Entrainment of the rat circadian clock controlling the pineal N-acetyltransferase rhythm depends on photoperiod

226

Brain Research, 584 (1992) 226-236 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 17910

Entrainment of the rat circadian clock controlling the pineal N-acetyltransferase rhythm depends on photoperiod Martina Humlovfi and Helena Illnerovfi Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czech and Slovak Federal Republic) (Accepted 25 February 1992)

Key words: Circadian pacemaker; Entrainment; Photoperiod; Pineal; N-Acetyltransferase rhythm; Rat

Entrainment of the circadian clock as a function of time when a light stimulus is presented has been studied in detail while little attention has been paid to a role photoperiod may play in the resetting. To find out whether and how photoperiod affects the entrainment, resetting of the rat circadian pacemaker by delays in the evening light offset and by advances in the morning light onset, respectively, was studied in rats maintained either under a short photoperiod, with 8 h of light and 16 h of darkness per day (LD 8 : 16) or under a long, LD 18 : 6 photoperiod. To assess phase shifts of the clock, the suprachiasmatic nucleus controlled rhythm in the pineal N-acetyltransferase (NAT), namely the time of the evening NAT rise and the time of the morning decline, were followed. One day after a delay in lights off, on LD 8:16 the NAT rhythm with a normal amplitude was retained following longer delays of the light offset and the maximum phase delay of the NAT rise was 3 times larger than on LD 18: 6. One day after an advance in lights on, the NAT decline was phase advanced under both photoperiods; on LD 8 : 16 the maximum shift was 3 times as large as on LD 18:6. On LD 8:16, the NAT rise was not shifted after shorter advances in lights on and became phase delayed only when the light onset was brought forward to before midnight while on LD 18:6 the NAT rise was phase delayed after any, even a mere 1 h, advance in lights on. The data show that magnitude and direction of phase shifts of the NAT rhythm depend not only on the time of light presentation but on photoperiod as well. Difference in resetting of the rhythm under various photoperiods may reflect photoperiod-dependent changes of an underlying pacemaker.

INTRODUCTION T h e m a m m a l i a n c i r c a d i a n clock is l o c a t e d in t h e suprachiasmatic nucleus (SCN) of the hypothalam u s 23'27'28. In a n o n - p e r i o d i c e n v i r o n m e n t , t h e clock f r e e - r u n s with its own p e r i o d , r , close to, b u t n o t e q u a l to 24 h 1. U n d e r n a t u r a l daylight, t h e clock is synchron i z e d to t h e 24-h d a y by daily cycles in light a n d d a r k n e s s : light in e a c h cycle resets t h e c i r c a d i a n p a c e m a k e r , e i t h e r by p h a s e a d v a n c i n g it w h e n r is l o n g e r t h a n 24 h o r by p h a s e d e l a y i n g it w h e n r is s h o r t e r t h a n 24 h 25. N u m e r o u s studies show t h a t m a g n i t u d e a n d d i r e c t i o n o f p h a s e shifts d e p e n d o n t h e t i m e w h e n a light stimulus is a p p l i e d . T h e s t u d i e s w e r e p e r f o r m e d mostly on a n i m a l s f r e e - r u n n i n g in c o n s t a n t d a r k n e s s 3'4'5 a n d occasionally on t h o s e m a i n t a i n e d u n d e r a lighting r e g i m e with 12 h o f light a n d 12 h o f d a r k n e s s p e r day ( L D 12 : 12) 12'~5'~6. R e c e n t d a t a suggest t h a t r e s e t t i n g o f t h e c i r c a d i a n clock m a y d e p e n d n o t only on t h e t i m e o f

light p r e s e n t a t i o n , b u t o n t h e d a y length, i.e. on p h o t o p e r i o d , as well 14 (Elliot a n d P i t t e n d r i g h , p e r s o n a l communication). T h e S C N p a c e m a k e r drives, b e s i d e s o t h e r rhythms, also t h e r h y t h m in t h e r a t p i n e a l N - a c e t y l t r a n s f e r a s e ( E C 2.3.1.87) 20 ( N A T ) which c o n t r o l s t h e r h y t h m i c m e l a t o n i n p r o d u c t i o n 17A9'21. Oscillatory i n f o r m a t i o n s p r o c e e d f r o m t h e S C N to s y m p a t h e t i c n e r v e t e r m i n a l s in t h e p i n e a l via n e u r o n a l pathways. A c c o r d i n g to t h e p a c e m a k e r ' s p r o g r a m m e , n o r e p i n e p h r i n e is r e l e a s e d at n i g h t in d a r k n e s s a n d t h r o u g h a d r e n e r g i c r e c e p t o r s a n d c A M P system i n d u c e s a n d activates N A T a n d i n c r e a s e s t h u s t h e m e l a t o n i n p r o d u c t i o n 19. T h e N A T r h y t h m is suitable for c i r c a d i a n studies as it exhibits a high a m p l i t u d e a n d has two w e l l - d e f i n e d p h a s e m a r k ers, n a m e l y t h e t i m e o f t h e e v e n i n g N A T rise a n d t h e t i m e o f t h e m o r n i n g N A T d e c l i n e 18. T h e r h y t h m p e r se is p h o t o p e r i o d d e p e n d e n t : t h e p h a s e r e l a t i o n s h i p bet w e e n t h e e v e n i n g N A T rise a n d t h e m o r n i n g d e c l i n e

Correspondence: H. Illnerovfi, Institute of Physiology, Czechoslovak Academy of Sciences, Videhskfi 1083, 14220 Prague 4, Czechoslovakia.

227 Phase shifting treatments

which determines the period of the elevated NAT activity and hence of the high nocturnal melatonin production, is short in long- and long in short-days s'9'13. A gradual extension of the phase relationship after a change from a long to a short photoperiod 1°'11 suggests that memory on long-days may be stored in the clock itself, i.e. properties of the pacemaker may change according to environmental lighting regimes. The present study was undertaken to find out whether and how photoperiod affects entrainment of the circadian pacemaker. Resetting of the clock after delays in the evening light offset or after advances in the morning light onset in short-days was compared with that in long-days. To assess phase shifts of the pacemaker, the NAT rhythm was used as hands of the clock.

When rats were subjected to a delay in the evening light offset, lights were switched off later than usual and from that time onwards the rats were maintained in darkness; control rats experienced the evening lights-off at the same time as usual. The NAT rhythm was followed during the same (night 0) and the next (night 1) night (Fig. 1B). When rats were subjected to bringing forward the morning light onset, they experienced the evening light offset at the usual time but the light onset occurred earlier; control rats experienced the morning lights-on at the usual time. The next day, lights were turned off already at 14.00 h in order to allow expression of an eventual advance of the evening NAT rise and the NAT rhythm was followed in the subsequent darkness (Fig. 1C, night 1).

Assay of N-acetyltransferase Pineal glands were removed rapidly and stored in Petri dishes on solid CO2 until assayed for NAT activity. Within 48 h of tissue collection, each gland was homogenized in 100 ~1 of 0.1 mol/l sodium phosphate buffer, pH 6.8, containing 0.25 mmol/l [1-14C]acetylCoA (spec. act. 37 MBq/mmol) and 10 mmol/l tryptamine, and NAT activity was determined by a modification24 of the method of Deguchi and Axelrod6. Blanks with boiled homogenates were processed simultaneously. [1-14C]acetylCoA (2.07 GBq/mmol) was purchased from the Radiochemicai Centre (Amersham, UK). Results were expressed as nanomoles of Nacetyltryptamine formed per 1 mg of pineal tissue per h (nmol. mg- 1 •h - l ) . Baseline day values were within the range of 0.05-0.10 nmol.mg- 1. h - 1.

MATERIALS AND METHODS Animals We used 50-60-day-old male Wistar rats from our own breeding colony. Rats were housed at a temperature of 23 + 2°C and had free access to water and commercial food pellets. Animals were maintained either under a short, LD 8:16, photoperiod, with the lights on from 08.00 to 16.00 h or under a long, LD 18:6, photoperiod, with the lights on from 03.00 to 21.00 h, for at least 3 weeks prior to all experiments. The intensity of illumination provided by overhead 40 W Tesla fluorescent tubes was between 50 and 200 1 × depending on position of cages in an animal room. Before being killed in darkness, rats were exposed to a very faint red light for less than 1 rain prior to decapitation. Other animals in the room which were to be killed later that night or the next night were shielded from the faint red light by a black curtain.

Data analysis Data were analysed using one-way analysis of variance. The t-test with Bonferroni probabilities (BMDP Statistical Software, University of California, Los Angeles) was employed for the post hoc comparison, with a = 0.05 required for significance. Heterogeneity of variances was reduced by log transformation of the data. Phase shifts of the evening NAT rise were determined by comparing the time when NAT during its evening rise reached the activity of 3 nmol- m g - 1. h - 1 in experimental animals with that in control animals, unless stated otherwise. Similarly, phase shifts of the morning NAT decline were

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Fig. 1. Paradigm of phase shifting treatments. A: rats were maintained in LD 8:16 or in LD 18:6. B: part of the rats was subjected to a delay in the usual evening light offset and thereafter released into darkness. The NAT rhythm was followed in the subsequent darkness still during the same night (night 0) and one day later (night 1). C: another part of the rats was subjected to the usual evening light offset and later that night to an advance in the morning light onset. The next day, the rats were released into darkness already at 14.00 h and the NAT rhythm was followed in the subsequent darkness (night 1). Closed bars represent dark periods, open bars light periode. Thick arrows indicate direction of shifts, thin arrows the beginning of monitoring of the NAT rhythm and broken lines beginnings and ends of original dark periods.

228 determined by comparing the time when NAT during its morning decline reached the activity of 3 n m o l - m g - l . h -~ in experimental animals with that in control animals. The value of 3 nmol. rag- 1. h 1 was chosen arbitrarilylZ']3; it is about 30 times higher than the baseline day activity.

RESULTS

was compressed considerably (Fig. 3A). Alter a 4-h and a 6-h delay in the light offset, only a residual, phase-delayed rhythm was present; the NAT activity increased significantly above baseline values at 05.00 h, 06.00 h and 08.00 h after the 4-h delay and at 06.00 h and 09.00 h after the 6-h delay (Fig. 3B,C).

Response to a delay in the evening light offset during the same night In LD 8: 16, after an ll-h and even after a 12-h delay in the evening light offset, the NAT rhythm with a normal amplitude was still present in the subsequent darkness, though in a compressed waveform, as the morning NAT decline was delayed less than the evening rise (Fig. 2A,B). After a 14-h delay in lights off, only a residual, low amplitude rhythm was expressed (Fig. 2C); the NAT rise occurred only close to the time of the decline and consequently a full rhythm could no more be expressed. After a 16-h delay in the evening light offset, no rhythm was retained (Fig. 2D). In LD 18:6, after a 2-h delay in lights off, only the evening NAT rise but not the morning decline was delayed in the subsequent darkness; consequently, the phase relationship between the rise and the decline

Response to a delay in the evening light offset the next night The next night after a 12-h (Fig. 4A) and a 14-h (Fig. 4B) delay of the evening light offset in LD 8 : 16, the evening NAT rise was phase delayed by 6.8 h and 6.4 h, respectively, and the morning decline by 4.6 h and 4.2 h, respectively, as compared with control rats (Fig. 4D). Hence the whole NAT rhythm was phaseshifted though its waveform was slightly compressed. Following a 16-h delay in lights off (Fig. 4C), the NAT rise was delayed by 8.4 h while the decline by only 2.6 h (Fig. 4D); consequently, the NAT rise occurred near the time of the decline and a full rhythm could not be expressed anymore. The next night after a 2-h (Fig. 5A) and a 4-h (Fig. 5B) delay of the evening light offset in LD 18:6, the

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Fig. 2. Response of the N-acetyltransferase rhythm under LD 8 : 16 to a delay in the evening light offset during the same night. Rats maintained in LD 8: 16 were either subjected to the usual evening light offset at 16.00 h (full symbols) or to a delay in the light offset till 03.00 h (A), 04.00 h

(B), 06.00 h (C) and 08.00 h (D), respectively (open symbols). Thereafter, they were released into darkness and the NAT rhythm was followed immediately in the subsequent darkness (night 0). Lines under the abscissa indicate periods of darkness. Data are expressed as means + ( - )S.E.M. of 4-8 animals. When S.E.M. are omitted, they were lower than 0.2 nmol. mg- 1. h - i.

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Fig. 3. Response of the N-acetyltransferase rhythm under LD 18: 6 to a delay in the evening light offset during the same night. Rats maintained in LD 18:6 were either subjected to the usual evening light offset at 21.00 h (full symbols) or to a delay in the light offset till 23.00 h (A), 01.00 h (B) and 03.00 h (C), respectively (open symbols). For further details see legend to Fig. 2.

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TIME( H ) Fig. 4. Response of the N-acetyltransferase rhythm under LD 8:16 to a delay in the evening light offset during the next night. Rats maintained in LD 8:16 were either subjected to the usual evening light offset at 16.00 h (full symbols) or to a delay in the light offset till 04.00 h (A), 06.00 h (B) and 08.00 h (C), respectively (open symbols). Thereafter, they were released into darkness and the NAT rhythm was followed during the next night (night 1). In D, phase delays of the evening NAT rise and of the morning decline, determined at the level of 3 n m o l . m g - 1 , h - 1 of NAT activity from A - C , are plotted as a function of the time of the light offset during the previous night. Lines under the abscissa indicate dark periods during the previous night. Data are expressed as m e a n s + ( - ) S . E . M , of 4-12 animals. When S.E.M. are omitted, they were lower than 0.25 nmol. m g - i. h - ].

230 evening NAT rise was phase-delayed by 1.0 h and 1.6 h, respectively, while the morning decline by 2.3 h and 3.3 h, respectively, as compared with control rats (Fig. 5F). Following a 6-h delay in lights off (Fig. 5C), phase shifts of the NAT rhythm were inconsistent. The rhythm exhibited two peaks; during the second peak, the activity at 07.00 h and 09.00 h was significantly higher than baseline values. The first NAT rise was phase-delayed by 2.9 h as compared with control rats. After an 8-h delay in the evening light offset (Fig. 5D), the evening NAT rise was phase-delayed by 3.0 h while the morning decline was not delayed at all; consequently, the NAT rise occurred near the time of the decline and the full rhythm could no more be expressed. After a 10-h delay in the light offset (Fig. 5E), the rhythm was not retained anymore; however, at 12.00 h, the NAT activity was significantly higher than baseline levels.

next night the morning NAT decline was phase-advanced while the evening rise was not phase shifted markedly; consequently, the phase relationship between the rise and the decline was compressed considerably. After a 9-h (Fig. 6E) and a 12-h (Fig. 6F) advance in lights on, the next night the morning NAT decline was phase-advanced as well but the evening rise was phase-delayed. Bringing forward the light onset to before midnight had thus a dual effect on the NAT rhythm, i.e. a phase advancing one on the NAT decline and at the same time a phase delaying one on the NAT rise. Due to the dual effect, the rise and the decline might occur close to one another and eventually amplitude of the NAT rhythm diminished. With the increasing advance in the morning light onset, the phase advances of the morning NAT decline increased at first as well, to almost 5 h; however, when the light onset was brought forward considerably to before midnight, the phase advance might decrease (Fig. 6F, Fig. 9B). At that time, the evening NAT rise became phase-delayed by more than 4 h in comparison with control rats and the phase delaying effect of a highly advanced light period might prevail.

Response to an advance in the morning light onset during the next night In LD 8 : 16, after a 3-h (Fig. 6A, 5-h (Fig. 6B) and 7-h (Fig. 6C) advance in the morning light onset, the

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Fig. 5. Response of the N-acetyltransferase rhythm under LD 18 : 6 to a delay in the evening light offset the next night. Rats maintained in LD 18:6 were either subjected to the usual evening light offset at 21.00 h (full symbols) or to a delay in the light offset till 23.00 h (A), 01.00 h (B), 03.00 h (C), 05.00 h (D) and 07.00 h (E), respectively (open symbols). Thereafter, they were released into darkness and the N A T rhythm was followed the next night (night 1). In F, phase delays of the evening N A T rise and of the morning decline, determined at the level of 3 nmol- m g - i. h - i of N A T activity from A - D , are plotted as a function of the time of the light offset during the previous night (full symbols): The open symbol indicates the phase shift of the first and the crossed one of the second decline of N A T activity after a delay in lights off till 03.00 h (read from C). For further details see legend to Fig. 4.

231 morning light onset, the phase delaying effect became more pronounced and phase delays of the evening NAT rise became larger; phase advances of the morning NAT decline increased at first as well, but when the light onset was brought forward to before midnight, they decreased again (Fig. 9A). At that time, the phase delaying effect of a highly advanced light period might already prevail. Due to the dual effect of an advanced light onset, the phase relationship between the evening NAT rise and the morning decline was compressed as compared with control rats; eventually the NAT rise occurred so close to the time of the NAT decline that diminution of the rhythm amplitude ensued. However, after all the advances in the light onset, the NAT rhythm was retained: at 00.00 h, 01.00 h and 02.00 h, NAT activity was always significantly higher than baseline values. On long days, not only an advance in the light onset but also a short light pulse exhibited the dual effect on the NAT rhythm. After a 30-min light pulse applied before (Fig. 10A) or past (Fig. 10B) midnight, the next night the evening NAT rise was phase-delayed by about

Following an 8-h advance in lights on, i.e. when the light onset was brought forward just to the middle of the night, the next day the evening NAT rise was once phase-advanced by 1 h and once not phase-shifted markedly (Fig. 6D). After bringing forward the light onset to only 00.30 h, the next day the evening NAT rise was phase-advanced by 0.9 h (Fig. 7); at 19.45 h and at 20.40 h, the NAT activity in experimental animals was significantly higher than in control ones. In LD 18:6, after any, even after a mere 1-h advance in the morning light onset, the next night not only the morning NAT decline was phase-advanced, but at the same time the evening NAT rise was phasedelayed (Fig. 8A-E). In such a long photoperiod, any bringing forward of the morning light onset had a dual effect on the NAT rhythm, i.e. a phase advancing one on the morning NAT decline and at the same time a phase delaying one on the evening NAT rise. Only when the lights were left on the whole night, with no dark period whatsoever, the next night just the evening NAT rise was phase-shifted but not the morning decline (Fig. 8F). With the increasing advance in the

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Fig. 6. Response of the N-acetyltransferase rhythm under LD 8:16 to an advance in the morning light onset during the next night. Rats maintained in LD 8: 16 were subjected to the evening light offset at the usual time of 16.00 h and later that night either to the morning light onset at the usual time of 08.00 h (full symbols) or to an advance in the light onsot to 05.00 h (A), 03.00 h (B), 01.00 h (C), 00.00 h (D), 23.00 h (E) and 20.00 h (F), respectively, (open symbols). The next day, light was turned off already at 14.00 h in order to allow expression of an eventual advance of the evening NAT rise, and the NAT rhythm was followed in the subsequent darkness (night 1). Bringing forward the light onset to 00.00 h was performed twice (D, open circles and stars). Lines under the abscissa indicate the times when dark periods ended during the previous night. Data are expressed as means + (-)S.E.M. of 4 animals; when S.E.M. were omitted, they were lower than 0.3 nmol-mg-1, h-1.

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Fig. 7. Response of the evening N-acetyltransferase rise under LD 8 : 16 to an advance in the light onset to 00.30 h. Rats maintained in LD 8:16 were subjected to the usual evening light offset and later that night either to the usual morning light onset at 08.00 h (full symbols) or to an advance in the light onset till 00.30 h (open symbols). The next day, light was turned off at 14.00 h and the evening NAT rise was followed. Data are expressed as m e a n s + ( - ) S . E . M . of 4 animals; when S.E.M. are omitted, they were lower than 0.1 n m o l ' m g - t ' h -I.

0.7 h and the morning decline was phase-advanced by 0.7 h (A) and 0.9 h (B), respectively; the phase relationship between the NAT rise and the decline was compressed. DISCUSSION The results indicate that resetting of the circadian NAT rhythm depends indeed on photoperiod. Under a short, LD 8:16, photoperiod, light extended into the night hours phase delays not only the evening NAT rise during the same night but the morning decline as well, though to a lesser extent than the rise, whereas under a long, LD 18:6, photoperiod, only the evening NAT rise but not the morning decline is delayed during the same night. Consequently, under the latter photoperiod, the NAT rise may occur just near or at the time of the decline and amplitude of the NAT

rhythm dampens or disappears after a 4-h or a longer delay in the light offset while under LD 8 : 16 a normal amplitude is still retained after a 12-h delay in lights off. The next night, the NAT rhythm with the normal amplitude is still retained and phase-shifted after a 14-h delay in lights off under LD 8:16, but no more after an 8-h delay under LD 18:6. It appears that the critical delay in the light offset for retaining of the normal NAT rhythm amplitude or for its temporal lowering or even disappearance is a 14- to 16-h delay under LD 8:16 but only a 6- to 8-h delay under LD 18 : 6. One day after extension of a light period into the night hours the longest phase delay of the NAT rise is more than 8 h under LD 8 : 16 but 3 h only under LD 18:6. In accordance with our previous data ~4, under the short photoperiod, magnitude of phase shifts is larger than under the long photoperiod and the NAT rhythm may adjust more rapidly to long delays in the evening light offset. Even greater differences in resetting of the NAT rhythm under various photoperiods exist after an advance in the morning light onset. The maximum phase advance of the morning NAT decline is 1.5 h under LD 18 : 6, but three times as much, around 5.0 h, under LD 8 : 16. While under LD 8 : 16 only bringing forward the light onset to before midnight but not a shorter advance in lights on has a dual effect on the NAT rhythm the next day, i.e. a phase advancing one on the NAT decline and at the same time a phase delaying one on the NAT rise, under LD 18: 6, any, albeit it a 1-h advance only in lights on, has the dual effect. Even a 30-min light pulse applied before or past midnight has the dual effect on the NAT rhythm. Actually, under such a long photoperiod, a 1-min light pulse presented one hour before or one hour after the middle of the night appears to have the dual effect as well ~4. Such a dual effect of a 1-min light pulse has never been observed under a shorter, LD 12:12, photoperio d 8"12'13'16. Resetting of the NAT rhythm by an advance in the light onset under various photoperiods may thus differ not only in magnitude but also in direction of transient phase shifts. Following an advance in the light onset, the next day the evening NAT rise is phase-delayed under LD 18 : 6 and either not phase-shifted or phase-delayed under LD 12:12 ~s'18, but never phase-advanced. Under LD 8:16, however, a narrow window for phase advancing of the NAT rise within one day may exist: when the light onset is brought close to the middle of the night, the next day the evening NAT rise may be slightly phase-advanced. Under such a short photoperiod, the NAT rhythm pattern may be completely decompressed and the rhythm may be entrained just by the phase-ad-

233 vancing effect of the morning light but not by the evening light 8'9'14. In the decompressed state of the rhythm, a slight advance of the NAT rise may occur. In humans during short winter days, a single exposure to a very early morning bright light may also, within one day, phase advance not only the morning melatonin decline but the evening rise as well 2. Difference in resetting of the NAT rhythm under various photoperiods is not due to a masking effect of light on the rhythm, i.e. to a suppressive effect of light on NAT activity7-9'22. Even under the long, LD 18:6, photoperiod, the NAT profile is shaped by the entraining rather than by the suppressive effect of light: the morning NAT decline occurs a few minutes ahead of the light onset 8. Following a delay in the evening light offset, animals were released into darkness; hence neither the NAT decline during the same night nor the NAT rise and decline during the next night might be affected by a masking effect of light• Following an advance in the morning light onset, the next day animals were released into darkness earlier than usual, i.e.

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at 14.00 h, in order to allow expression of an eventual advance of the NAT rise. The maximum phase advance of the morning NAT decline was 1.5 h on LD 18 : 6 and 5.0 h on LD 8:16. If the NAT rise had been also advanced by the same amount, it would have occurred 7.5 h after the light offset on LD 18 : 6 and 2 h after the light offset on LD 8 : 16. Hence, in neither case could the eventual NAT rise be suppressed by light. At 14 h, at the time of the advanced lights off, control and experimental animals might be in different subjective times due to the previous exposure of the experimental animals to an advance in lights on. Therefore, it cannot be excluded that the end of the light exposure might contribute to the final phase shift observed in the experimental rats. This seems, however, unlikely, as the light offset preceded the actual NAT rise by more than 7 h 8'14. The difference in resetting of the NAT rhythm under various photoperiods is neither due to a changed sensitivity of the pineal system to norepinephrine released according to the pacemaker's programme at night in darkness from sympathetic nerve

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Fig. 8. Response of the N-acetyltransferase rhythm under LD 18:6 to an advance in the morning light onset the next night. Rats maintained in LD 18:6 were subjected to the usual evening light offset at 21.00 h and later that night either to the usual morning light onset at 03.00 h (full symbols) or to an advance in the light onset to 02.00 h (A), 01.00 h (B), 00.00 h (C), 23.00 h (D) and to 22.00 h (E), respectively; eventually, light was left on the whole night (F) (open symbols). The next day, light was turned off already at 14.00 h and the NAT rhythm was followed in the subsequent darkness (night 1). Lines under the abscissa indicate dark periods during the previous night. Data are expressed as means + ( - ) S . E . M . of 4 animals. When S.E.M. are omitted, they were lower than 0.3 nmol- m g - 1. h - 1.

234 endings in the pineal ]9. On long days, animals experience less darkness and should be more sensitive and respond more to norepinephrine by the NAT activity rise 2°'29 if the neurotransmitter were actually released in sufficient quantity; however, diminution of the NAT rhythm amplitude occurs more easily just in the long days. Rather, the difference in entrainment under various photoperiods might be due to photoperiod-dependent changes in the pacemaker function. The data on the adjustment of the NAT and melatonin rhythms to a change from a long to a short photoperiod suggest that memory on long days may be actually stored in the pacemaker ]0.]]. Entrainment of the NAT rhythm under various photoperiods may thus reflect entrainment of the changed pacemaker itself. Occasionally, resetting of the NAT rhythm proceeds via a temporal diminution of amplitude. Such a diminution or even disappearance occurs when the NAT rise and the decline become so close that the rhythm cannot be fully expressed. This may happen in two ways. First, the evening NAT rise may be phase-delayed to a much larger extent than the morning decline, as is the case, for example, one day after a 16-h

delay in the light offset under LD 8:16 or one day after an 8-h delay in lights-off under LD 18 : 6. Second, the morning NAT decline may be phase-advanced while the evening NAT rise may be phase delayed as is the case, for example, one day after a 12-h advance in the light onset under LD 8:16 or one day after a 4-h advance in lights on under LD 18:6; the response of the NAT rise to such a long advance in the light onset may thus be temporarily antidromic. In conclusion, resetting of the circadian rhythm in the rat pineal NAT activity and of its underlying pacemaker under a short photoperiod differs from that under a long photoperiod in magnitude of phase shifts, in ability to adjust to long delays in the evening light offset within a short time and in responses to advances in the morning light onset. The data strongly suggest that not only the time of light presentation may be important in resetting of the circadian pacemakei" but a photoperiod under which animals are maintained and a state of the pacemaker as well. Acknowledgements. We thank Mrs. Irena Slavikovfi for her excellent technical assistance, Mrs. Jana Koldovfi for kindly typing the

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Fig. 9. Phase shifts of the evening N A T rise and of the morning N A T decline under LD 18:6 (A) and LD 8:16 (B) the next night after an advance in the light onset. Phase shifts (full symbols) determined from Figs. 6 and 8 at the level of 3 nmol. m g - z. h - 1 of N A T activity, are plotted as a function of time when light was turned on during the previous night; + indicates phase advances, - phase delays. The open symbols in A represent hypothesized phase shifts which would be determined from Fig. 8D if the evening N A T rise and the morning decline in the shifted rats ran in parallel with those in control rats. T h e open symbol and the star in B represent phase shifts read from two different experiments in Fig. 6D; the crossed symbol read from Fig. 6F may not represent the true phase shift. Lines u n d e r the abscissa indicate dark periods during the previous night corresponding to inidividual phase shifts.

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Fig. 10. Response of the N-acetyltransferase rhythm under LD 18:6 to a 30-min light pulse applied before (A) or past (B) midnight. Rats maintained in LD 18:6 were subjected to a 30-min light pulse between 23.00 h and 23.30 h (A) and between 00.130 h and 00.30 h (B), respectively (open symbols) or were left unpulsed (full symbols), thereafter were released into darkness and the NAT rhythm was followed the next night. Data are expressed as means + ( - )S.E.M. of 4-12 animals; when S.E.M. are omitted, they were lower than 0.15 nmol- m g - 1. h - 1. Bars under the abscissa indicate the dark periods during the previous night.

manuscript and Mrs. JiHna Slejmarov~ for managing the animal care facilities. The work was supported by the Czechoslovak Academy of Sciences Grant 71118.

8 9

REFERENCES

1 Aschoff, J., Freerunning and entrained circadian rhythms. In J. Aschoff (Ed.), Handbook of Behacioral Neurobiology, Vol. 4, Biological Rhythms, Plenum Press, New York London, 1981, pp. 81-93. 2 Bure~ovfi, M., Dvo~ikov~i, M., Zvolsk~, P. and Illnerovfi, H., Early morning bright light phase advances the human circadian pacemaker within one day, Neurosci. Lett., 121 (1991) 47-50. 3 Daan, S. and Pittendrigh, C.S., A functional analysis of circadian pacemaker in nocturnal rodents: II. The variability of phase-response curves, J. Comp. Physiol., 106 (1976) 252-266. 4 Davis, F.C., Darrow, J.M. and Menaker, M., Sex differences in the circadian control of hamster wheel-running activity, Am. J. Physiol., 244 (Reg. Integr. Comp. Physiol. 13) (1983) R93-R105. 5 De Coursey, P.J., Daily light sensitivity rhythm in a rodent, Science, 131 (1960) 33-35. 6 Deguehi, T. and Axelrood, J., Sensitive assay for serotonin Nacetyltransferase activity in the rat pineal, Anal Biochern., 50 (1972) 174-179. 7 Deguchi, T. and Axelrood, H., Control of circadian change in serotonin N-acetyltransferase activity in the pineal organ by the

10

11

12

13

14

15

/3-adrenergic receptor, Proc. Natl. Acad. Sci. USA, 69 (1972) 2547-2550. Illnerovfi, H., Circadian Rhythms in the Mammalian Pineal Gland, Academia, Prague, 1986. Illnerov~i, H., Entrainment of mammalian circadian rhythms in melatonin production by light, Pineal Res., 6 (1988) 173-217. IIInerovfi, H., Hoffmann, K. and Van~ek, J., Adjustment of pineal melatonin and N-acetyltransferase rhythms to change from long to short photoperiod in the Djungarian hamster Phodopus sungorus, Neuroendocrinology, 38 (1984) 226-231. Illnerovfi, H., Hoffmann, K. and Van~ek, J., Adjustment of the rat pineal N-acetyltransferase rhythm to change from long to short photoperiod depends on the direction of extension of the dark period, Brain Res., 362 (1986) 403-408. Illnerovfi, H. and Van~ek, J., Two-oscillator structure of the pacemaker controlling the circadian rhythm of N-acetyltransferase in the rat pineal gland, J. Comp. Physiol. A, 145 (1982) 533-548. Illnerovfi, H. and Van~ek, J., Complex control of the circadian rhythm in N-acetyltransferase activity in the rat pineal gland. In J. Aschoff, S. Daan and G. Groos (Eds.), Vertebrate Circadian Systems: Structure and Physiology, Springer-Verlag, Berlin, 1982, pp. 285-296. Illnerovfi, H. and Van~ek, J., Entrainment of the circadian rhythm in rat pineal N-acetyltransferase activity under extremely long and short photoperiods, J. Pineal Res., 2 (1985) 67-78. Illnerovfi, H. and Van~ek, J. Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity by prolonged periods of light, J. Comp. Physiol. A, 161 (1987) 495-510.

236 16 lllnerovfi, H. and Van~Eek, J., Dynamics of discrete entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity during transient cycles, J. Biol. Rhythms, 1 (1987) 95-108. 17 lllnerov~, H., Van~ek, J. and Hoffmann, K., Regulation of the pineal melatonin concentration in the rat (Rattus norvegicus) and in the Djungarian hamster (Phodopus sungorus ), Comp. Biochem. Physiol., 74A (1983) 155-159. 18 Illnerovfi, H., Van~Eek, J. and Hoffmann, K., Different mechanisms of phase delays and phase advances of the circadian rhythm in rat pineal N-acetyltransferase activity, J. Biol. Rhythms, 4 (1989) 187-200. 19 Klein, D.C., The pineal gland: a model of neuroendocrine regulation. In S. Reichlin, R.J. Baldessarini and J.B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 303-327. 20 Klein, D.C. and Moore, R.Y., Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: control by the retinohypothalamic tract and the suprachiasmatic nucleus, Brain Res., 174 (1979) 245-262. 21 Klein, D.C. and Weller, J.L., Indole metabolism in the pineal gland. A circadian rhythm in N-acetyltransferase activity, Science, 169 (1970) 1093-1095. 22 Klein, D.C. and Weller, J.L., Rapid light-induced decrease in pineal serotonin N-acetyltransferase, Science, 177 (1972) 532-533.

23 Moore, R.Y. and Eichler, V.B., Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Res., 42 (1972) 201-206. 24 Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K. and Marks, B.H., Blockade by oubain or elevated potassium ion concentration of the adrenergic and adenosine cyclic 3',5'-monophosphate induced stimulation of pineal serotonin N-acetyltransferase activity, MoL Pharmacol., 11 (1975) 241-255. 25 Pittendrigh, C.S., Circadian systems: entrainment. In J. Aschoff (Ed.), Handbook of Behavioral Neurobiology, Vol. 4, Biological Rhythms, Plenum Press, New York, 1981, pp. 95-124. 26 Romero, J.A., Zatz, M., Kebabian, J.W. and Axelrood, J., Circadian cycles in binding of 3H-alprenolol to/3-adrenergic receptor sites in the rat pineal, Nature, 258 (1975) 435-436. 27 Rusak, B. and Zucker, J., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 28 Stephan, F.K. and Zucker, J., Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions, Proc. Natl. Acad. Sci. USA, 69 (1972) 15831586. 29 Strada, S. and Weiss, B., Increased response to catecholamines of the cyclic AMP system of rat pineal gland induced by decreased sympathetic activity, Arch. Biochem. Biophys., 160 (1974) 197-204.