). No bird molted during that time. Postnuptial molt began during July under natural daylight in all canaries but was terminated in three of the nine birds (Nos. 2, 3, and 5) before the end of the second test series. Locomotor activity recordings of three canaries are presented in Fig. 5. Canaries Nos. 2 and 6 showing free-running rhythms in LL did not entrain to the LD regimes at T 24.5 and 25.0 during the first test. Their rhythms were relatively coordinated with the zeitgeber (see the Method section). Canary No. 8, which was arrhythmic in LL, relatively entrained to the LD regimes at T 25.0 but desynchronized from the zeitgeber shortly before the end of the test. All three canaries were molting at the beginning of the second test, and their activity patterns and/or periods differed from those during the first test. Canary No. 8, that was previously arrhythmic, showed two significant periods in the periodogram (22.0 and 25.7 h). The previously rhythmic bird (No. 2) was arrhythmic during molt, and canary No. 6 shortened its ~from 23.7 to 23.2 h. Canaries Nos. 6 and 8 entrained to the LD regimes at T 24.5 and 25.0. Bird No. 2, that had already terminated molt, was relatively entrained at T 24.5 and 25.0. Table 3 presents the results of all birds tested during this experiment. In the first test, when all birds were in reproductive state, only three birds were not entrained (Nos. 2 and 6) or relatively entrained (No. 8) to the LD regime at T 25.0, whereas six canaries were entrained regardless of whether they were rhythmic or arrhythmic in LL. In the second test all molting birds as well as two of three birds that had already completed molt entrained at T 25.0. The amount of locomotor activity was greatly reduced during molt in six of eight birds measured in both tests (p <
CIRCADIAN ENTRAINMENT AND REPRODUCTION
No 9
L
V
F
Q(p) 4o0
2OO
LL
T 25.0 h
T 25.5 h
LL
T 25.0 h
T 25.5 h
LL
807
ness, molt) and in properties of the circadian pacemaking system, it was necessary to determine the physiological state of each individual bird during the test conditions. Normal seasonal changes in cloacal protuberance, molt, and vocalization were observed in about 30% of the birds tested. In the majority of birds seasonality of physiological and behavioral functions was interrupted. These birds might have interpreted the relatively high light intensities during the test periods (see Table 1) as constant long days and photorefractoriness might not have broken. W h e n male domestic canaries were kept under constant long days (LD 16:8) for 3 years, about 20% of the birds tested showed circannual rhythms in wing feather molt and associated changes in plasma L H (unpublished data). All 15 birds that were molting (i.e., in photorefractory state) during one or two entrainment tests of both experiments were entrained to either T 25.5 (Experiment 1) or T 25.0 (Experiment 2), including only one case of relative entrainment. However, it must be taken into account that a relatively large percentage (ca. 60%) of nonmolting canaries was also entrained to the respective light regimes. It has recently been found that the circadian system of the domestic canary is highly sensitive to small changes in light intensity (29). Thus, the experimental light conditions used in the present tests might not have been subtle enough to force more birds' circadian rhythms out of the range of entrainment (1). From the results of the two experiments, it is indicated that the ability of the circadian system of the canary to entrain to lowamplitude LD cycles with periods longer than 24.0 h changes in relation to the transition from the reproductive to photorefractory state and from photorefractoriness to a state in which the birds are again photosensitive. Changes in entrainment properties were not correlated with systematic changes in r or with changes between rhythmicity and arrhythmia, though nearly all birds that were arrhythmic in LL entrained to T 25.5 (Experiment 1) or T 25.0 (Experiment 2). Only one of 11 birds (involving seven individuals) that did not entrain to, or were relatively entrained with, the respective LD regimes was arrhythmic in LL (see Tables 2 and 3). Thus, it seems likely that the occurrence of ar-
TABLE 2
T 25.0 h
CIRCADIAN PERIOD (7") IN LL*, ENTRAINMENT TO T 25.5, A N D O C C U R R E N C E OF W I N G FEATHER M O L T IN MALE C A N A R I E S D U R I N G EXPERIMENT 1 Test No. T 25.5 h 1
Bird No. LL Period
18
22
26 30
10
22
26
30
18
22 26 30 h
FIG. 4. Chi-square periodograms of male canary No. 9 (cf. Fig. 2). Details as in Fig. 3. L, locomotion; F, feeding; V, total vocalization (calls and song).
0.01; Wilcoxon signed rank test; Fig. 6), but there were no systematic changes in either 7- or between rhythmicity and arrhythmia. DISCUSSION
To evaluate corresponding changes in the endocrine system controlling seasonal functions (reproduction, photorefractori-
1 2 3 3a 4 5 6 7 8 9
2
7" (h)
E
M
ar 23.5 23.3
+ + (+)
+ +
24.1 23.3 23.6 ar ar 23.4
+ + + + + (+)
+ + + -
3
7" (h)
E
M
7" (h)
E
M
ar 23.2
+ (+)
-
ar 23.8
+ (+)
(+) -
23.5 ar ar 23.5 23.3 ar 23.4
+ + + + + +
(+) +
23.5 ar ar 23.3 23.4
+ + + +
(+) (+) -
23.5
-
-
* Measured after T 25.5. ar = arrhythmia; E+ = entrained, E(+) = relatively entrained, E - = not entrained, M+ = complete molt, M(+) = partial molt, M = no molt.
808
POHL
No 6
No 2
No 8
1
LL
20
T 24.5
40
T 25.0
60 1
t,L
O "O ID
20 T 24.5
E I---
40 T 25.0 60
LL 80 I
i
0
2z
i
I
t
48 0
2~
I
I
I
48 0
2~
.J
~8
Time of doy (h) FIG. 5. Original double-plotted recordings of locomotion of three male canaries (Nos. 2, 6, and 8) under various light regimes of Experiment 2 from March 1 to April 27 (upper diagrams) and from A u g u s t 1 to Ocober 26 (lower diagrams). Lines indicate onsets and ends of light time. Mean illumination levels were 55:24 lx in L D and 24 lx in LL. Indices on the right margin refer to canary No. 8.
rhythmia in LL facilitated entrainment to low-amplitude LD cycles. It w a s f o u n d e a r l i e r i n f r i n g i l l i d s (Fringilla coelebs, Pyrrhula that temporal dissociation (splitting) of two circadian activity components after inversion of the LD cycle accelerates the rate of reentrainment (25). Comparable results were reported o f t h e d i u r n a l S i b e r i a n c h i p m u n k , Eutamias sibiricus ( 2 3 , 2 5 ) .
pyrrhula)
TABLE
More recently, differences in responses to daily melatonin injections were observed in nocturnal rats (5). Individuals that lengthen r after being transferred from total darkness to continuous light (LL) did not entrain to the melatonin zeitgeber, although individuals with disrupted patterns of activity or arrhythmia in LL did entrain. This remarkable congruence in responses to different zeitgeber modalities of birds and diurnal as well as
100 %
3
CIRCADIAN PERIOD (r) 1N LL, ENTRAINMENT AT T 25.0, AND OCCURRENCE OF WING FEATHER MOLT IN MALE CANARIES DURING EXPERIMENT 2 Test No.
g,
1
2
T
o
E 8
T
Bird No.
(h)
E
M
1 la 2 3 4 5 6 7 8 9
23.3
+
-
23.2 23.7 ar 23.5 23.7 23.3 ar ar
+ + + + (+) +
-
5 8
(h)
ar 23.2* ar 23.2 ar 23.2 23.6 ar 23.4
E
+ (+) + + + + + + +
M
+ + + + + +
ar = arrhythmia, E + = entrained, E ( + ) = relatively entrained, E = not entrained, M + = molt, M - = no molt. * Measured after T 25.0.
3
o
"6
50
~
9 6 7 4 2
0
[ March
I August
FIG. 6. Percentage change in the amount of locomotion per circadian period, measured in LL, between March and A u g u s t (Experiment 2). N u m b e r s refer to i n d i v i d u a l s (see Table 3).
CIRCADIAN ENTRAINMENT AND REPRODUCTION
nocturnal mammals with disrupted or arrhythmic activity patterns in LL strongly suggests that changes in the organization of the circadian system influence the ability and the range of entrainment of the rhythms to external and internal zeitgebers. Based on experiments on greenfinches (Carduelis chloris) and house sparrows (Passer domesticus), a hypothesis was proposed that explained photorefractoriness by seasonal differences in the circadian organization of the photoperiodic timing mechanism (18-20). Different states of the annual reproductive cycle are accompanied, or caused, by changes in phase relations between circadian rhythms of photosensitivity that trigger secretion of LH and the follicle stimulating hormone (FSH), both controlling testicular development (21). Although this specific hypothesis was not substantiated by experiments on domestic canaries (31), phase shifts between photoinducible phases of sensitivity rhythms regulating other hormones are still likely (14,32). The observed changes in the organization of the circadian system and related changes in entrainment properties may be associated with alterations of the phase relations between circadian rhythms within the endocrine and neuroendocrine systems as well as between behavioral rhythms. Such changes may derive from different causes: for example, from temporary desynchronization of subsidiary oscillators as a result of uncoupling from the main pacemaker(s), or from a reduction of the degree (or loss) of self-sustainment of the circadian pacemaker, that is, by a decrease (or inhibition) of periodic release of melatonin from the pineal (2,3). Both possibilties extend the range of entrainment of circadian oscillators with an LD zeitgeber (1,38). The role of hormones controlling these changes still remains to be clarified. In the majority of birds tested in the two experiments, the periods and phases of the rhythms of locomotor activity, feeding, and vocalization were similar in LL and under the LD regimes.
809
In less than 50% of all birds tested, activity time (a) of the locomotor rhythm was between 1 and 5 h longer than that of the feeding rhythm. Activity time of the rhythm of vocalization (song and calls) was shorter than that of locomotion or feeding, and its temporal pattern was more variable in most birds. Stable phase relationships between rhythms of locomotor activity and feeding have been reported from finches, sparrows, and pigeons exposed to a variety of experimental light conditions, suggesting that timing of both functions is controlled by the same pacemaker (4), but independent control by two pacemakers is more likely (8,27). Close synchrony between locomotor activity and vocalization (crowing) was also observed in Japanese quail, Coturnix coturnix japonica (37). Entrainment patterns of the rhythms of locomotion, feeding, and vocalization, estimated by their periodograms, were also similar in most birds of this study. Some exceptions, however, are instructive with respect to the organization of the circadian system. Significant differences in the periodograms between locomotion and feeding were found in birds Nos. 7, 8, and 9 of Experiment 1. In canaries Nos. 7 and 8 only one significant peak was present at ca. 25.0 h (T 25.0) or 25.5 h (T 25.5) in the periodograms for feeding, and three or two significant peaks, respectively, were shown in the periodograms for locomotion. In canary No. 9, locomotion was arrhythmic during molt and feeding was still rhythmic. After the low-amplitude LD cycle was reintroduced, the rhythmic pattern of locomotion immediately reappeared (see Figs. 2 and 4). These findings support previous hypotheses: (a) that behavioral functions (e.g., locomotion, feeding) are independently controlled by separate oscillators (or groups of ocscillators), and (b) that internal desynchronization of these subsidiary oscillators may occur as a result of uncoupling from the main pacemaker(s) (11,34).
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22. Nicholls, T. J. Changes in plasma LH levels during a photoperiodically controlled reproductive cycle in the canary (Serinus canarius). Gen. Comp. Endocrinol. 24:442-445; 1974. 23. Pohl, H. Die Aktivit~itsperiodik yon zwei tagaktiven Nagern, Funambulus palmarum und Eutamias sibiricus, unter Dauerlichtbedingnngen. J. Comp. Physiol. 78:60-74; 1972. 24. Pohl, H. Circadian rhythms of metabolism in Cardueline finches as function of light intensity and season. Comp. Biochem. Physiol. [A] 56:145-153; 1977. 25. Pohl, H. Comparative aspects of circadian rhythms in homeotherms: Re-entrainment after phase shifts of the zeitgeber. Int. J. Chronobiol. 5:493-517; 1978. 26. Pohl, H. Properties of the circadian clock of the redpoll Carduelis flammea. I. Stability of period and ranges of entrainment. Physiol. Zool. 53:186-198; 1980. 27. Pohl, H. Properties of the circadian clock of the redpoll Carduelis flammea. II. Overt functions in relation to the pacemaker. Physiol. Zool. 53:199-209; 1980. 28. Pohl, H. Circadian pacemaker does not arrest in deep hibernation: Evidence for desynchronization from the light cycle. Experientia 43:293-294; 1987. 29. Pohl, H. Optimal light sensitivity--its significance for measurement of daylength. Second Meeting of SRBR, Abstract; 1990. 30. Sokolove, P. G.; Bushell, W. N. The chi-square periodogram: Its utility for analysis of circadian rhythms. J. Theor. Biol. 72:131-160; 1978. 31. Storey, C. R. Circadian photosensitivity in the photoperiodic control of gonadotropin secretion in the canary, Serinus canarius. Gen. Comp. Endocrinol. 30:198-203; 1976.
POHL
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