Neuroscience Research, 13 (1992) 147-153 © 1992 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/92/$05.00
147
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Resetting of the rat circadian clock after a shift in the light/dark cycle depends on the photoperiod Martina Humlovfi and Helena Illnerovfi Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 22 April 1991; Revised version received 19 August 1991; Accepted 19 October 1991) Key words: Rat; Circadian pacemaker; Resetting; Light/dark cycle; Photoperiod; Pineal; Melatonin; N-Acetyltransferase rhythm
SUMMARY Adjustment of the circadian clock to shifts in the light/dark (LD) cycle was assessed from the rat pineal N-acetyltransferase (NAT) rhythm which is controlled by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Re-entrainment to an 8-h delay in the LD cycle took more than 3 days in rats maintained under a regime with 18 h of light and 6 h of darkness per day (LD 18:6) whereas it was completed within 3 days in those maintained under LD 12: 12. Re-entrainment to an advance in the LD cycle proceeded through a transient diminution or almost disappearance of the NAT rhythm amplitude following a 5-h, 3-h and even a mere 2-h advance shift under LD 18:6, whereas no such diminution occurred under LD 12:12 even after a 5-h advance shift. Altogether, the data indicate that resetting of the circadian clock after shifts in the LD cycle depends on the photoperiod.
I n a n o n - p e r i o d i c e n v i r o n m e n t , the m a m m a l i a n c i r c a d i a n clock f r e e r u n s w i t h a p e r i o d c l o s e to, b u t n o t e q u a l to, 24 h I. U n d e r n a t u r a l d a y l i g h t or in a 24-h artificial l i g h t / d a r k ( L D ) cycle, t h e clock is s y n c h r o n i z e d to t h e 24-h day by p e r i o d i c c h a n g e s in light a n d d a r k n e s s Is. F o l l o w i n g shifts in the L D cycle w h i c h m i m i c t r a n s m e r i d i a n flights, t h e c i r c a d i a n p a c e m a k e r m u s t r e - a d j u s t to a n e w e n v i r o n m e n t a l time. In p r e v i o u s p a p e r s we s t u d i e d r e - e n t r a i n m e n t o f t h e clock a f t e r shifts in t h e L D cycle in rats m a i n t a i n e d u n d e r o n l y o n e p h o t o p e r i o d , n a m e l y in a r e g i m e w i t h 12 h o f light a n d 12 h o f d a r k n e s s p e r d a y ( L D 12 : 12) 4'6'7'12. A s r e c e n t d a t a suggest t h a t r e s e t t i n g of the c i r c a d i a n p a c e m a k e r m a y d e p e n d on t h e p h o t o p e r i o d s'9, we d e c i d e d to study r e - e n t r a i n m e n t of the c l o c k a f t e r shifts in t h e L D cycle in rats m a i n t a i n e d u n d e r a n o t h e r p h o t o p e r i o d , this t i m e u n d e r a l o n g e r one, in o r d e r to c o m p a r e the r e - a d j u s t m e n t u n d e r d i f f e r e n t p h o t o p e r i o d s . T o assess t h e p h a s e s o f t h e c i r c a d i a n p a c e m a k e r , we u s e d t h e overt r h y t h m in p i n e a l N - a c e t y l t r a n s f e r a s e ( N A T ) (arylalkylamine: acetyl C o A N - a c e t y l t r a n s f e r a s e , E C 2.3.1.87) activity which drives 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 in t h e rat 11"15, as h a n d s o f t h e clock. T h e N A T r h y t h m is c o n t r o l l e d by a p a c e m a k e r l o c a t e d in t h e s u p r a c h i a s m a t i c n u c l e i o f t h e h y p o t h a l a m u s 14, as a r e o t h e r c i r c a d i a n r h y t h m s 16,2°. A t night, a c c o r d i n g to t h e
Correspondence: RNDr. Helena Illnerovfi, DrSc., Institute of Physiology, Czechoslovak Academy of Sciences, Vide~sk~ 1083, 14220 Prague 4, Czechoslovakia.
148 pacemaker's program, norepinephrine is released from sympathetic nerve endings in thu pineal gland at a higher rate and induces and activates NAT ~. The NAT rhythm i.~ it good model for circadian studies, as it exhibits a high amplitude and has two well-defined phase markers, namely the time of the evening NAT rise and the time of the morning decline. We used 50-day-old male Wistar rats from our own breeding colony housed under LD 18 : 6 and at a temperature of 23 + 2°C for at least 3 weeks prior to the experiments. The rats had free access to water and commercial food pellets. Light provided by overhead 40 W Tesla fluorescent tubes was automatically turned on at 03.00 h and off at 21.00 h. A delay in the LD cycle was accomplished by lengthening of one light period, an advance by shortening of one light period or, alternatively, by shortening of one dark period (see legends to Figs. 2 and 3); thereafter the light and dark periods alternated again regularly. When rats were to be killed in darkness, they were exposed for less than 1 min prior to decapitation to a red light weaker than 1 lx; such a light does not affect nocturnal NAT activity 2~. Pineal glands were removed rapidly and stored in Petri dishes on solid CO 2. Within 48 h after decapitation, NAT activity was determined by a modification w of the method of Deguchi and Axelrod e. [1-~4C]Acetyl-CoA (2.07 G B q / m m o l ) was purchased from the Radiochemical Centre (Amersham, U.K.). Units of NAT activity were defined as nmol N-acetyltryptamine formed in 1 h / r a g of pineal tissue (nmol • mg ~ • h ~). Baseline day values were within the range of 0.05-0.15 n m o l - m g -~- h -L. Data were analysed using one-way analysis of variance. The t-test with Bonferroni probabilities (BMDP Statistical Software, University of California, Los Angeles) was used for the post hoc comparison, with a = 0.05 required for significance. Heterogeneity of variance was reduced by log transformation of the data. Folllowing an 8-h delay in the LD 18 : 6 cycle, no NAT rhythm was expressed during night 0, i.e. after the prolonged light period (Fig. 1A). However, already during the next night, i.e. during night 1, the NAT activity was significantly higher than the baseline day value at 08.00, 09.00, 10.00 and 10.45 h, respectively ( P < 0.001). Though the rhythm was already expressed during night 1 and thereafter, its amplitude was temporarily lowered. The phase relationship between the evening NAT rise and the morning decline was shortened as compared to the pre-shift pattern even after 3 days following the shift. After an 8-h delay in the LD 12 : 12 cycle, no significant diminution of the NAT rhythm amplitude occurs (Fig. 1B) ~2. The phase relationship between the NAT rise and the decline is shortened considerably only during night 0; during nights 1 and 2, the phase relationship is shortened just slightly, and within 3 days it is the same as before the delay shift. After a 5-h advance of the LD 18:6 regime, the NAT rhythm with a normal amplitude temporarily disappeared (Fig. 2A). However, during night 1, the NAT activity was significantly higher than the baseline day value at 18.00 h and 20.00 h, respectively ( P < 0.05) and during night 2 at 19.00 h ( P < 0.05), 20.00 h ( P < 0.01) and 21.00 h ( P < 0.001), respectively. During night 3, the amplitude in individual animals occasionally increased to a pre-shift level. After a 5-h advance in the LD 12:12 regime, the N A T rhythm persists with a normal amplitude, though at the beginning in a compressed waveform (Fig. 2B). The 5-h advance in the LD 18:6 cycle was accomplished in a different way from that in the LD 12 : 12 cycle, i.e. by shortening of one light instead of one dark period, in order not to limit the dark period during the shift to 1 h only. Thc difference in re-entrainment under the two photoperiods does not appear, however, to be due to various ways of advancing a light/dark regime: when the 5-h advance in the
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Fig. 1. The effect of an 8-h delay in the LD cycle on NAT rhythm. Rats adapted to LD 18:6, with lights on from 03.00 to 21.00 h (A), or to LD 12:12, with lights on from 06.00 to 18.00 h (B), were subjected to the 8-h delay in the LD cycle accomplished by lengthening one light period by 8 h. The NAT rhythm was followed before the delay shift (night -1), during the first dark period after prolongation of the light period (night 0), and during nights + 1, + 2 and + 3 after the delay shift. Lines under the abcissa indicate periods of darkness. Data are expressed as means_+SEM of 4 animals. When SEM are omitted, they were lower than 0.2 nmol.mg I.h-]. (B) After Illnerovfiet al? 2. L D 12:12 cycle is accomplished also by the shortening of one light period, re-entrainment of the N A T rhythm proceeds in a similar way as after the advance shift accomplished by the shortening of one dark period, i.e. a normal amplitude persists and the phase relationship between N A T onset and offset is shortened at the beginning (Illnerovfi, unpublished observations). It appears that re-entrainment of the N A T rhythm to an advance in the LD cycle depends rather on the timing of the new advanced light period than on the way of shifting the cycle. Under LD 18 : 6, the amplitude temporarily diminished not only after the 5-h advance in the LD cycle, but also after a 3-h advance and even after a mere 2-h advance (Fig. 3). The 3-h advance was accomplished by the shortening of one light rather than of one dark period in order not to limit the dark period during the shift to 3 h only whereas the 2-h advance was accomplished by the shortening of one dark period. During both advance shifts, i.e. during night 0, the evening N A T rise was similar and the activity increased significantly above the baseline day level at 22.00 h ( P < 0.05). After the 3-h advance, a residual rhythm persisted during the next 3 days: NAT activity was significantly higher than the baseline day level at 23.50 h ( P < 0.05) during night 1, at 21.00 h ( P < 0.05), 23.00 h ( P < 0.01) and 23.50 h ( P < 0.05), respectively, during night 2, and at 23.50 h ( P < 0.01) during night 3. However, even during the third night, the amplitude of the N A T rhythm did not yet reach the pre-shift level (Fig. 3A). After the 2-h advance, the N A T rhythm with a normal amplitude persisted during night 1; however, the evening
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Fig. 2. The effect of a 5-h advance in the LD cycle on NAT rhythm. Rats adapted to LD 18:6, with lights on from 03.00 h to 21.00 h (A), or to LD 12:12, with lights on from 06.00 h to I8.00 h (B), were subjected to the 5-h advance in the LD cycle accomplished either by shortening of one light period by 5 h (A) or by shortening of one dark period by 5 h (B). The NAT rhythm was followed before the advance shift (night -l), during the shift (night 0), and during nights + 1, +2 and +3 after the advance shift. Occasionally, NAT activity was checked at intervals longer than 3 h throughout the light periods; as it never exceeded baseline values, the data are not shown. Lines under the abcissa indicate dark periods. Data are expressed as means + SEM of 4 animals. When SEM are omitted, they were lower than 0.2 nmol.mg I.h t. (B) After lllnerov~i and Humlovfi 7. N A T rise above the baseline level o c c u r r e d only at 23.00 h ( P < 0.001), i.e. by 1 h later t h a n during night 0 (Fig. 3B). D u r i n g nights 2 and 3, the amplitude temporarily diminished, but the rhythm was expressed: N A T activity rose significantly above the baseline day level at 00.00 h and 00.45 h, respectively ( P < 0,001) during night 2, and at 23.00 h, 00.00 h and 00.45 h, respectively ( P < 0.05), during night 3. Only during night 4 did the N A T r h y t h m with a normal amplitude reappear; however, the p h a s e relationship b e t w e e n the evening N A T rise and the m o r n i n g decline still remained shortened. T h e a b o v e - m e n t i o n e d results indicate that r e - e n t r a i n m e n t of the N A T rhythm to a shift in the L D cycle d e p e n d s actually on the p h o t o p e r i o d . While after an 8-h delay in the L D 18 : 6 cycle accomplished by the lengthening o f one light period no N A T rhythm is expressed in the subsequent d a r k period, after the delay in the L D 1 2 : 1 2 cycle the r h y t h m during night 0 is expressed, t h o u g h the phase relationship b e t w e e n the N A T rise a n d the decline is shortened. T h e difference m a y be due to the fact that u n d e r L D 1 2 : 1 2 , after an extension of the light period into the night hours, the m o r n i n g N A T decline is delayed in the subsequent darkness, t h o u g h to a lesser d e g r e e than the evening rise 1° w h e r e a s u n d e r L D 18 : 6 the decline is not phase-delayed at all (Humlovfi a n d Illnerovfi, in preparation). Consequently, u n d e r L D 18 : 6, the N A T rise might occur only at the time o f the decline and hence no r h y t h m is expressed whatsoever. Alterna-
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Fig. 3. The effect of a 3-h (A) and 2-h (B) advance in the LD 18:6 regime on NAT rhythm. Rats adapted to LD 18 : 6 were subjected either to the 3-h advance in the LD cycle accomplished by the shortening of one light period by 3 h (A) or to the 2-h advance in the LD cycle accomplished by the shortening of one dark period by 2 h (B). The NAT rhythm was followed before the advance shift (night -1), during the shift (night 0), during nights +1, + 2 and + 3 after the advance shift, and eventually during night +4. For further details, see legend to Fig. 2.
tively, under LD 12: 12, the NAT rhythm is already partly delayed during night 0, but the first part of the night-time activity is suppressed by light, while under LD 18:6 the whole nocturnal activity expression is still suppressed by light. Under LD 18:6, re-entrainment of the NAT rhythm to the 8-h delay shift takes more than 3 days while under LD 12 : 12 it is completed within this period. Even greater differences exist in re-entrainment under various photoperiods following an advance in the LD cycle. Under LD 18:6, the amplitude of the NAT rhythm temporarily diminishes after a 5-h, 3-h and even after a mere 2-h advance in the LD cycle, while under LD 12 : 12 the rhythm still retains its original amplitude after a 5-h, but no more after a 7-h advance shift 7. More processes may be involved in the different way of re-entrainment under LD 18 : 6. First, the masking, i.e. suppressing, effect of light and the phase relationship between the onset and end of nocturnal NAT activity must be considered. After the 5-h advance in the LD 18:6 cycle, the advanced light period suppresses high nocturnal NAT activity and may phase-advance the NAT offset whereas the NAT onset is not phase-advanced. Consequently, the NAT rise may occur close to or at the time of the NAT decline, the rhythm cannot be fully expressed, and the amplitude dampens immediately. Second, the advanced light period, besides the suppressing effect and the phase-advancing effect on the NAT offset, may temporarily phase-delay the NAT onset. Following a 3-h advance in the LD 18:6 cycle, the NAT activity rises significantly above the baseline value at 22.00 h during night 0, but only at
152 23.50 h during night 1. Following a 2-h advance shift, the activity rises significantly al 22.00 h during night 0, but only at 23.(10 h during night 1 and at 00.00 h during night 2. Due to the suppressing and to the dual entraining effect of light, i.e. the phase-advancing effect on the NAT offset and at the same time the phase-delaying effect on thc NAT onset, the time of NAT onset approaches gradually that of the offset and damping ensues. It appears, therefore, that following the 3-h and the 2-h advance shift, the NAT onset response may be temporarily antidromic. Recent data on the mechanism of NAT rhythm re-entrainment to an 8-h advance in a LD 12:12 cycle reveal that, due to an advance in the NAT offset and an antidromic response of the NAT onset, the phase relationship between the onset and offset may become so compressed that the rhythm cannot be expressed even in continuous darkness ~'. Under such a compressed state, N A T activity may phase-jump into a new advanced dark period. Under LD 18:6 any, even a mere l-h, advance in the morning light onset may have a dual entraining effect on the NAT rhythm the next day (Humlovfi and Illnerovfi, in preparation) whereas under LD 12 : 12 only an advance longer than 6 h, i.e. bringing forward the light onscl to before the middle of the night, has such a dual effect ~. In a long photoperiod, the time interval between the evening NAT rise and the morning decline, i.e. the phase relationship between the evening and the morning component of the NAT rhythm, is shortened considerably ~. In such a compressed phase relationship, any advanced light period may have the dual effect on the NAT rhythm as it may easily hit both components of the rhythm, i.e. phase-delay the NAT rise and phase-advance the decline. The shortened phase relationship between the NAT rise and decline may reflect a changed state of an underlying complex pacemaker 3'5"~'~. In long days, under the changed state, phase shifts in the pacemaker are of smaller magnitude '~ (Humlovfi and Illnerovfi, in preparation) and, as our present results indicate, adjustment to shifts in the LD cycle proceeds in a more complicated way than in shorter days. In conclusion, re-entrainment of the circadian rhythm of rat pineal NAT activity and of its underlying pacemaker to shifts in the LD cycle in a long photoperiod differs from that in a shorter photoperiod as regards the rate and way of adjustment to the shift and the critical advance shift for retaining of the NAT rhythm amplitude or for its temporal lowering. The data strongly suggest that adjustment of the pacemaker to a shift in the LD cycle depends on the photoperiod and probably on the state of the pacemaker as well. ACKNOWLEDGEMENTS
This work was supported by the Czechoslovak Academy of Sciences Grant No. 71118. We wish to thank Mrs. I. Slavlkovfi for her excellent technical assistance, Mrs. J. Koldovfi for kindly typing the manuscript, and Mrs. J. Slejmarovfi for managing the animal care facilities. REFERENCES 1 Aschoff, J., Freerunning and entrained circadian rhythms. In J. Aschoff (Ed.), Biological Rhythms, Handbook of Behavioral Neurobiology, Vol. 4, Plenum Press, New York-London, 1981, pp. 81-93. 2 Deguchi, T. and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in the rat pineal gland, Anal Biochem., 50 (1972) 174-179. 3 Honma, K., Honma, S. and Hiroshige, T., Response curve, free-running period and activity time in circadian locomotor rhythm of rats, Jpn. J. PhysioL, 35 (1985) 643-658.
153 4 Humlovfi, M. and Illnerov~t, H., Rate of re-entrainment of circadian rhythms to advances of light-dark cycles may depend on ways of shifting the cycles, Brain Res., 531 (1990) 304-306. 5 Illnerov~i, H., Circadian Rhythms in the Mammalian Pineal Gland, Academia, Prague, 1986, 105 pp. 6 Illnerov~., H., Mechanism of re-entrainment of the circadian rhythm in the rat pineal N-acetyltransferase to an eight-hour advance of the light-dark cycle: phase jump is involved, Brain Res., 434 (1989) 365-368. 7 Illnerov~i, H. and Humlovfi, M., The rat pineal N-acetyltransferase rhythm persists after a five-hour, but disappears temporarily after a seven-hour advance of the light-dark cycle: a six-hour shift may be a turning point, Neurosci. Lett., 110 (1990) 77-81. 8 Illnerovfi, H. and Humlovfi, M., Entrainment of the pacemaker controlling the rhythmic melatonin production depends on photoperiod. In J. Arendt (Ed.), Advances in Pineal Research, Vol. 5, John Libbey, London-Paris, 1991, pp. 267-272. 9 Illnerovfi, H. and Van~zek, J., Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity under extremely long and short photoperiods, J. Pineal Res., 2 (1985) 67-78. 10 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). 11 Illnerovfi, H., Van~fiek, 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. 12 lllnerovfi, H., Van~Eek, J. and Hoffmann, K., Adjustment of the rat pineal N-acetyltransferase rhythm to eight-hour shifts of the light-dark cycle: advance of the cycle disturbs the rhythm more than delay, Brain Res., 417 (1987) 167-171. 13 Klein, D.C., The pineal gland: a model of neuroendocrine regulation. In S. Reichlin, R.R. Baldessarini and J.B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 303-327. 14 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. 15 Klein, D.C. and Weller, J.L., Indole metabolism in the pineal gland. A circadian rhythm in Nacetyltransferase activity, Science, 169 (1970) 1093-1095. 16 Moore, R.J. and Eichler, V.B., Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Res., 42 (1972) 201-206. 17 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. 18 Pittendrigh, C.S., Circadian systems: entrainment. In J. Aschoff (Ed.), Biological Rhythms, Handbook of Behavioral Neurobiology, Vol. 4, Plenum Press, New York, 1981, pp. 95-124. 19 Pittendrigh, C.S. and Daan, S., A functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: A clock for all seasons, J. Comp. Physiol., 106 (1976) 333-355. 20 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) 1583-1586. 21 Vanfi6ek, J. and Illnerovfi, H., Night pineal N-acetyltransferase activity in rats exposed to white or red light pulses of various intensity and duration, Experientia, 38 (1982) 1318-1319.
147
NEURES 00522
Resetting of the rat circadian clock after a shift in the light/dark cycle depends on the photoperiod Martina Humlovfi and Helena Illnerovfi Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 22 April 1991; Revised version received 19 August 1991; Accepted 19 October 1991) Key words: Rat; Circadian pacemaker; Resetting; Light/dark cycle; Photoperiod; Pineal; Melatonin; N-Acetyltransferase rhythm
SUMMARY Adjustment of the circadian clock to shifts in the light/dark (LD) cycle was assessed from the rat pineal N-acetyltransferase (NAT) rhythm which is controlled by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Re-entrainment to an 8-h delay in the LD cycle took more than 3 days in rats maintained under a regime with 18 h of light and 6 h of darkness per day (LD 18:6) whereas it was completed within 3 days in those maintained under LD 12: 12. Re-entrainment to an advance in the LD cycle proceeded through a transient diminution or almost disappearance of the NAT rhythm amplitude following a 5-h, 3-h and even a mere 2-h advance shift under LD 18:6, whereas no such diminution occurred under LD 12:12 even after a 5-h advance shift. Altogether, the data indicate that resetting of the circadian clock after shifts in the LD cycle depends on the photoperiod.
I n a n o n - p e r i o d i c e n v i r o n m e n t , the m a m m a l i a n c i r c a d i a n clock f r e e r u n s w i t h a p e r i o d c l o s e to, b u t n o t e q u a l to, 24 h I. U n d e r n a t u r a l d a y l i g h t or in a 24-h artificial l i g h t / d a r k ( L D ) cycle, t h e clock is s y n c h r o n i z e d to t h e 24-h day by p e r i o d i c c h a n g e s in light a n d d a r k n e s s Is. F o l l o w i n g shifts in the L D cycle w h i c h m i m i c t r a n s m e r i d i a n flights, t h e c i r c a d i a n p a c e m a k e r m u s t r e - a d j u s t to a n e w e n v i r o n m e n t a l time. In p r e v i o u s p a p e r s we s t u d i e d r e - e n t r a i n m e n t o f t h e clock a f t e r shifts in t h e L D cycle in rats m a i n t a i n e d u n d e r o n l y o n e p h o t o p e r i o d , n a m e l y in a r e g i m e w i t h 12 h o f light a n d 12 h o f d a r k n e s s p e r d a y ( L D 12 : 12) 4'6'7'12. A s r e c e n t d a t a suggest t h a t r e s e t t i n g of the c i r c a d i a n p a c e m a k e r m a y d e p e n d on t h e p h o t o p e r i o d s'9, we d e c i d e d to study r e - e n t r a i n m e n t of the c l o c k a f t e r shifts in t h e L D cycle in rats m a i n t a i n e d u n d e r a n o t h e r p h o t o p e r i o d , this t i m e u n d e r a l o n g e r one, in o r d e r to c o m p a r e the r e - a d j u s t m e n t u n d e r d i f f e r e n t p h o t o p e r i o d s . T o assess t h e p h a s e s o f t h e c i r c a d i a n p a c e m a k e r , we u s e d t h e overt r h y t h m in p i n e a l N - a c e t y l t r a n s f e r a s e ( N A T ) (arylalkylamine: acetyl C o A N - a c e t y l t r a n s f e r a s e , E C 2.3.1.87) activity which drives 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 in t h e rat 11"15, as h a n d s o f t h e clock. T h e N A T r h y t h m is c o n t r o l l e d by a p a c e m a k e r l o c a t e d in t h e s u p r a c h i a s m a t i c n u c l e i o f t h e h y p o t h a l a m u s 14, as a r e o t h e r c i r c a d i a n r h y t h m s 16,2°. A t night, a c c o r d i n g to t h e
Correspondence: RNDr. Helena Illnerovfi, DrSc., Institute of Physiology, Czechoslovak Academy of Sciences, Vide~sk~ 1083, 14220 Prague 4, Czechoslovakia.
148 pacemaker's program, norepinephrine is released from sympathetic nerve endings in thu pineal gland at a higher rate and induces and activates NAT ~. The NAT rhythm i.~ it good model for circadian studies, as it exhibits a high amplitude and has two well-defined phase markers, namely the time of the evening NAT rise and the time of the morning decline. We used 50-day-old male Wistar rats from our own breeding colony housed under LD 18 : 6 and at a temperature of 23 + 2°C for at least 3 weeks prior to the experiments. The rats had free access to water and commercial food pellets. Light provided by overhead 40 W Tesla fluorescent tubes was automatically turned on at 03.00 h and off at 21.00 h. A delay in the LD cycle was accomplished by lengthening of one light period, an advance by shortening of one light period or, alternatively, by shortening of one dark period (see legends to Figs. 2 and 3); thereafter the light and dark periods alternated again regularly. When rats were to be killed in darkness, they were exposed for less than 1 min prior to decapitation to a red light weaker than 1 lx; such a light does not affect nocturnal NAT activity 2~. Pineal glands were removed rapidly and stored in Petri dishes on solid CO 2. Within 48 h after decapitation, NAT activity was determined by a modification w of the method of Deguchi and Axelrod e. [1-~4C]Acetyl-CoA (2.07 G B q / m m o l ) was purchased from the Radiochemical Centre (Amersham, U.K.). Units of NAT activity were defined as nmol N-acetyltryptamine formed in 1 h / r a g of pineal tissue (nmol • mg ~ • h ~). Baseline day values were within the range of 0.05-0.15 n m o l - m g -~- h -L. Data were analysed using one-way analysis of variance. The t-test with Bonferroni probabilities (BMDP Statistical Software, University of California, Los Angeles) was used for the post hoc comparison, with a = 0.05 required for significance. Heterogeneity of variance was reduced by log transformation of the data. Folllowing an 8-h delay in the LD 18 : 6 cycle, no NAT rhythm was expressed during night 0, i.e. after the prolonged light period (Fig. 1A). However, already during the next night, i.e. during night 1, the NAT activity was significantly higher than the baseline day value at 08.00, 09.00, 10.00 and 10.45 h, respectively ( P < 0.001). Though the rhythm was already expressed during night 1 and thereafter, its amplitude was temporarily lowered. The phase relationship between the evening NAT rise and the morning decline was shortened as compared to the pre-shift pattern even after 3 days following the shift. After an 8-h delay in the LD 12 : 12 cycle, no significant diminution of the NAT rhythm amplitude occurs (Fig. 1B) ~2. The phase relationship between the NAT rise and the decline is shortened considerably only during night 0; during nights 1 and 2, the phase relationship is shortened just slightly, and within 3 days it is the same as before the delay shift. After a 5-h advance of the LD 18:6 regime, the NAT rhythm with a normal amplitude temporarily disappeared (Fig. 2A). However, during night 1, the NAT activity was significantly higher than the baseline day value at 18.00 h and 20.00 h, respectively ( P < 0.05) and during night 2 at 19.00 h ( P < 0.05), 20.00 h ( P < 0.01) and 21.00 h ( P < 0.001), respectively. During night 3, the amplitude in individual animals occasionally increased to a pre-shift level. After a 5-h advance in the LD 12:12 regime, the N A T rhythm persists with a normal amplitude, though at the beginning in a compressed waveform (Fig. 2B). The 5-h advance in the LD 18:6 cycle was accomplished in a different way from that in the LD 12 : 12 cycle, i.e. by shortening of one light instead of one dark period, in order not to limit the dark period during the shift to 1 h only. Thc difference in re-entrainment under the two photoperiods does not appear, however, to be due to various ways of advancing a light/dark regime: when the 5-h advance in the
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Fig. 1. The effect of an 8-h delay in the LD cycle on NAT rhythm. Rats adapted to LD 18:6, with lights on from 03.00 to 21.00 h (A), or to LD 12:12, with lights on from 06.00 to 18.00 h (B), were subjected to the 8-h delay in the LD cycle accomplished by lengthening one light period by 8 h. The NAT rhythm was followed before the delay shift (night -1), during the first dark period after prolongation of the light period (night 0), and during nights + 1, + 2 and + 3 after the delay shift. Lines under the abcissa indicate periods of darkness. Data are expressed as means_+SEM of 4 animals. When SEM are omitted, they were lower than 0.2 nmol.mg I.h-]. (B) After Illnerovfiet al? 2. L D 12:12 cycle is accomplished also by the shortening of one light period, re-entrainment of the N A T rhythm proceeds in a similar way as after the advance shift accomplished by the shortening of one dark period, i.e. a normal amplitude persists and the phase relationship between N A T onset and offset is shortened at the beginning (Illnerovfi, unpublished observations). It appears that re-entrainment of the N A T rhythm to an advance in the LD cycle depends rather on the timing of the new advanced light period than on the way of shifting the cycle. Under LD 18 : 6, the amplitude temporarily diminished not only after the 5-h advance in the LD cycle, but also after a 3-h advance and even after a mere 2-h advance (Fig. 3). The 3-h advance was accomplished by the shortening of one light rather than of one dark period in order not to limit the dark period during the shift to 3 h only whereas the 2-h advance was accomplished by the shortening of one dark period. During both advance shifts, i.e. during night 0, the evening N A T rise was similar and the activity increased significantly above the baseline day level at 22.00 h ( P < 0.05). After the 3-h advance, a residual rhythm persisted during the next 3 days: NAT activity was significantly higher than the baseline day level at 23.50 h ( P < 0.05) during night 1, at 21.00 h ( P < 0.05), 23.00 h ( P < 0.01) and 23.50 h ( P < 0.05), respectively, during night 2, and at 23.50 h ( P < 0.01) during night 3. However, even during the third night, the amplitude of the N A T rhythm did not yet reach the pre-shift level (Fig. 3A). After the 2-h advance, the N A T rhythm with a normal amplitude persisted during night 1; however, the evening
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tively, under LD 12: 12, the NAT rhythm is already partly delayed during night 0, but the first part of the night-time activity is suppressed by light, while under LD 18:6 the whole nocturnal activity expression is still suppressed by light. Under LD 18:6, re-entrainment of the NAT rhythm to the 8-h delay shift takes more than 3 days while under LD 12 : 12 it is completed within this period. Even greater differences exist in re-entrainment under various photoperiods following an advance in the LD cycle. Under LD 18:6, the amplitude of the NAT rhythm temporarily diminishes after a 5-h, 3-h and even after a mere 2-h advance in the LD cycle, while under LD 12 : 12 the rhythm still retains its original amplitude after a 5-h, but no more after a 7-h advance shift 7. More processes may be involved in the different way of re-entrainment under LD 18 : 6. First, the masking, i.e. suppressing, effect of light and the phase relationship between the onset and end of nocturnal NAT activity must be considered. After the 5-h advance in the LD 18:6 cycle, the advanced light period suppresses high nocturnal NAT activity and may phase-advance the NAT offset whereas the NAT onset is not phase-advanced. Consequently, the NAT rise may occur close to or at the time of the NAT decline, the rhythm cannot be fully expressed, and the amplitude dampens immediately. Second, the advanced light period, besides the suppressing effect and the phase-advancing effect on the NAT offset, may temporarily phase-delay the NAT onset. Following a 3-h advance in the LD 18:6 cycle, the NAT activity rises significantly above the baseline value at 22.00 h during night 0, but only at
152 23.50 h during night 1. Following a 2-h advance shift, the activity rises significantly al 22.00 h during night 0, but only at 23.(10 h during night 1 and at 00.00 h during night 2. Due to the suppressing and to the dual entraining effect of light, i.e. the phase-advancing effect on the NAT offset and at the same time the phase-delaying effect on thc NAT onset, the time of NAT onset approaches gradually that of the offset and damping ensues. It appears, therefore, that following the 3-h and the 2-h advance shift, the NAT onset response may be temporarily antidromic. Recent data on the mechanism of NAT rhythm re-entrainment to an 8-h advance in a LD 12:12 cycle reveal that, due to an advance in the NAT offset and an antidromic response of the NAT onset, the phase relationship between the onset and offset may become so compressed that the rhythm cannot be expressed even in continuous darkness ~'. Under such a compressed state, N A T activity may phase-jump into a new advanced dark period. Under LD 18:6 any, even a mere l-h, advance in the morning light onset may have a dual entraining effect on the NAT rhythm the next day (Humlovfi and Illnerovfi, in preparation) whereas under LD 12 : 12 only an advance longer than 6 h, i.e. bringing forward the light onscl to before the middle of the night, has such a dual effect ~. In a long photoperiod, the time interval between the evening NAT rise and the morning decline, i.e. the phase relationship between the evening and the morning component of the NAT rhythm, is shortened considerably ~. In such a compressed phase relationship, any advanced light period may have the dual effect on the NAT rhythm as it may easily hit both components of the rhythm, i.e. phase-delay the NAT rise and phase-advance the decline. The shortened phase relationship between the NAT rise and decline may reflect a changed state of an underlying complex pacemaker 3'5"~'~. In long days, under the changed state, phase shifts in the pacemaker are of smaller magnitude '~ (Humlovfi and Illnerovfi, in preparation) and, as our present results indicate, adjustment to shifts in the LD cycle proceeds in a more complicated way than in shorter days. In conclusion, re-entrainment of the circadian rhythm of rat pineal NAT activity and of its underlying pacemaker to shifts in the LD cycle in a long photoperiod differs from that in a shorter photoperiod as regards the rate and way of adjustment to the shift and the critical advance shift for retaining of the NAT rhythm amplitude or for its temporal lowering. The data strongly suggest that adjustment of the pacemaker to a shift in the LD cycle depends on the photoperiod and probably on the state of the pacemaker as well. ACKNOWLEDGEMENTS
This work was supported by the Czechoslovak Academy of Sciences Grant No. 71118. We wish to thank Mrs. I. Slavlkovfi for her excellent technical assistance, Mrs. J. Koldovfi for kindly typing the manuscript, and Mrs. J. Slejmarovfi for managing the animal care facilities. REFERENCES 1 Aschoff, J., Freerunning and entrained circadian rhythms. In J. Aschoff (Ed.), Biological Rhythms, Handbook of Behavioral Neurobiology, Vol. 4, Plenum Press, New York-London, 1981, pp. 81-93. 2 Deguchi, T. and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in the rat pineal gland, Anal Biochem., 50 (1972) 174-179. 3 Honma, K., Honma, S. and Hiroshige, T., Response curve, free-running period and activity time in circadian locomotor rhythm of rats, Jpn. J. PhysioL, 35 (1985) 643-658.
153 4 Humlovfi, M. and Illnerov~t, H., Rate of re-entrainment of circadian rhythms to advances of light-dark cycles may depend on ways of shifting the cycles, Brain Res., 531 (1990) 304-306. 5 Illnerov~i, H., Circadian Rhythms in the Mammalian Pineal Gland, Academia, Prague, 1986, 105 pp. 6 Illnerov~., H., Mechanism of re-entrainment of the circadian rhythm in the rat pineal N-acetyltransferase to an eight-hour advance of the light-dark cycle: phase jump is involved, Brain Res., 434 (1989) 365-368. 7 Illnerov~i, H. and Humlovfi, M., The rat pineal N-acetyltransferase rhythm persists after a five-hour, but disappears temporarily after a seven-hour advance of the light-dark cycle: a six-hour shift may be a turning point, Neurosci. Lett., 110 (1990) 77-81. 8 Illnerovfi, H. and Humlovfi, M., Entrainment of the pacemaker controlling the rhythmic melatonin production depends on photoperiod. In J. Arendt (Ed.), Advances in Pineal Research, Vol. 5, John Libbey, London-Paris, 1991, pp. 267-272. 9 Illnerovfi, H. and Van~zek, J., Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity under extremely long and short photoperiods, J. Pineal Res., 2 (1985) 67-78. 10 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). 11 Illnerovfi, H., Van~fiek, 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. 12 lllnerovfi, H., Van~Eek, J. and Hoffmann, K., Adjustment of the rat pineal N-acetyltransferase rhythm to eight-hour shifts of the light-dark cycle: advance of the cycle disturbs the rhythm more than delay, Brain Res., 417 (1987) 167-171. 13 Klein, D.C., The pineal gland: a model of neuroendocrine regulation. In S. Reichlin, R.R. Baldessarini and J.B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 303-327. 14 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. 15 Klein, D.C. and Weller, J.L., Indole metabolism in the pineal gland. A circadian rhythm in Nacetyltransferase activity, Science, 169 (1970) 1093-1095. 16 Moore, R.J. and Eichler, V.B., Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Res., 42 (1972) 201-206. 17 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. 18 Pittendrigh, C.S., Circadian systems: entrainment. In J. Aschoff (Ed.), Biological Rhythms, Handbook of Behavioral Neurobiology, Vol. 4, Plenum Press, New York, 1981, pp. 95-124. 19 Pittendrigh, C.S. and Daan, S., A functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: A clock for all seasons, J. Comp. Physiol., 106 (1976) 333-355. 20 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) 1583-1586. 21 Vanfi6ek, J. and Illnerovfi, H., Night pineal N-acetyltransferase activity in rats exposed to white or red light pulses of various intensity and duration, Experientia, 38 (1982) 1318-1319.
