- Email: [email protected]
Extension of the rat pineal N-acetyltransferase rhythm in continuous darkness and on short photoperiod
176
Brain Research, 261 (1983) 176-179 Elsevier Biomedical Press
Extension of the rat pineal N-acetyttransferase rh~hm in con.nuous d a ~ n n s and on short pi',otqzeriod HELENA ILLNEROV,~ and Jl~,I VANECEK
Institute of Physiology, Czechoslovak Academy of Sciences, Videhska 1083, 142 20 Prague 4 (Czechoslovakia) (Accepted September 28th, 1982)
Key words: pineal - N-acetyltransferase - circadian rhythm - phase-shift - light pulse - photoperiod
Extension of the rat pineal N-acetyltransferase rhythm after transition from LD 12:12 to LD 8:16 or to constant darkness proceeds into the morning hours. The rhythm is more extended in continuous darkness than in LD 8:16. After a compression caused by 1 min light pulse at 03.00 h the rhythm gradually decompresses in darkness for more than 4 days.
The pineal gland of rats exhibits a circadian rhythm in the activity of N-acetyltransferase (acetyl CoA: arylamine N-acetyltransferase, EC 2.3.1.5) (NAT), the enzyme responsible for the rhythmic production of melatonin8.9. Our previous data are consistent with a hypothesis that the NAT rhythm is regulated by a two-oscillator pacemaker 6,7. The evening NAT rise is controlled by an evening oscillator coupled to dusk, the morning NAT decline is controlled by a morning oscillator coupled to dawn. The phaserelation between both oscillators may determine the period of high night NAT activity and hence the length of time for which melatonin levels are elevated. The period of high NAT activity is extended more on shorter than on longer days, as on long days light intruding in the late evening and early morning hours compresses the NAT rhythm 5,7. Besides the effect of long days, the period of high NAT activity may be also compressed by brief light pulses at night~. After transition of rats from short to long days or after 1 min light pulses at night, the NAT rhythm is compressed almost instantaneouslyS-L Information about the time-course and about the extent of the NAT rhythm decompression after transition of rats to short photoperiods are, however, still missing. As knowledge of the duration of high NAT activity and of its changes is important since the period of elevated melatonin forflOO6-Rqq'~/1~'~/ flOOO_f)O00/%03.00© 1983 Elsevier Biomedical Press
mation may provide photoperiodic species with information about environmental day length2-4, we studied the decompression of the NAT rhythm both in darkness and in short days. Male Wistar rats from our own breeding colony were used. They were housed at 23 __. 2 °C and fed ad libitum. Rats killed in LD 12:12 or released into darkness were maintained from the age of 30 days under the artificial lighting regime LD 12:12 till the age of 60 days. Thereafter they were either killed in LD 12:12 or they were released into constant darkness or they were pulsed and then released into darkness. These, which were pulsed, were exposed to light of intensity of 100 Lux at the cage level for 1 min either at 21.00 h or at 03.00 h. Thereafter they were released into constant darkness as the unpulsed controls and they were killed during the next night or the fourth night after the pulses. Rats killed after one week in LD 8:16 were maintained since the age of 3 days in LD 12:12. At the age of 50 days they were transferred to LD 8:16 and killed one week later. Rats killed after 11 weeks in LD 8:16 were also maintained since the age of 3 days in LD 12: 12. At the age of 30 days, they were transferred to LD 8:16 and killed 11 weeks later. The animal room was lit by 40 W Tesla fluorescent tubes, in LD 12:12 from 06.00 h to 18.00 h, in LD 8:16 from 08.00 h to 16.00 h. When rats were to be killed in darkness, they
177 were exposed to a very faint red light for less than 1 min prior to decapitation. Other animals in the room were sheltered from the faint red light by a black curtain. Pineal glands were removed rapidly and stored in petri dishes on solid CO 2. Within 48 h after decapitation, NAT activity was determined b y a modification ]° of the method of Deguchi and Axelrodz. Units of NAT activity were defined as nmol N-acetyltryptamine formed in 1 h/l mg of pineal tissue. Baseline day values were in the range of 0.05-0.10 nmol.h-l.mg -~. The time when NAT activity reached the value of 3 nmol.mg -~.h-] during its increase was arbitrarily chosen as the phase-reference point for the evening NAT rise and hence for the phase of the evening oscillator (E). Similarly, the time when NAT activity declined to 3 nmol.mg-t.h -~ during its decrease was chosen as the phase-reference point for the morning NAT decline and hence for the phase of the morning oscillator (M). The time distance between the phase-reference point for E and M, i.e. the phase-relation between E and M, determined thus the period when NAT activity was equal to 3 nmol.mg-~.h -1 or greater. This period was called the period of high NAT activity. When comparing two groups of animals, a time-distance between phase-reference points for E was termed a phase-shift in the evening NAT rise and the distance between points for M was termed a phase-shift in the morning NAT decline. After 4 and 8 days in darkness, the evening NAT rise in unpulsed rats occurred at approximately the same time as after one day in darkness; the morning NAT decline, however, was phase-delayed by 1 h after 4 days and by 2 h after 8 days as compared to the decline after one day in darkness (Fig. IA). Consequently, after 8 days, the period of high NAT activity was extended by 2 h (Table I). The decompression of the rhythm occurred thus only into the morning hours as if the morning oscillator were phase-delayed in darkness. Following the pulse at 21.00 h, the period of the high NAT activity was slightly compressed after one day in darkness as compared with the value of the unpulsed controls because the NAT rise was more phase-delayed
than the decline (Fig. IB, Table I). However, after 4 days in darkness, both the evening NAT rise and the morning decline were phase-delayed by the same extent as compared to the phases of the unpulsed rats and hence the period of high NAT acitivity was already the same as in the unpulsed controls. Following the pulse at 03.00 h, the NAT rise after one day in darkness occurred at about the same time as in the unpulsed controls, while the NAT decline was phase-advanced by 2.5 h (Fig. 1C). Consequently, the period of high NAT activity was drastically reduced. After 4 days in darkness, the morning decline was still phase-advanced by 2.5 h as compared with the decline in the unpulsed rats,
'r
12-
I
z
I
1'8
20
2'2
0'0
02
04 0'~ TIME OF DAY /I-I/
08
Fig. 1. Extensionof the N-acetyltransferaserhythm in darkness. Rats maintained for 4 weeks in LD 12:12 were either unpulsed (A) or they were pulsed by 1 min pulse at 21.00 h (B) or at 03.00h (C) and from that time on they were released into constant darkness. They were killed after 1 (O----O), 4 (O - - - O) and 8 (A ... A) days followingthe release into darkness. Data are expressed as means + S.E,M. of 4 animals. When S.E. are omitted they were lower than 0.2 nmol.mg-l.h-l.
178 TABLE I
Phase-reference points for evening N-ace(vltransferase rise andfor the morning decline and period of high N-acetyltransJbrase activi(v in darkness (after the values in Fig. l) Days in darkness
Nopulse
Pulse at 21. O0 h
Pulse at 03. O0h
Time of the evening N-acetyltransferase rise to 3 nmol.mg-m-h I(h)
1 4 8
20.52 21.08 20.48
22.28 22.52
21.12 19.28
Time of the morning N-acetyltransferase decline to 3 nmol.mg l . h l ( h )
I 4 8
05.20 06.16 07.20
06.28 08.08
02.48 03.48
Period of high N-acetyltransferase activity (3 nmol.mg-l.h l(h)
1 4 8
8.5h 9.1h 10.4h
8.0h 9.2h
5.6h 8.3h
but the evening rise was also phase-advanced, at that time by more than 1.5 h. The period of high NAT activity extended by 2.7 h as compared to the period one day after the pulse to a value which was about 1 h shorter than the period of the unpulsed controls. A slow time-course of the
,! z :i
t I
5-
l*li
/
,i
z ,( ..r
j..
~ v)4 z
I.
'ii
!
~f :
it
U ,4 !
li!..
z
16
M
20
22
O0
i
i
i
i
02
04
06
08
T I M E OF D A Y / H I
Fig. 2. Extension of the N-acetyltransferase rhythm in LD 8:16. Rats were either killed in LD 12:12 ( 0 - - - 0 ) or after 1 ((3 - - - ©) or 11 ( A . . . A) weeks following transition from LD 12:12 to LD 8:16. Data are expressed as means + S.E.M. of 4 animals. When S.E. are omitted they were lower than 0.1 nmol.mg-l.h-L
phase-advance of E after the pulse at 03.00 h is in accordance with observations in golden hamsters ~. In these animals, after the light pulse applied late in the subjective night, it takes several transient cycles before a phase-advance in the evening onset of locomotor activity is completed. After 1 and 11 weeks in LD 8: 16, the evening NAT rise occurred at almost the same time as in LD 12:12, while the morning NAT decline was phase-delayed by about 1 h (Fig. 2). Thus, in short days, the decompression proceeded into the morning hours as in constant darkness. The extension of the NAT rhythm was completed almost within one week. The period of high NAT activity after 11 weeks in LD 8: 16, which lasted 9.1 h, was about 1 h shorter than the period after 8 days in darkness or the period found in rats killed in natural daylight on December 197. In conclusion, we found that decompression of the pineal NAT rhythm in rats after transition from LD 12:12 to LD 8:16 or to constant darkness proceeds into the morning hours. The rhythm is more extended in continuous darkness than in LD 8:16. The effect of I min light pulses on the compression of the rhythm lasts for at least 4 days if the pulse is applied at 03.00 h, but not if it is applied at 21.00 h. We thank Mrs. M. Svobodov~i for excellent technical assistance and Mrs. M. Sumov~i for typing the manuscript.
179 1 Carter, D. S. and Goldman, B. D., Antigonadal and progonadal effects of programmed melatonin infusion into juvenile Djungarian hamsters (Phodopussungorus), Biol. Reprod., 24, Suppl. 1 (1981) 23A. 2 Deguchi, T. and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in rat pineal, Analyt. Biochem., 50 (1972) 174-179. 3 Hoffmann, K., Photoperiodic function of the mammalian pineal organ. In A. Oksche and P. Pevet (Eds.), The
Pineal Organ: Photobiology-Biochronometry Endocrinology, Elsevier, North-Holland Biomedical Press, Amsterdam, 1981, pp. 123-138. 4 Hoffmann, K., Illnerov/t, H. and Van~ek, J., Effect of photoperiod and of one minute light at night-time on the pineal rhythm of N-acetyltransferase activity in the Djungarian hamster Phodopus sungorus, Biol. Reprod., 24(1981) 551-556. 5 Illnerov~i, H. and Van~ek, J., Pineal rhythm in N-acetyltransferase activity in rats under different artificial photoperiods and in natural daylight in the course of a year, Neuroendocrinology, 31 (1980) 316-320. 6 Illnerov~i, H. and Van~ek, J., Two-oscillator structure of the pacemaker controlling the circadian rhythm of Nacetyltransferase in the rat pineal gland, J. comp. Phy-
siol., 145 (1982) 539-548. 7 Illnerov~i, 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.), Circadian Systems: Structure and Function, Springer, Heidelberg, 1982, pp. 285-296. 8 Illnerov/t, H., Van~dek, J. and Hoffmann, K., Regulation of the pineal melatonin concentration in the rat (Rattus norvegicus) and in the Djungarian hamster (Phodopus sungorus), Comp. Physiol. Biochem., in press. 9 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. 10 Parfitt, A., Weller, J. L., Klein, D. C., Sakai, K. K. and Marks, B. H., Blockade by ouabain or elevated potassium ion concentration of the adrenergic and adenosine cyclic Y,5'-monophosphate induced stimulation of pineal serotonin N-acetyltransferase activity, Molec. Pharmacol., 11 (1975)241 255. 11 Pittendrigh, C. S., Circadian clocks: what are they? In J. Woodward Hastings and H. C. Sweiger (Eds.), The Molecular Basis of Circadian Rhythms, Dahlem Konferenzen, Berlin, 1976, pp. 11-48.
Brain Research, 261 (1983) 176-179 Elsevier Biomedical Press
Extension of the rat pineal N-acetyttransferase rh~hm in con.nuous d a ~ n n s and on short pi',otqzeriod HELENA ILLNEROV,~ and Jl~,I VANECEK
Institute of Physiology, Czechoslovak Academy of Sciences, Videhska 1083, 142 20 Prague 4 (Czechoslovakia) (Accepted September 28th, 1982)
Key words: pineal - N-acetyltransferase - circadian rhythm - phase-shift - light pulse - photoperiod
Extension of the rat pineal N-acetyltransferase rhythm after transition from LD 12:12 to LD 8:16 or to constant darkness proceeds into the morning hours. The rhythm is more extended in continuous darkness than in LD 8:16. After a compression caused by 1 min light pulse at 03.00 h the rhythm gradually decompresses in darkness for more than 4 days.
The pineal gland of rats exhibits a circadian rhythm in the activity of N-acetyltransferase (acetyl CoA: arylamine N-acetyltransferase, EC 2.3.1.5) (NAT), the enzyme responsible for the rhythmic production of melatonin8.9. Our previous data are consistent with a hypothesis that the NAT rhythm is regulated by a two-oscillator pacemaker 6,7. The evening NAT rise is controlled by an evening oscillator coupled to dusk, the morning NAT decline is controlled by a morning oscillator coupled to dawn. The phaserelation between both oscillators may determine the period of high night NAT activity and hence the length of time for which melatonin levels are elevated. The period of high NAT activity is extended more on shorter than on longer days, as on long days light intruding in the late evening and early morning hours compresses the NAT rhythm 5,7. Besides the effect of long days, the period of high NAT activity may be also compressed by brief light pulses at night~. After transition of rats from short to long days or after 1 min light pulses at night, the NAT rhythm is compressed almost instantaneouslyS-L Information about the time-course and about the extent of the NAT rhythm decompression after transition of rats to short photoperiods are, however, still missing. As knowledge of the duration of high NAT activity and of its changes is important since the period of elevated melatonin forflOO6-Rqq'~/1~'~/ flOOO_f)O00/%03.00© 1983 Elsevier Biomedical Press
mation may provide photoperiodic species with information about environmental day length2-4, we studied the decompression of the NAT rhythm both in darkness and in short days. Male Wistar rats from our own breeding colony were used. They were housed at 23 __. 2 °C and fed ad libitum. Rats killed in LD 12:12 or released into darkness were maintained from the age of 30 days under the artificial lighting regime LD 12:12 till the age of 60 days. Thereafter they were either killed in LD 12:12 or they were released into constant darkness or they were pulsed and then released into darkness. These, which were pulsed, were exposed to light of intensity of 100 Lux at the cage level for 1 min either at 21.00 h or at 03.00 h. Thereafter they were released into constant darkness as the unpulsed controls and they were killed during the next night or the fourth night after the pulses. Rats killed after one week in LD 8:16 were maintained since the age of 3 days in LD 12:12. At the age of 50 days they were transferred to LD 8:16 and killed one week later. Rats killed after 11 weeks in LD 8:16 were also maintained since the age of 3 days in LD 12: 12. At the age of 30 days, they were transferred to LD 8:16 and killed 11 weeks later. The animal room was lit by 40 W Tesla fluorescent tubes, in LD 12:12 from 06.00 h to 18.00 h, in LD 8:16 from 08.00 h to 16.00 h. When rats were to be killed in darkness, they
177 were exposed to a very faint red light for less than 1 min prior to decapitation. Other animals in the room were sheltered from the faint red light by a black curtain. Pineal glands were removed rapidly and stored in petri dishes on solid CO 2. Within 48 h after decapitation, NAT activity was determined b y a modification ]° of the method of Deguchi and Axelrodz. Units of NAT activity were defined as nmol N-acetyltryptamine formed in 1 h/l mg of pineal tissue. Baseline day values were in the range of 0.05-0.10 nmol.h-l.mg -~. The time when NAT activity reached the value of 3 nmol.mg -~.h-] during its increase was arbitrarily chosen as the phase-reference point for the evening NAT rise and hence for the phase of the evening oscillator (E). Similarly, the time when NAT activity declined to 3 nmol.mg-t.h -~ during its decrease was chosen as the phase-reference point for the morning NAT decline and hence for the phase of the morning oscillator (M). The time distance between the phase-reference point for E and M, i.e. the phase-relation between E and M, determined thus the period when NAT activity was equal to 3 nmol.mg-~.h -1 or greater. This period was called the period of high NAT activity. When comparing two groups of animals, a time-distance between phase-reference points for E was termed a phase-shift in the evening NAT rise and the distance between points for M was termed a phase-shift in the morning NAT decline. After 4 and 8 days in darkness, the evening NAT rise in unpulsed rats occurred at approximately the same time as after one day in darkness; the morning NAT decline, however, was phase-delayed by 1 h after 4 days and by 2 h after 8 days as compared to the decline after one day in darkness (Fig. IA). Consequently, after 8 days, the period of high NAT activity was extended by 2 h (Table I). The decompression of the rhythm occurred thus only into the morning hours as if the morning oscillator were phase-delayed in darkness. Following the pulse at 21.00 h, the period of the high NAT activity was slightly compressed after one day in darkness as compared with the value of the unpulsed controls because the NAT rise was more phase-delayed
than the decline (Fig. IB, Table I). However, after 4 days in darkness, both the evening NAT rise and the morning decline were phase-delayed by the same extent as compared to the phases of the unpulsed rats and hence the period of high NAT acitivity was already the same as in the unpulsed controls. Following the pulse at 03.00 h, the NAT rise after one day in darkness occurred at about the same time as in the unpulsed controls, while the NAT decline was phase-advanced by 2.5 h (Fig. 1C). Consequently, the period of high NAT activity was drastically reduced. After 4 days in darkness, the morning decline was still phase-advanced by 2.5 h as compared with the decline in the unpulsed rats,
'r
12-
I
z
I
1'8
20
2'2
0'0
02
04 0'~ TIME OF DAY /I-I/
08
Fig. 1. Extensionof the N-acetyltransferaserhythm in darkness. Rats maintained for 4 weeks in LD 12:12 were either unpulsed (A) or they were pulsed by 1 min pulse at 21.00 h (B) or at 03.00h (C) and from that time on they were released into constant darkness. They were killed after 1 (O----O), 4 (O - - - O) and 8 (A ... A) days followingthe release into darkness. Data are expressed as means + S.E,M. of 4 animals. When S.E. are omitted they were lower than 0.2 nmol.mg-l.h-l.
178 TABLE I
Phase-reference points for evening N-ace(vltransferase rise andfor the morning decline and period of high N-acetyltransJbrase activi(v in darkness (after the values in Fig. l) Days in darkness
Nopulse
Pulse at 21. O0 h
Pulse at 03. O0h
Time of the evening N-acetyltransferase rise to 3 nmol.mg-m-h I(h)
1 4 8
20.52 21.08 20.48
22.28 22.52
21.12 19.28
Time of the morning N-acetyltransferase decline to 3 nmol.mg l . h l ( h )
I 4 8
05.20 06.16 07.20
06.28 08.08
02.48 03.48
Period of high N-acetyltransferase activity (3 nmol.mg-l.h l(h)
1 4 8
8.5h 9.1h 10.4h
8.0h 9.2h
5.6h 8.3h
but the evening rise was also phase-advanced, at that time by more than 1.5 h. The period of high NAT activity extended by 2.7 h as compared to the period one day after the pulse to a value which was about 1 h shorter than the period of the unpulsed controls. A slow time-course of the
,! z :i
t I
5-
l*li
/
,i
z ,( ..r
j..
~ v)4 z
I.
'ii
!
~f :
it
U ,4 !
li!..
z
16
M
20
22
O0
i
i
i
i
02
04
06
08
T I M E OF D A Y / H I
Fig. 2. Extension of the N-acetyltransferase rhythm in LD 8:16. Rats were either killed in LD 12:12 ( 0 - - - 0 ) or after 1 ((3 - - - ©) or 11 ( A . . . A) weeks following transition from LD 12:12 to LD 8:16. Data are expressed as means + S.E.M. of 4 animals. When S.E. are omitted they were lower than 0.1 nmol.mg-l.h-L
phase-advance of E after the pulse at 03.00 h is in accordance with observations in golden hamsters ~. In these animals, after the light pulse applied late in the subjective night, it takes several transient cycles before a phase-advance in the evening onset of locomotor activity is completed. After 1 and 11 weeks in LD 8: 16, the evening NAT rise occurred at almost the same time as in LD 12:12, while the morning NAT decline was phase-delayed by about 1 h (Fig. 2). Thus, in short days, the decompression proceeded into the morning hours as in constant darkness. The extension of the NAT rhythm was completed almost within one week. The period of high NAT activity after 11 weeks in LD 8: 16, which lasted 9.1 h, was about 1 h shorter than the period after 8 days in darkness or the period found in rats killed in natural daylight on December 197. In conclusion, we found that decompression of the pineal NAT rhythm in rats after transition from LD 12:12 to LD 8:16 or to constant darkness proceeds into the morning hours. The rhythm is more extended in continuous darkness than in LD 8:16. The effect of I min light pulses on the compression of the rhythm lasts for at least 4 days if the pulse is applied at 03.00 h, but not if it is applied at 21.00 h. We thank Mrs. M. Svobodov~i for excellent technical assistance and Mrs. M. Sumov~i for typing the manuscript.
179 1 Carter, D. S. and Goldman, B. D., Antigonadal and progonadal effects of programmed melatonin infusion into juvenile Djungarian hamsters (Phodopussungorus), Biol. Reprod., 24, Suppl. 1 (1981) 23A. 2 Deguchi, T. and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in rat pineal, Analyt. Biochem., 50 (1972) 174-179. 3 Hoffmann, K., Photoperiodic function of the mammalian pineal organ. In A. Oksche and P. Pevet (Eds.), The
Pineal Organ: Photobiology-Biochronometry Endocrinology, Elsevier, North-Holland Biomedical Press, Amsterdam, 1981, pp. 123-138. 4 Hoffmann, K., Illnerov/t, H. and Van~ek, J., Effect of photoperiod and of one minute light at night-time on the pineal rhythm of N-acetyltransferase activity in the Djungarian hamster Phodopus sungorus, Biol. Reprod., 24(1981) 551-556. 5 Illnerov~i, H. and Van~ek, J., Pineal rhythm in N-acetyltransferase activity in rats under different artificial photoperiods and in natural daylight in the course of a year, Neuroendocrinology, 31 (1980) 316-320. 6 Illnerov~i, H. and Van~ek, J., Two-oscillator structure of the pacemaker controlling the circadian rhythm of Nacetyltransferase in the rat pineal gland, J. comp. Phy-
siol., 145 (1982) 539-548. 7 Illnerov~i, 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.), Circadian Systems: Structure and Function, Springer, Heidelberg, 1982, pp. 285-296. 8 Illnerov/t, H., Van~dek, J. and Hoffmann, K., Regulation of the pineal melatonin concentration in the rat (Rattus norvegicus) and in the Djungarian hamster (Phodopus sungorus), Comp. Physiol. Biochem., in press. 9 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. 10 Parfitt, A., Weller, J. L., Klein, D. C., Sakai, K. K. and Marks, B. H., Blockade by ouabain or elevated potassium ion concentration of the adrenergic and adenosine cyclic Y,5'-monophosphate induced stimulation of pineal serotonin N-acetyltransferase activity, Molec. Pharmacol., 11 (1975)241 255. 11 Pittendrigh, C. S., Circadian clocks: what are they? In J. Woodward Hastings and H. C. Sweiger (Eds.), The Molecular Basis of Circadian Rhythms, Dahlem Konferenzen, Berlin, 1976, pp. 11-48.