304
Brain Research, 531 (1990)3(t4-3t1(~
EIscvicl BRES 24351
Rate of re-entrainment of circadian rh hms to adv ces of light-dark cycles may depend on ways of shifting the cycles Martina Humlov i and Helena Illnerov i Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
(Accepted 17 July 1990) Key words: Circadian rhythm; Suprachiasmatic nucleus; Entrainment; Light-dark cycle; Rat; Pineal; N-acetyltransferase
When an 8-h advance of a light-dark (LD) cyclewas accomplishedby shortening of one dark period by 8 h, the rat pineal N-acetyltransferase rhythm was abolished during the first 3 subsequent cycles and reappeared in its original waveform during the fifth cycle only. When the 8-h advance shift was accomplished by lengthening of two consecutive light periods by 8 h, the N-acetyltransferase rhythm persisted and attained its original waveform by 2 days earlier than under the former shift. Re-entrainment is thus more rapid when the advance shift is accomplished by twice delaying than by once advancing of the LD cycle by 8 h. Circadian rhythm in the pineal N-acetyltransferase (EC 2.3.1.87) (NAT) activity is responsible for the rhythmic melatonin production in the rat 7"it. As other circadian rhythms 12'1s, the NAT rythm is controlled by a pacemaker located in the suprachiasmatic nuclei of the hypothalamus 1°. Phase delays of the NAT rhythm, similarly as delays of the locomotor activity rhythm 15 are usually accomplished within one cycle, while it takes more transient cycles before phase advances of the whole NAT rhythm are completed as well 3"9. After an advance of a light-dark (LD) cycle longer than 6 h the NAT rhythm may even temporarily disappear before it phase advances 5. Different rates of phase delaying and of phase advancing of the NAT rhythm suggest that adjustment of the rhythm to an advance of a LD cycle might proceed more rapidly when the advance shift is accomplished by delaying than by directly advancing the LD cycle. To test this hypothesis, we subjected rats maintained under the lighting regime of 12 h of light and 12 h of darkness per day (LD 12:12) either to an 8-h advance of the LD cycle or to a 16-h delay of the LD cycle accomplished by twice delaying the cycle by 8 h and we followed the NAT rhythm during subsequent days. We used 50-day-old male Wistar rats from our breeding colony housed under LD 12:12 for 3 weeks prior to experiments. Light provided by overhead Tesla fluorescent tubes was automatically turned on at 06.00 h and off at 18.00 h. When the LD cycle was advanced by 8 h, one dark period (night 0) was shortened by 8 h and thereafter the light and dark period alternated again regularly (Fig. 1A). When the LD cycle was advanced by 8 h by twice
delaying the cycle by 8 h, a light period was lengthened by 8 h during the first day and once more during the second day, and only then the light and dark period alternated again regularly (Fig. 1B). When rats were to be killed in darkness, they were exposed prior to decapitation to a very faint red light for tess than 1 min. 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 t4 of the method of Deguchi and Axelrod 2. [1-~4C]Acetyl CoA (2.076 Bq/mmol) was purchased from the Radiochemical Centre (Amersham, U.K.). Units of NAT activity were defined as nmol N-acetyltryptamine formed in 1 h/mg of pineal tissue (nmol.mg-Lh-1). Baseline day values were within the range of 0.1-0.2 nmd-mg-Lh -~. Data were analyzed using one-way ANOVA. 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. After the 8-h advance of the LD cycle, the NAT rhythm was completely abolished during nights 1 and 2 (Fig. 1A). During night 3, NAT activity occasionally increased, but only at 21.00 h was the activity significantly elevated above the baseline value at 10.00 h (P < 0.01). The rhythm reappeared only during night 4, though still in a changed waveform. At 12.00 h (P < 0.001), 14.00 h (P < 0.05) and at 15.00 h to 22.00 h (P < 0.001), respectively, the activity was significantly higher than the baseline value at 10.00 h. However, only
Correspondence: H. lllnerov~i, Institute of Physiology, CzechoslovakAcademy of Sciences, Vfdefisk~ 1083, 142 20 Prague 4, Czechoslovakia.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
305
6
-1
E
0
+|
+2
+3
+4
NIGHT +S
12-~ 6-
o6 -!
;s
tlh
d6~
0
A fi +!
0'6 ~
Ik fi
+2
.
m
tl 22
÷3
TIME(H) NIGHT
Fig. 1. Adjustment of the N-acetyltransferase rhythm to an 8-h advance of the LD cycle accomplished either by advancing once (A) or by delaying twice (B) the cycle. Open bars represent light periods, full bars dark periods. Alternating open and full bars indicate the schedule of advancing the LD cycle by shortening of one dark period by 8 h (A) or by lengthening of two consecutive light periods by 8 H (B). Rats adapted to LD 12:12 were killed during the night before shifts (night -1), in the course of the shift (night 0), and during the subsequent nights (night +1, +2 etc.). Data are expressed as means + S.E.M. of 4-32 animals. When S.E.M. are omitted, they were lower than 0.3 nmol.mg-l.h -l.
during night 5 was the NAT rhythm similar to the pre-shift one. After the first delay of the LD cycle by 8 h, during night 0, as well as after the second delay, during night 1, the NAT rhythm was present, though in a changed waveform (Fig. 1B). During night 0, the activity was significantly higher at 04.00 h to 07.00 h (P < 0.001) and at 09.00 h (P < 0.01), respectively, than the baseline value at 02.00 h, during night 1, the activity was significantly higher at 13.00 h (P < 0.01), 14.00 h (P < 0.001), 15.00 h (P < 0.05) and at 17.00 h (P < 0.01), respectively, than the baseline value at 10.00 h. However, only during night 2, the NAT rhythm was similar to the pre-shift one. Re-entrainment to the 8-h advance of the LD cycle was thus completed by about 2 days earlier when the shift was accomplished by twice delaying the LD cycle than when it was accomplished by once advancing the cycle (Fig. 1). Different rates of the adjustment may be explained by different mechanisms of phase delaying and phase ad-
vancing of the NAT rhythm and of its underlying pacemaker 3'4'8'9. During the 8-h delay of the LD cycle, the delayed light period phase shifts the evening NAT rise and the morning decline in the same direction 8. Within one cycle, the NAT rhythm is phase delayed and its waveform does not change. During the 8-h advance of the LD cycle, the advanced light period phase shifts the NAT decline and the rise in opposite directions: while the morning decline phase advances, the evening rise phase delays 4. Eventually, the phase relationship between the rise and the decline may become so compressed that the NAT rhythm may not be even expressed in continuous darkness. Under such a compressed state of the rhythm and of its underlying circadian pacemaker, the NAT activity suddenly phase jumps into the advanced dark period. Adjustment of the hamster locomotor activity rhythm to an 8-h advance of a LD cycle is accelerated by running in the beginning of the advanced dark period 13'17. Adjustment of the rat locomotor 16 and NAT 6 activity
306 rhythms to the 8-h advance of the LD cycle is accelerated by administration of melatonin at the start of the new dark period. Similarly, adjustment of the h u m a n cortisol and melatonin rhythms to transition across 8 time zones eastward is accelerated by melatonin administered before the new bed time ~. It seems that there may exist another way how to accelerate r e - e n t r a i n m e n t to the 8-h advance
1 Arendt, J., Aldhous, M., English, J., Marks, V. and Arendt, J.H., Some effects of jet-lag and their alleviation by melatonin, Ergonomics, 30 (1987) 1379-1393. 2 Deguchi, T. and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in rat pineal, Anal. Biochem., 50 (1972) 174-179. 3 Ilinerov~i, H., Orcadian Rhythms in the Mammalian Pineal Gland, Academia, Prague, 1986. 4 Illnerovfi, H., Mechanism of the re-entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity to an eight-hour advance of the light-dark cycle: phase jump is involved, Brain Research, 494 (1989) 365-368. 5 Illnerov~i, H. and Humlov~i, 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. 6 Illnerovfi, H., Trentini, G.P. and Maslova, L., Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle, J. Comp. Physiol. A., 166 (1989) 97-102. 7 Illnerov~l, H., Van~.C~k,J. and Hoffman, K., Regulation of the pineal melatonin concentration in the rat (Rattus norvegicus) and in the Djungarian hamster (Phodopus sungorus), Comp. Biochem. Physiol., 74 (1983) 155-159. 8 Illnerov~i,H., V a n ~ k , 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 Research, 417 (1987) 167-171. 9 Illnerov~l, H., Van~ek, 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.
of the LD cycle - - strategy of shifting the cycle. Apparently, a d j u s t m e n t is more rapid when the advance shift is accomplished by twice delaying than by once advancing the LD cycle by 8 h. We thank Mrs. Irena Slav~ov~i for her excellent technical assistance, Mrs. Jana Koldov~ifor kindly typing the manuscript and Mrs. Jifina ~;lejmarov~for managing the animal care facilities.
10 Klein, D.C. and Moore, R.J., Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: control by the retinal hypothalamic tract and the suprachiasmatic nucleus, Brain Research, 174 (1979) 245-262. 11 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. 12 Moore, R.Y. and Eichler, V.B., Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Research, 42 (1972) 201-206. 13 Mrosovski, N. and Salmon, P.A., A behavioral method for accelerating re-entrainment of rhythms to new light-dark cycles, Nature, 330 (1988) 372-373. 14 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 adrenerglc and adenosine cyclic 3",5"-monophosphate induced stimulation of pineal serotonin N-acetyltransferase activity, Mol. Pharmacol., 11 (1975) 241-245. 15 Pittendrigh, C.S., Circadian clocks: What are they? In J.W. Hastings and H.G. Schweiger (Eds.), The Molecular Basis of Circadian Rhythms, Dahlem Konferenzen, Berlin, 1975, pp. 11-43. 16 Redman, J.R. and Armstrong, S.M., Reentrainment of rat circadian activity rhythms: effect of melatonin, J. Pineal Res., 5 (1988) 203-215. 17 Turek, F.W., Effects of stimulated physical activity on the circadan pacemaker of vertebrates, J. Biol. Rhythms, 4 (1989) 135-147. 18 Stephan, F.K. and Zucker, J., Circadian rhythms in drinking behaviour and locomotor activity af rats are eliminated by hypothalamic lesions, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 1583-1586.
Brain Research, 531 (1990)3(t4-3t1(~
EIscvicl BRES 24351
Rate of re-entrainment of circadian rh hms to adv ces of light-dark cycles may depend on ways of shifting the cycles Martina Humlov i and Helena Illnerov i Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
(Accepted 17 July 1990) Key words: Circadian rhythm; Suprachiasmatic nucleus; Entrainment; Light-dark cycle; Rat; Pineal; N-acetyltransferase
When an 8-h advance of a light-dark (LD) cyclewas accomplishedby shortening of one dark period by 8 h, the rat pineal N-acetyltransferase rhythm was abolished during the first 3 subsequent cycles and reappeared in its original waveform during the fifth cycle only. When the 8-h advance shift was accomplished by lengthening of two consecutive light periods by 8 h, the N-acetyltransferase rhythm persisted and attained its original waveform by 2 days earlier than under the former shift. Re-entrainment is thus more rapid when the advance shift is accomplished by twice delaying than by once advancing of the LD cycle by 8 h. Circadian rhythm in the pineal N-acetyltransferase (EC 2.3.1.87) (NAT) activity is responsible for the rhythmic melatonin production in the rat 7"it. As other circadian rhythms 12'1s, the NAT rythm is controlled by a pacemaker located in the suprachiasmatic nuclei of the hypothalamus 1°. Phase delays of the NAT rhythm, similarly as delays of the locomotor activity rhythm 15 are usually accomplished within one cycle, while it takes more transient cycles before phase advances of the whole NAT rhythm are completed as well 3"9. After an advance of a light-dark (LD) cycle longer than 6 h the NAT rhythm may even temporarily disappear before it phase advances 5. Different rates of phase delaying and of phase advancing of the NAT rhythm suggest that adjustment of the rhythm to an advance of a LD cycle might proceed more rapidly when the advance shift is accomplished by delaying than by directly advancing the LD cycle. To test this hypothesis, we subjected rats maintained under the lighting regime of 12 h of light and 12 h of darkness per day (LD 12:12) either to an 8-h advance of the LD cycle or to a 16-h delay of the LD cycle accomplished by twice delaying the cycle by 8 h and we followed the NAT rhythm during subsequent days. We used 50-day-old male Wistar rats from our breeding colony housed under LD 12:12 for 3 weeks prior to experiments. Light provided by overhead Tesla fluorescent tubes was automatically turned on at 06.00 h and off at 18.00 h. When the LD cycle was advanced by 8 h, one dark period (night 0) was shortened by 8 h and thereafter the light and dark period alternated again regularly (Fig. 1A). When the LD cycle was advanced by 8 h by twice
delaying the cycle by 8 h, a light period was lengthened by 8 h during the first day and once more during the second day, and only then the light and dark period alternated again regularly (Fig. 1B). When rats were to be killed in darkness, they were exposed prior to decapitation to a very faint red light for tess than 1 min. 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 t4 of the method of Deguchi and Axelrod 2. [1-~4C]Acetyl CoA (2.076 Bq/mmol) was purchased from the Radiochemical Centre (Amersham, U.K.). Units of NAT activity were defined as nmol N-acetyltryptamine formed in 1 h/mg of pineal tissue (nmol.mg-Lh-1). Baseline day values were within the range of 0.1-0.2 nmd-mg-Lh -~. Data were analyzed using one-way ANOVA. 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. After the 8-h advance of the LD cycle, the NAT rhythm was completely abolished during nights 1 and 2 (Fig. 1A). During night 3, NAT activity occasionally increased, but only at 21.00 h was the activity significantly elevated above the baseline value at 10.00 h (P < 0.01). The rhythm reappeared only during night 4, though still in a changed waveform. At 12.00 h (P < 0.001), 14.00 h (P < 0.05) and at 15.00 h to 22.00 h (P < 0.001), respectively, the activity was significantly higher than the baseline value at 10.00 h. However, only
Correspondence: H. lllnerov~i, Institute of Physiology, CzechoslovakAcademy of Sciences, Vfdefisk~ 1083, 142 20 Prague 4, Czechoslovakia.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
305
6
-1
E
0
+|
+2
+3
+4
NIGHT +S
12-~ 6-
o6 -!
;s
tlh
d6~
0
A fi +!
0'6 ~
Ik fi
+2
.
m
tl 22
÷3
TIME(H) NIGHT
Fig. 1. Adjustment of the N-acetyltransferase rhythm to an 8-h advance of the LD cycle accomplished either by advancing once (A) or by delaying twice (B) the cycle. Open bars represent light periods, full bars dark periods. Alternating open and full bars indicate the schedule of advancing the LD cycle by shortening of one dark period by 8 h (A) or by lengthening of two consecutive light periods by 8 H (B). Rats adapted to LD 12:12 were killed during the night before shifts (night -1), in the course of the shift (night 0), and during the subsequent nights (night +1, +2 etc.). Data are expressed as means + S.E.M. of 4-32 animals. When S.E.M. are omitted, they were lower than 0.3 nmol.mg-l.h -l.
during night 5 was the NAT rhythm similar to the pre-shift one. After the first delay of the LD cycle by 8 h, during night 0, as well as after the second delay, during night 1, the NAT rhythm was present, though in a changed waveform (Fig. 1B). During night 0, the activity was significantly higher at 04.00 h to 07.00 h (P < 0.001) and at 09.00 h (P < 0.01), respectively, than the baseline value at 02.00 h, during night 1, the activity was significantly higher at 13.00 h (P < 0.01), 14.00 h (P < 0.001), 15.00 h (P < 0.05) and at 17.00 h (P < 0.01), respectively, than the baseline value at 10.00 h. However, only during night 2, the NAT rhythm was similar to the pre-shift one. Re-entrainment to the 8-h advance of the LD cycle was thus completed by about 2 days earlier when the shift was accomplished by twice delaying the LD cycle than when it was accomplished by once advancing the cycle (Fig. 1). Different rates of the adjustment may be explained by different mechanisms of phase delaying and phase ad-
vancing of the NAT rhythm and of its underlying pacemaker 3'4'8'9. During the 8-h delay of the LD cycle, the delayed light period phase shifts the evening NAT rise and the morning decline in the same direction 8. Within one cycle, the NAT rhythm is phase delayed and its waveform does not change. During the 8-h advance of the LD cycle, the advanced light period phase shifts the NAT decline and the rise in opposite directions: while the morning decline phase advances, the evening rise phase delays 4. Eventually, the phase relationship between the rise and the decline may become so compressed that the NAT rhythm may not be even expressed in continuous darkness. Under such a compressed state of the rhythm and of its underlying circadian pacemaker, the NAT activity suddenly phase jumps into the advanced dark period. Adjustment of the hamster locomotor activity rhythm to an 8-h advance of a LD cycle is accelerated by running in the beginning of the advanced dark period 13'17. Adjustment of the rat locomotor 16 and NAT 6 activity
306 rhythms to the 8-h advance of the LD cycle is accelerated by administration of melatonin at the start of the new dark period. Similarly, adjustment of the h u m a n cortisol and melatonin rhythms to transition across 8 time zones eastward is accelerated by melatonin administered before the new bed time ~. It seems that there may exist another way how to accelerate r e - e n t r a i n m e n t to the 8-h advance
1 Arendt, J., Aldhous, M., English, J., Marks, V. and Arendt, J.H., Some effects of jet-lag and their alleviation by melatonin, Ergonomics, 30 (1987) 1379-1393. 2 Deguchi, T. and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in rat pineal, Anal. Biochem., 50 (1972) 174-179. 3 Ilinerov~i, H., Orcadian Rhythms in the Mammalian Pineal Gland, Academia, Prague, 1986. 4 Illnerovfi, H., Mechanism of the re-entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity to an eight-hour advance of the light-dark cycle: phase jump is involved, Brain Research, 494 (1989) 365-368. 5 Illnerov~i, H. and Humlov~i, 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. 6 Illnerovfi, H., Trentini, G.P. and Maslova, L., Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle, J. Comp. Physiol. A., 166 (1989) 97-102. 7 Illnerov~l, H., Van~.C~k,J. and Hoffman, K., Regulation of the pineal melatonin concentration in the rat (Rattus norvegicus) and in the Djungarian hamster (Phodopus sungorus), Comp. Biochem. Physiol., 74 (1983) 155-159. 8 Illnerov~i,H., V a n ~ k , 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 Research, 417 (1987) 167-171. 9 Illnerov~l, H., Van~ek, 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.
of the LD cycle - - strategy of shifting the cycle. Apparently, a d j u s t m e n t is more rapid when the advance shift is accomplished by twice delaying than by once advancing the LD cycle by 8 h. We thank Mrs. Irena Slav~ov~i for her excellent technical assistance, Mrs. Jana Koldov~ifor kindly typing the manuscript and Mrs. Jifina ~;lejmarov~for managing the animal care facilities.
10 Klein, D.C. and Moore, R.J., Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: control by the retinal hypothalamic tract and the suprachiasmatic nucleus, Brain Research, 174 (1979) 245-262. 11 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. 12 Moore, R.Y. and Eichler, V.B., Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Research, 42 (1972) 201-206. 13 Mrosovski, N. and Salmon, P.A., A behavioral method for accelerating re-entrainment of rhythms to new light-dark cycles, Nature, 330 (1988) 372-373. 14 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 adrenerglc and adenosine cyclic 3",5"-monophosphate induced stimulation of pineal serotonin N-acetyltransferase activity, Mol. Pharmacol., 11 (1975) 241-245. 15 Pittendrigh, C.S., Circadian clocks: What are they? In J.W. Hastings and H.G. Schweiger (Eds.), The Molecular Basis of Circadian Rhythms, Dahlem Konferenzen, Berlin, 1975, pp. 11-43. 16 Redman, J.R. and Armstrong, S.M., Reentrainment of rat circadian activity rhythms: effect of melatonin, J. Pineal Res., 5 (1988) 203-215. 17 Turek, F.W., Effects of stimulated physical activity on the circadan pacemaker of vertebrates, J. Biol. Rhythms, 4 (1989) 135-147. 18 Stephan, F.K. and Zucker, J., Circadian rhythms in drinking behaviour and locomotor activity af rats are eliminated by hypothalamic lesions, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 1583-1586.
