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Uncoupling of the pineal melatonin synthesis of rats from the circadian regulation
ELSEVIER
Neuroscience Letters 179 (1994) 5-8
NEUROSCIENCE LETTERS
Uncoupling of the pineal melatonin synthesis of rats from the circadian regulation Maija-Liisa Laakso*, Taina H~it6nen, Aino Alila Institute of Biomedicine, Department of Physiology, University of Helsinki, PO Box 9, 00014 Helsinki, Finland Received 29 June 1994; Revised version received 27 July 1994; Accepted 27 July 1994
Abstract We show that the pineal melatonin synthesis of rats can be uncoupled from the circadian regulation by exposing the animals to abnormally long light periods. Male rats were kept 7 days under 22.5/1.5-h light/dark conditions and then exposed to darkness at different times of the day. After a 60-rain dark exposure, the melatonin synthesis increased independently of the time of the day (Expt. 1). During 22 h in darkness, the mean melatonin content did not return to the low daytime level (Expt. 2). A dark-induced, time-independent increase of melatonin was also found after the rats had been 3-7 days under constant light (Expts. 3 and 4). Key words: Circadian rhythm; Constant light; Dark pulse; Entrainment; Melatonin; Pineal gland
Melatonin (5-methoxy-N-acetyltryptamine) is synthetized in the pineal gland from serotonin which is acetylated by the rate-limiting enzyme N-acetyltransferase (NAT) and further methylated by hydroxyindole-Omethyltransferase [10,15]. The formation of active NAT depends on the activity of the sympathetic nerves originating from the superior cervical ganglia [4]. Combined fl-l-adrenergic and a-l-adrenergic mechanisms produce a synergistic increase in cAMP which is a precondition for the synthesis of NAT (or its regulator) both at the transcriptional and translational level, and for the maintenance of NAT in an active form [5,6]. The sympathetic superior cervical ganglia are under the control of a circadian oscillator located in the hypothalamic suprachiasmatic nuclei (SCN). The endogenous rhythm of the SCN is entrained by the light/ dark cycles which are signalled by nerve impulses from the retina [4]. Abundant melatonin synthesis is possible only at a certain phase of the circadian oscillator and only if illuminance is low enough. In constant light, the melatonin rhythm is inhibited; in constant darkness the rhythm continues flee-running according to the endogenous rhythm of the oscillator. In laboratory rats,
*Corresponding author. Fax: (358) (0) 1918681. 0304-3940/94l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00602-4
the melatonin synthesis can be stimulated even during the normal light period by fl-adrenergic drugs [1,2,13]. However, if the rats are exposed to darkness during the light phase of the day, the melatonin synthesis does not increase. The circadian regulatory system inhibits the rise by a mechanism which probably comprises a feedback control of the suprachiasmatic oscillator by melatonin [12] and intrapineal regulation of the expression of genes [14]. Our previous experiments showed that the pineal melatonin synthesis in rats could be adapted to 22.5/1.5h light/dark (LD) conditions; the synthesis increased at 24-h intervals at the beginning of each short dark period [7]. Simultaneously, however, the locomotor activity rhythm was free-running with a period o f - 2 5 h [9]. The dissociation of the two rhythms suggested that they were somewhat differently controlled. This finding made us explore whether the rhythmic melatonin synthesis was a sign of a 24-h rhythm of a neural oscillator or whether it only reflected the rhythmic changes of environmental lighting. It was rationalized that, if the melatonin rhythm was uncoupled from the neural oscillator, the synthesis should start at any time of the day if darkness interrupts the light period. In Expt. l, male Wistar rats (Han:Wist, age 2-3 months, weight 270-320 g) were kept for 7 days in
6
M.-L. Laakso et al./Neuroscience Letters 179 (1994) 5 ~
3 "0 C
13)
"1
ILl
C
-12
-6 0 SHIFT h
+6
+12
Fig. 1. Pineal melatonin contents (mean and S.D. values, n = 9-10) in male rats kept for 1 week under 22.5/1.5-h light/dark conditions and then exposed to darkness at different times of the day. The first seven 90-min dark pulses were from 18:00 to 19:30, then the 8th light-off time was advanced or delayed 6 or 12 h. One group of rats was exposed to darkness at the expected time at 18:00 (group 0). The rats were left in the dark for 60 min and then killed with a guillotine under dim red light within 15 min. The control values of melatonin (group C) were obtained from rats not exposed to darkness. Kruskal-Wallis non-parametric ANOVA P < 0.001. Dunn's multiple comparisons test: *different from the control, P < 0.05; **P < 0.01; the groups exposed to darkness did not differ from each other.
22.5/1.5-h LD conditions (illuminance 100-150 lx at the level of the cages, cool white fluorescent tubes, complete darkness from 18:00 to 19:30). On day 8, the time of the dark pulse was advanced or delayed by 6 or 12 h. The pineal glands were collected after the rats had been in the dark for 60 min and the melatonin contents were measured by RIA [7,8,16]. Fig. 1 shows that the rise of the pineal melatonin content was similar at all times of the day. We concluded that the increase of melatonin was under the direct control of light and dark, and not under the control of the circadian clock which continued to maintain a free-running locomotor activity rhythm for at least 10 days [9]. In Expt. 2, we explored whether the circadian regulation of the melatonin synthesis could be restored by darkness long enough to enable a melatonin peak of a normal duration (-10 h). The rats were kept 7 days in 22.5/1.5-h LD conditions and then in continuous darkness. The pineal samples were collected at 3-h intervals. Fig. 2 shows that the average pineal melatonin content did not return to the normal low level during a 22-h dark period. The circadian system was not able to take control of the melatonin synthesis or the rhythms in individual rats were quite asynchronous. The average melatonin contents reached within 1 h or maintained during the 22-h follow-up period were 2030% of the normal peak levels measured in our strain of rats with the same reagents and procedures [7]. The interindividual variation was large: 18% of the rats killed during darkness in Expt. 2 had melatonin contents of <0.7 ng/gland (the practical upper limit of the normal daytime content) and 6% > 3.0 ng/gland (the practical lower limit of the normal peak content). However, in
most rats (76%), the pineal melatonin content was between these limits, suggesting that even in individual rats the rate of the melatonin synthesis remained at an intermediate level dispite prolonged darkness. The reason for the abnormally low melatonin levels cannot be explained on the basis of this study. In Expt. 3, we examined whether the uncoupling of the melatonin synthesis from the circadian regulation was solely due to abnormally long light periods or whether it required intermittent dark pulses. The rats were 7 days under continuous light and then 60 min in complete darkness beginning at 18:00, 24:00, 06:00 or 12:00. Again the rise of the melatonin synthesis was independent of the time of the day (Fig. 3A). We concluded that the dark pulses were not necessary for the uncoupling of the pineal glands from the circadian regulation. Too long light periods 'stopped the melatonin clock at the evening phase'. The length of the light period required for the uncoupling of the rat's melatonin synthesis from the circadian regulation was studied in Expt. 4. After being kept in constant light for 1-5 days, the rats were taken into the dark at 12:00 and killed at 13:00. In some rats, the melatonin contents exceeded the usual daytime levels after 1 or 2 days of constant light. The group average was, however, increased significantly only after 3 days under light (Fig. 3B). An equal period was required under the 22.5/1.5-h LD conditions in our earlier experiment to produce a statistically significant increase of melatonin during the dark pulse [7]. "0 C
3 ¥¥¥
01 C
2 Z m Z
0
41 3Z
16
19
22
1 4 TIME h
7
10
13
16
Fig. 2. Pineal melatonin contents (mean and S.E.M. values, n -- 7-10) in male rats kept 7 days under 22.5/1.5-h light/dark conditions and then in constant darkness during the sampling. The pineal glands were collected at 3-h intervals. Darkness is indicated by the hatched bars on the abscissa: the lower bar indicates the week preceding the sampling, the upper bar the day of sampling. One-way ANOVA for the logarithmic values: P < 0.0001. Tukey-Kramer multiple comparisons test: *different from the first value under light P < 0.05, **P < 0.01, ***P < 0.001; the melatonin contents during the dark did not differ significantly from each other.
M.-L. Laakso et al. I Neuroscience Letters 179 (1994) 5-8
night phase of the circadian oscillator is well known. Our results show that a direct stimulating effect of darkness may pass a free-running circadian regulatory system even during the day. The differences between the circadian and photic control of the pineal melatonin synthesis are unknown. In addition to the sympathetic innervation, the pineal gland receives other peripheral fibers and even central input from the lateral hypothalamus, habenula and paraventricular regions of the brain [11]. In addition to the adrenergic receptors, the pineal gland is rich in other receptors for regulatory substances [3] which may modulate the adrenergic regulation. The present finding offers a strategy to study the circadian and photic regulations separately both at the neural and pineal levels.
A
c
19
01
07
13
h
z
"~ 3 0
B
.J "'
7
2
C
1
2 3 days
4
5
Fig. 3. Pineal melatonin contents (mean and S.D. values, n = 8-10) in male rats kept under constant light for 7 days (A) or 1-5 days (B) and then exposed to darkness for 60 min. The rats kept under light for 1 week were released into darkness at 18:00, 24:00, 06:00 or 12:00 on the 8th day. The control group C was not exposed to darkness. The rats kept under light over 1-5 nights were released the following day into darkness at 12:00. Kruskal-Wallis non-parametric ANOVA both for A and B P < 0.001; Dunn's multiple comparisons test: *different from the control, P < 0.05; **P < 0.01; ***P < 0.001; in A the groups exposed to darkness did not differ from each other, in B the group kept 2 days under light differed almost significantly (P < 0.05) from the group kept 3 days under light.
A latency of 1-2 h precedes the initiation of melatonin rise in rats when the synthesis is stimulated by fl-adrenergic agonists during the daytime or by turning off the lights after a normal light period. Inhibitors of protein synthesis block the induction of NAT by adrenergic drugs, indicating that the lag is required for the substitution of degraded NAT molecules by new ones [1,2,13]. In the 22.5/1.5-h LD conditions, the pineal melatonin content increased in some rats within 15 min [7]. Thus, when the pineal gland is uncoupled from circadian regulation by abnormally long light periods, a sufficient amount of synthetized NAT seems to be ready for the dark-elicited activation. Our findings suggest that the synthesis of NAT (or its regulator) and the activation of NAT are separately controlled. The circadian clock in its certain phase may initiate and complete the synthesis while the neural information originating from the retina is responsible for the inhibition and activation of the enzyme. The direct inhibitory effect of light on the melatonin synthesis during the
[1] Deguchi, T., Role of the beta adrenergic receptor in the elevation of adenosine cyclic 3',5'-monophosphate and induction of serotonin N-acetyltransferase in rat pineal glands, Mol. Pharmacol., 9 (1973) 184-190. [2] Deguchi, T. and Axelrod, J., Control of circadian change of serotonin N-acetyltransferase activity in the pineal organ by the betaadrenergic receptor, Proc. Natl. Acad. Sci. USA, 69 (1972) 25472550. [3] Ebadi, M. and Govitrapong, P., Orphan transmitters and their receptor sites in the pineal gland. In R.J. Reiter (Ed.), Pineal Research Reviews, Vol. 4, Alan R. Liss, New York, NY, 1986, pp. 1-54,
[4] 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. [5] Klein, D.C., Schaad, N.L., Namboordiri, M.A.A., Yu, L. and Weller, J.L., Regulation of pineal serotonin N-acetyltransferase activity, Biochem. Soc. Trans., 20 (1992) 299-304. [6] Klein, D.C., Sugden, D. and Weller, J.L., Postsynaptic alphaladrenergic receptors potentiate the beta-adrenergic stimulation of serotonin N-acetyltransferase, Proc. Natl. Acad. Sci. USA, 80 (1983) 599~503. [7] Laakso, M.-L., H/it6nen, T. and Alila, A., The adjustment of the melatonin rhythm of rats by 90-min dark pulses, Neurosci. Lett., 166 (1994) 13-16. [8] Laakso, M.-L., Porkka-Heiskanen, T., Alila, A., Peder, M. and Johansson, G., Twenty-four-hour patterns of pineal melatonin, and pituitary and plasma prolactin in male rats under 'natural' and artificial lighting conditions, Neuroendocrinology, 48 (1988) 308313. [9] Laakso, M.-L., Porkka-Heiskanen, T., Leinonen, L., Joutsiniemi, S.L. and M~innist6, P.T., Hormonal and locomotor activity rhythms in rats under 90-rain dark-pulse conditions, Am. J. Physiol., 264 (1993) R1058-R1064. [10] Reiter, R.J., Pineal melatonin - cell biology of its synthesis and of its physiological interactions, Endocr. Rev., 12 (1991) 151-180. [11] Reuss, S., Workings of the internal clock - neuroanatomy of the circadian system of mammals, Naturwissenschaften, 80 (1993) 501-510. [12] Rietveld, W.J., The circadian network are feedback loops to the circadian oscillator of any functional importance to its control function, J. Interdisc. Cycle Res., 23 (1992) 140-142. [13] Romero, J.A., Zatz, M. and Axelrod, J., Beta-adrenergic stimulation of pineal N-acetyltransferase: adenosine 3":5'-cyclic monophosphate stimulates both RNA and protein synthesis, Proc. Natl. Acad. Sci. USA, 72 (1975)2107-2111.
8
M.-L. Laakso et aL/Neuroscience Letters 179 (1994) 5~8
[14] Stehle, J.H., Foulkes, N.S., Molina, C.A., Simonneaux, V., P6vet, P. and Sassone-Corsi, P., Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland, Nature (London), 365 (1993) 314-320. [15] Sugden, D., Melatonin biosynthesis in the mammalian pineal gland, Experientia, 45 (1989) 922-932.
[16] Vakkuri, O., Lepp/iluoto, J. and Vuolteenaho, O., Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinol. (Copenhagen), 106 (1984) 152 157.
Neuroscience Letters 179 (1994) 5-8
NEUROSCIENCE LETTERS
Uncoupling of the pineal melatonin synthesis of rats from the circadian regulation Maija-Liisa Laakso*, Taina H~it6nen, Aino Alila Institute of Biomedicine, Department of Physiology, University of Helsinki, PO Box 9, 00014 Helsinki, Finland Received 29 June 1994; Revised version received 27 July 1994; Accepted 27 July 1994
Abstract We show that the pineal melatonin synthesis of rats can be uncoupled from the circadian regulation by exposing the animals to abnormally long light periods. Male rats were kept 7 days under 22.5/1.5-h light/dark conditions and then exposed to darkness at different times of the day. After a 60-rain dark exposure, the melatonin synthesis increased independently of the time of the day (Expt. 1). During 22 h in darkness, the mean melatonin content did not return to the low daytime level (Expt. 2). A dark-induced, time-independent increase of melatonin was also found after the rats had been 3-7 days under constant light (Expts. 3 and 4). Key words: Circadian rhythm; Constant light; Dark pulse; Entrainment; Melatonin; Pineal gland
Melatonin (5-methoxy-N-acetyltryptamine) is synthetized in the pineal gland from serotonin which is acetylated by the rate-limiting enzyme N-acetyltransferase (NAT) and further methylated by hydroxyindole-Omethyltransferase [10,15]. The formation of active NAT depends on the activity of the sympathetic nerves originating from the superior cervical ganglia [4]. Combined fl-l-adrenergic and a-l-adrenergic mechanisms produce a synergistic increase in cAMP which is a precondition for the synthesis of NAT (or its regulator) both at the transcriptional and translational level, and for the maintenance of NAT in an active form [5,6]. The sympathetic superior cervical ganglia are under the control of a circadian oscillator located in the hypothalamic suprachiasmatic nuclei (SCN). The endogenous rhythm of the SCN is entrained by the light/ dark cycles which are signalled by nerve impulses from the retina [4]. Abundant melatonin synthesis is possible only at a certain phase of the circadian oscillator and only if illuminance is low enough. In constant light, the melatonin rhythm is inhibited; in constant darkness the rhythm continues flee-running according to the endogenous rhythm of the oscillator. In laboratory rats,
*Corresponding author. Fax: (358) (0) 1918681. 0304-3940/94l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00602-4
the melatonin synthesis can be stimulated even during the normal light period by fl-adrenergic drugs [1,2,13]. However, if the rats are exposed to darkness during the light phase of the day, the melatonin synthesis does not increase. The circadian regulatory system inhibits the rise by a mechanism which probably comprises a feedback control of the suprachiasmatic oscillator by melatonin [12] and intrapineal regulation of the expression of genes [14]. Our previous experiments showed that the pineal melatonin synthesis in rats could be adapted to 22.5/1.5h light/dark (LD) conditions; the synthesis increased at 24-h intervals at the beginning of each short dark period [7]. Simultaneously, however, the locomotor activity rhythm was free-running with a period o f - 2 5 h [9]. The dissociation of the two rhythms suggested that they were somewhat differently controlled. This finding made us explore whether the rhythmic melatonin synthesis was a sign of a 24-h rhythm of a neural oscillator or whether it only reflected the rhythmic changes of environmental lighting. It was rationalized that, if the melatonin rhythm was uncoupled from the neural oscillator, the synthesis should start at any time of the day if darkness interrupts the light period. In Expt. l, male Wistar rats (Han:Wist, age 2-3 months, weight 270-320 g) were kept for 7 days in
6
M.-L. Laakso et al./Neuroscience Letters 179 (1994) 5 ~
3 "0 C
13)
"1
ILl
C
-12
-6 0 SHIFT h
+6
+12
Fig. 1. Pineal melatonin contents (mean and S.D. values, n = 9-10) in male rats kept for 1 week under 22.5/1.5-h light/dark conditions and then exposed to darkness at different times of the day. The first seven 90-min dark pulses were from 18:00 to 19:30, then the 8th light-off time was advanced or delayed 6 or 12 h. One group of rats was exposed to darkness at the expected time at 18:00 (group 0). The rats were left in the dark for 60 min and then killed with a guillotine under dim red light within 15 min. The control values of melatonin (group C) were obtained from rats not exposed to darkness. Kruskal-Wallis non-parametric ANOVA P < 0.001. Dunn's multiple comparisons test: *different from the control, P < 0.05; **P < 0.01; the groups exposed to darkness did not differ from each other.
22.5/1.5-h LD conditions (illuminance 100-150 lx at the level of the cages, cool white fluorescent tubes, complete darkness from 18:00 to 19:30). On day 8, the time of the dark pulse was advanced or delayed by 6 or 12 h. The pineal glands were collected after the rats had been in the dark for 60 min and the melatonin contents were measured by RIA [7,8,16]. Fig. 1 shows that the rise of the pineal melatonin content was similar at all times of the day. We concluded that the increase of melatonin was under the direct control of light and dark, and not under the control of the circadian clock which continued to maintain a free-running locomotor activity rhythm for at least 10 days [9]. In Expt. 2, we explored whether the circadian regulation of the melatonin synthesis could be restored by darkness long enough to enable a melatonin peak of a normal duration (-10 h). The rats were kept 7 days in 22.5/1.5-h LD conditions and then in continuous darkness. The pineal samples were collected at 3-h intervals. Fig. 2 shows that the average pineal melatonin content did not return to the normal low level during a 22-h dark period. The circadian system was not able to take control of the melatonin synthesis or the rhythms in individual rats were quite asynchronous. The average melatonin contents reached within 1 h or maintained during the 22-h follow-up period were 2030% of the normal peak levels measured in our strain of rats with the same reagents and procedures [7]. The interindividual variation was large: 18% of the rats killed during darkness in Expt. 2 had melatonin contents of <0.7 ng/gland (the practical upper limit of the normal daytime content) and 6% > 3.0 ng/gland (the practical lower limit of the normal peak content). However, in
most rats (76%), the pineal melatonin content was between these limits, suggesting that even in individual rats the rate of the melatonin synthesis remained at an intermediate level dispite prolonged darkness. The reason for the abnormally low melatonin levels cannot be explained on the basis of this study. In Expt. 3, we examined whether the uncoupling of the melatonin synthesis from the circadian regulation was solely due to abnormally long light periods or whether it required intermittent dark pulses. The rats were 7 days under continuous light and then 60 min in complete darkness beginning at 18:00, 24:00, 06:00 or 12:00. Again the rise of the melatonin synthesis was independent of the time of the day (Fig. 3A). We concluded that the dark pulses were not necessary for the uncoupling of the pineal glands from the circadian regulation. Too long light periods 'stopped the melatonin clock at the evening phase'. The length of the light period required for the uncoupling of the rat's melatonin synthesis from the circadian regulation was studied in Expt. 4. After being kept in constant light for 1-5 days, the rats were taken into the dark at 12:00 and killed at 13:00. In some rats, the melatonin contents exceeded the usual daytime levels after 1 or 2 days of constant light. The group average was, however, increased significantly only after 3 days under light (Fig. 3B). An equal period was required under the 22.5/1.5-h LD conditions in our earlier experiment to produce a statistically significant increase of melatonin during the dark pulse [7]. "0 C
3 ¥¥¥
01 C
2 Z m Z
0
41 3Z
16
19
22
1 4 TIME h
7
10
13
16
Fig. 2. Pineal melatonin contents (mean and S.E.M. values, n -- 7-10) in male rats kept 7 days under 22.5/1.5-h light/dark conditions and then in constant darkness during the sampling. The pineal glands were collected at 3-h intervals. Darkness is indicated by the hatched bars on the abscissa: the lower bar indicates the week preceding the sampling, the upper bar the day of sampling. One-way ANOVA for the logarithmic values: P < 0.0001. Tukey-Kramer multiple comparisons test: *different from the first value under light P < 0.05, **P < 0.01, ***P < 0.001; the melatonin contents during the dark did not differ significantly from each other.
M.-L. Laakso et al. I Neuroscience Letters 179 (1994) 5-8
night phase of the circadian oscillator is well known. Our results show that a direct stimulating effect of darkness may pass a free-running circadian regulatory system even during the day. The differences between the circadian and photic control of the pineal melatonin synthesis are unknown. In addition to the sympathetic innervation, the pineal gland receives other peripheral fibers and even central input from the lateral hypothalamus, habenula and paraventricular regions of the brain [11]. In addition to the adrenergic receptors, the pineal gland is rich in other receptors for regulatory substances [3] which may modulate the adrenergic regulation. The present finding offers a strategy to study the circadian and photic regulations separately both at the neural and pineal levels.
A
c
19
01
07
13
h
z
"~ 3 0
B
.J "'
7
2
C
1
2 3 days
4
5
Fig. 3. Pineal melatonin contents (mean and S.D. values, n = 8-10) in male rats kept under constant light for 7 days (A) or 1-5 days (B) and then exposed to darkness for 60 min. The rats kept under light for 1 week were released into darkness at 18:00, 24:00, 06:00 or 12:00 on the 8th day. The control group C was not exposed to darkness. The rats kept under light over 1-5 nights were released the following day into darkness at 12:00. Kruskal-Wallis non-parametric ANOVA both for A and B P < 0.001; Dunn's multiple comparisons test: *different from the control, P < 0.05; **P < 0.01; ***P < 0.001; in A the groups exposed to darkness did not differ from each other, in B the group kept 2 days under light differed almost significantly (P < 0.05) from the group kept 3 days under light.
A latency of 1-2 h precedes the initiation of melatonin rise in rats when the synthesis is stimulated by fl-adrenergic agonists during the daytime or by turning off the lights after a normal light period. Inhibitors of protein synthesis block the induction of NAT by adrenergic drugs, indicating that the lag is required for the substitution of degraded NAT molecules by new ones [1,2,13]. In the 22.5/1.5-h LD conditions, the pineal melatonin content increased in some rats within 15 min [7]. Thus, when the pineal gland is uncoupled from circadian regulation by abnormally long light periods, a sufficient amount of synthetized NAT seems to be ready for the dark-elicited activation. Our findings suggest that the synthesis of NAT (or its regulator) and the activation of NAT are separately controlled. The circadian clock in its certain phase may initiate and complete the synthesis while the neural information originating from the retina is responsible for the inhibition and activation of the enzyme. The direct inhibitory effect of light on the melatonin synthesis during the
[1] Deguchi, T., Role of the beta adrenergic receptor in the elevation of adenosine cyclic 3',5'-monophosphate and induction of serotonin N-acetyltransferase in rat pineal glands, Mol. Pharmacol., 9 (1973) 184-190. [2] Deguchi, T. and Axelrod, J., Control of circadian change of serotonin N-acetyltransferase activity in the pineal organ by the betaadrenergic receptor, Proc. Natl. Acad. Sci. USA, 69 (1972) 25472550. [3] Ebadi, M. and Govitrapong, P., Orphan transmitters and their receptor sites in the pineal gland. In R.J. Reiter (Ed.), Pineal Research Reviews, Vol. 4, Alan R. Liss, New York, NY, 1986, pp. 1-54,
[4] 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. [5] Klein, D.C., Schaad, N.L., Namboordiri, M.A.A., Yu, L. and Weller, J.L., Regulation of pineal serotonin N-acetyltransferase activity, Biochem. Soc. Trans., 20 (1992) 299-304. [6] Klein, D.C., Sugden, D. and Weller, J.L., Postsynaptic alphaladrenergic receptors potentiate the beta-adrenergic stimulation of serotonin N-acetyltransferase, Proc. Natl. Acad. Sci. USA, 80 (1983) 599~503. [7] Laakso, M.-L., H/it6nen, T. and Alila, A., The adjustment of the melatonin rhythm of rats by 90-min dark pulses, Neurosci. Lett., 166 (1994) 13-16. [8] Laakso, M.-L., Porkka-Heiskanen, T., Alila, A., Peder, M. and Johansson, G., Twenty-four-hour patterns of pineal melatonin, and pituitary and plasma prolactin in male rats under 'natural' and artificial lighting conditions, Neuroendocrinology, 48 (1988) 308313. [9] Laakso, M.-L., Porkka-Heiskanen, T., Leinonen, L., Joutsiniemi, S.L. and M~innist6, P.T., Hormonal and locomotor activity rhythms in rats under 90-rain dark-pulse conditions, Am. J. Physiol., 264 (1993) R1058-R1064. [10] Reiter, R.J., Pineal melatonin - cell biology of its synthesis and of its physiological interactions, Endocr. Rev., 12 (1991) 151-180. [11] Reuss, S., Workings of the internal clock - neuroanatomy of the circadian system of mammals, Naturwissenschaften, 80 (1993) 501-510. [12] Rietveld, W.J., The circadian network are feedback loops to the circadian oscillator of any functional importance to its control function, J. Interdisc. Cycle Res., 23 (1992) 140-142. [13] Romero, J.A., Zatz, M. and Axelrod, J., Beta-adrenergic stimulation of pineal N-acetyltransferase: adenosine 3":5'-cyclic monophosphate stimulates both RNA and protein synthesis, Proc. Natl. Acad. Sci. USA, 72 (1975)2107-2111.
8
M.-L. Laakso et aL/Neuroscience Letters 179 (1994) 5~8
[14] Stehle, J.H., Foulkes, N.S., Molina, C.A., Simonneaux, V., P6vet, P. and Sassone-Corsi, P., Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland, Nature (London), 365 (1993) 314-320. [15] Sugden, D., Melatonin biosynthesis in the mammalian pineal gland, Experientia, 45 (1989) 922-932.
[16] Vakkuri, O., Lepp/iluoto, J. and Vuolteenaho, O., Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer. Acta Endocrinol. (Copenhagen), 106 (1984) 152 157.