Steinlechner S, Champney TH, Houston ML, Reiter RJ (1984). Simultaneous determination of N-acetyltransferase activity, hydroxyindole-O-methyl-transferase ...
Journal of Sensory, Comparative and .o.,a,.
J Comp Physiol A (1987) 160:593-597
Physiology A
Physiology ~~
9 Springer-Verlag 1987
Circadian rhythms of pineal N-acetyltransferase activity in the Djungarian hamster, Phodopus sungorus, in response to seasonal changes of natural photoperiod* Stephan Steinlechner, Astrid Buchberger, and Gerhard Heldmaier Department of Biology/Zoology, Philipps-University, D-3550 Marburg, Federal Republic of Germany Accepted February 4, 1987
Summary. The aim of this study was to describe the regular annual pattern of the daily melatonin synthesis in Djungarian hamsters, Phodopus sungorus sungorus. The hamsters were maintained from birth in natural photoperiodic conditions and in bimonthly intervals the day/night rhythms of pineal N-acetyltransferase (NAT) were measured. Analysis of the circadian profiles of NAT activity showed that the duration of elevated melatonin synthesis closely reflects the duration of the scotophase throughout the seasons. Thus the duration of elevated melatonin seems to represent a direct humoral signal transmitting the photoperiodic message. The duration of the nightly melatonin pulse appears to be influenced mainly by the time of dawn rather than by the time of dusk. Additional information about the time of year might be encoded in the total amount of melatonin synthesized per day, whereas the amplitude of the nightly melatonin peak seems to be of minor importance.
Introduction The Djungarian hamster, Phodopus sungorus sungorus, is known to be a strongly photoperiodic species. This small rodent, native to the Siberian steppe, shows conspicuous seasonal rhythms in a number of physiological and morphological functions such as gonadal size and activity, body weight, pelage color and several thermoregulatory parameters. For all these rhythms photoperiodic control has been demonstrated (Hoffmann /973, 1981; Heldmaier et al. 1981; Steinlechner and Abbreviation: N A T N-acetyltransferase
* Dedicated to Dr. Klaus Hoffmann on the occasion of his 60th birthday
Heldmaier 1982). All these d a t a imply that this hamster is able to use the information about the photoperiod to adaptively anticipate other seasonal changes in its environment. Information on day length is thought to be transduced via the pineal gland and its pattern of melatonin secretion (Axelrod 1974). A unique feature of pineal melatonin synthesis is its circadian rhythmicity with high levels found during the night and very low levels during the daylight hours (Wurtman et al. 1963). This circadian rhythm is driven by a rhythm in the activity of the rate limiting enzyme of melatonin synthesis, namely N-acetyltransferase (NAT), which is synchronized with the daily light/dark cycle (Klein and Weller 1970). According to Goldman (/983) there are four primary properties of the melatonin rhythm which might comprise information about the day length. These properties are (1) the amplitude of the nocturnal melatonin peak, (2) the phase relationship of the peak to the light/ dark cycle or other physiological rhythms, (3) the duration of the nocturnal melatonin secretion, and (4) the total amount of melatonin secreted. In a series of very elegant experiments, Goldman and coworkers (Carter and Goldman 1983a, b; Goldman et al. 1984) have recently demonstrated that in Phodopus the duration of melatonin production is probably the critical parameter conveying the photoperiodic information. This result is in agreement with the findings that the nocturnal elevation of pineal melatonin in Phodopus spans 5-6 hours in an artificial photoperiod of 16L:8D and 1112 hours in 10L:14D (Goldman et al./981). Using pineal NAT activity as an index for melatonin synthesis, Hoffmann and coworkers (/981) found marked differences in the temporal pattern of pineal melatonin formation of Phodopus kept in either long or short photoperiod. Similarly, Illnerovfi and Van~6ek (1980) reported that in rats housed under
594
S. Steinlechner et al. : Seasonal changes of circadian N A T activity rhythms
different artificial photoperiods the duration of high night NAT activity was shortest under 16L:8D and longest under 8L:I6D. However, these authors also showed that there are obvious differences between NAT rhythms of rats kept in natural daylight and those exposed to artificial photoperiods. They contend that these differences might be the result of the previous light history of the animals (Illnerovfi and V a n ~ e k 1980). In order to see how the profile of melatonin synthesis is influenced by the seasonal change of natural photoperiod, we measured daily rhythms of pineal NAT activity in hamsters maintained continuously under natural photoperiod throughout the course of a year.
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48 h using a combination of the method of Deguchi and Axelrod (1972) and a micro method described by Witte and Matthaei (~980). Our modification of these assays has been described in detail elsewhere (Steinlechner et al. 1984). All samples were measured in duplicates. For the entire series of enzyme determination of the present study a single batch of t4C-acetylcoenzyme A was used.
Data analysis. Results were expressed as means+_1 standard error of the mean (x _+1 SEM). Daytime levels of enzyme activity were found to be consistently low (mean 0.556 • 0.03 nmoles pineal- ~ h - t ) throughout the year. Therefore, the amplitude of the N A T rhythm ( = p e a k amplitude) of each daily profile was determined by simply subtracting this mean value from the corresponding N A T peak activity. Similarly, the duration of elevated N A T activity ( = pulse duration) was arbitrarily determined by measuring the time interval between the two time points where N A T activity was significantly elevated (i.e. mean daytime level + 99% confidence interval). In the case When the end point of this time interval fell outside the dark period (e.g. on June 30, Fig. 1) only the time interval until the end of darkness was used. Since pineal N A T activity declines very rapidly after animals are exposed to light at night (halflife between 3 and 8 min; Klein and Weller 1972; Illnerovfi et al. 1979; Rollag et al. 1980; Brainard et al. 1982) it can be assumed that
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N-acetyltransferase assay. N A T activity was measured within
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All hamsters used in the present study were obtained from a breeding colony maintained in our laboratory. The colony is kept under natural photoperiodic conditions (Marburg 50 ~ 49'N, 09~ and thus breeding occurs only between March through September. Illumination of the animal room is provided by windows facing north and overhead fluorescent lighting controlled by a timer that was reset weekly according to sunrise and sunset. Ambient temperature was held constant throughout the year (23 ~ C) by air condition. Litters were weaned at 20 days of age and subsequently housed in individual cages. Hamsters received food (Altromin pellets) and water ad libitum, supplemented weekly with slices of apple. Only adult hamsters of both sexes aged 3 to 9 months were used in this study. At the dates and times indicated in Fig. 1 five animals per point were killed by decapitation. Each pineal was quickly removed, immediately frozen on dry ice, and subsequently stored at - 6 0 ~ During the dark period the pineal glands were collected with the aid of a dim red light.
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Fig. 1. Day/night profiles of N-acetyltransferase activity in bimonthly intervals throughout the course of a year. Shaded area represents time of darkness for each particular date. Values represent mean + 1 SEM for 5 hamster pineal glands each. Open circles : animals killed during daylight hours; closed circles : animals killed in darkness
S. Steinlechner et al. : Seasonal changes of circadian N A T activity rhythms
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Fig. 2. Seasonal course of a amplitude, b integral ( = t o t a l amount), e time of peak N A T activity relative to true midnight, and d pulse duration of the day/night profiles of N-acetyltransferase activity as shown in Fig. 1
NAT activity promptly drops to low daytime values at sunrise. The area enclosed by a straight line between these two points delimiting the duration of elevated N A T activity, and the N A T profile is used as an index for the total amount of melatonin synthesis per night.
Results
The day/night-profiles of pineal N A T activity as measured every other month throughout the course of a year are shown in Fig. 1. N A T activities during the daylight hours are consistently low with only very little variation (mean 0.556 __0.03 nmoles pineal-1 h - l , n=24). The nocturnal increase in N A T activity is consistent as well, however, the pattern of the daily N A T profiles changes with the seasons. In February there is a relatively small increase in N A T activity until midnight and subsequently a gradual decrease to the low day time level. During spring (April) and summer (June, August) peak N A T activities are considerably higher, but the duration of elevated enzyme activi-
595
ties is reduced. Most notable in June high N A T activity seems to be cut off at the onset of daylight. N A T activities in October and December are again characterized by ever broader but at the same time flatter profiles. In Fig. 2 single characteristics of the day/night profiles are plotted as a function of the time of year. Although the amplitude of N A T activity doubles between December and August, the overall seasonal changes of the amplitude are not very prominent (Fig. 2a). The area underneath the nightly elevated levels of N A T activity (i.e. the integral) as a measure of total synthetic activity shows an apparent asymmetry towards the end of the year (Fig. 2b). The peak of N A T activity is reached in December two hours before midnight whereas in summer this peak occurs two hours after midnight. It appears that the seasonal course of peak N A T activity parallels the seasonal shift of sunset with a delay of 6 to 7 hours. As a consequence, there is a large seasonal change in the interval between peak N A T activity and sunrise (Fig. 2c). Pulse duration, as well, shows a strong seasonal rhythm which additionally appears to be very closely correlated with the seasonal change of the daily scotophase (dotted line in Fig. 2 d). Discussion
Illnerovfi and coworkers (1983), have demonstrated a close correlation between the pineal N A T activity and the amount of melatonin within the pineal gland of Phodopus, showing that N A T activity can indeed be used as an index of melatonin production. Daily profiles of N A T activity and/or melatonin levels have already been measured under various photoperiods in a number of different mammalian species (Rollag et al. 1978; Illnerovfi and Van6~ek 1980; Panke et al. 1980; Arendt et al. 1981 ; Goldman et al. 1981 ; Hoffmann et al. 1981 ; Lynch et al. 1982; Yellon et al. 1982; Brainard et al. 1982; Bittmann et al. 1983). However, the present study is the first one to describe a complete seasonal cycle of the daily profiles of pineal melatonin synthesis in hamsters maintained from birth under natural photoperiod. Our results show that the duration of nocturnal melatonin synthesis varies with changes in day length in an inverse relationship: the longer the day, the shorter the melatonin peak. This finding is in good agreement with the conclusion of Goldman and coworkers (Goldman 1983; Carter and Goldman 1983a) that the changes in duration of the melatonin peak may constitute a hormonal signal transmitting day length information to the reproductive system and
596
s. Steinlechneret al. : Seasonalchanges of circadian NAT activityrhythms
to other systems which respond to changes in photoperiod. Under the conditions of the present study and the criteria used for determining the duration of elevated NAT activity, the duration of melatonin synthesis is only slightly shorter than the scotophase in June, but more than two hours shorter than the scotophase in December. This corresponds to the finding that in hamsters living in continuous darkness the pineal melatonin level was elevated for not more than approximately 14 hours (Yellon et al. 1982). The interval between sunset and peak N A T activity shows only little variation around a mean of 6 to 7 hours, i.e. N A T activity increases with a relatively constant rate throughout the year, reaches a peak at about 6 to 7 hours after sunset and starts to decline before dawn. The large seasonal change of the interval between peak N A T activity and sunrise indicates that the duration of the nocturnal melatonin peak is influenced mainly by the seasonal change of the time of dawn rather than by the seasonal fluctuation of dusk. Illnerovfi and coworkers (1986) found that after transition from long day (16L:SD) to short day (8L:16D) photoperiod the extension of the periods of high N A T activity proceeded more rapidly when the dark period was prolonged into the morning hours than when darkness was extended into the evening hours. These results are consistent with a hypothesis that the N A T rhythm may be regulated by a two-oscillator pacemaker (Illnerov/t and Van~6ek 1982, 1984). Despite its seasonal variability, the amplitude of N A T activity does not seem to play a major role in transmitting the photoperiodic information. This parameter seems to be primarily influenced by the age of the animals (Hoffmann et al. 1985; Reiter et al. 1980, 1981). These studies have shown that the nightly peak of N A T activity is considerably reduced in old animals without a change in the temporal pattern of the N A T activity rhythm. Such an age-associated reduction in N A T activity could explain the seasonal differences found in the present study since the animals killed on December 21st and February 16th were the oldest (7 to 9 months). Even though the melatonin pulse duration corresponds very well with the duration of the scotophase, the information about the duration per se is not sufficient for a proper seasonal response. It is not sufficient because similar photoperiods - and consequently similar pulse durations - are experienced by the hamsters twice during one seasonal cycle, prior to and after the summer solstice. However, a similar photoperiod may be stimula-
tory for the gonads (or inhibitory for thermoregulation) in spring and inhibitory for the gonads (stimulatory for thermoregulation) in the autumn. From the results of the present study one might speculate, that in addition to pulse duration the total amount of the hormone synthesized might be involved in transmitting the photoperiodic message. E.g. the same pulse duration of about 13 hours is associated on Feb. 16 with a relatively low amount of melatonin synthesized ( = integral, Fig. 2b), whereas on Oct. 27 this pulse duration corresponds with a high amount of hormone produced. In a recent study H o f f m a n n et al. (1986) reported that a photoperiod of 14L: 10D induced regression of testes in adult Djungarian hamsters maintained originally in 16L:8D, but recrudescence in hamsters kept originally in 8L: 16D. Irrespective of whether the animals had come from 16L:8D or from 8L:16D the duration and amplitude as well as the integral of the nocturnal melatonin pulse were identical. Similar data gathered on the ewe have been reported by Robinson (1985). Further in the present study, the amplitude and duration of the daily NAT-profiles were the same in photoperiods close to 14L : 10D, i.e. on April 27 and August 24. H o f f m a n n and coworkers (1986), therefore, suggest that the change in duration of the nighttime melatonin peak and its direction rather than absolute duration may be the hormonal signal driving photoperiodic responses in adult Djungarian hamsters. Acknowledgements. This work has been supported by a grant of the Deutsche Forschungsgemeinschaft(SFB 305). We thank Prof. Dr. R.J. Reiter and Dr. PeggyD. Rismillerfor criticaUy reading the manuscript. Technical assistance by Ms. S. St6hr and Mr. P. Hebblethwaite,and secretarial help by Ms. A. Drosihn and Ms. A. J6rg are gratefullyacknowledged. References Arendt ], Symons AM, Laud C (1981) Pineal function in the sheep: evidence for a possible mechanism mediating seasonal reproductive activity.Experientia 37 : 584-586 Axelrod J (1974) The pineal gland: a neurochemicaltransducer. Science 184:/341-1348 Baum MJ, Lynch HJ, GallagherCA, Deng MH (1986) Plasma and pineal melatonin levels in female ferrets housed under long or short photoperiods. Biol Reprod 34:96-100 Bittmann EL, DempseyRJ, Karsch FJ (1983) Pinealmelatonin secretion drives the reproductive response to daylength in the ewe. Endocrinology113:2276-2283 Brainard GC, Petterborg LJ, Richardson BA, Reiter RJ (1982) Pineal melatoninin Syrianhamsters: circadianand seasonal rhythms in animalsmaintainedunder laboratory and natural conditions.Neuroendocrinology35 : 342-348 Brainard GC, Richardson BA, Petterborg LJ, Reiter RJ (/982) The effect of differentlight intensities on pineal melatonin content. Brain Res 233 :75-81 Carter DS, Goldman BD (1983a) Antigonadaleffectsof timed
S. Steinlechner et al. : Seasonal changes of circadian NAT activity rhythms melatonin infusion in pinealectomized male Djungarian hamsters (Phodopussungorus sungorus) : duration is the critical parameter. Endocrinology 113 : 1261-1267 Carter DS, Goldman BD (1983 b) Progonadal role of the pineal in the Djungarian hamster (Phodopus sungorus sungorus): mediation by melatonin. Endocrinology 113 : 1268-1273 Deguchi T, Axelrod J (1972) Sensitive assay for serotonin Nacetyltransferase activity in rat pineal. Anal Biochem 50:174-179 Goldman BD (1983) The physiology of melatonin in mammals. Pineal Res Rev 1:145-182 Goldman B, Hall V, Hollister C, Reppert S, Roychoudhury P, Yellon S, Tamarkin L (1981) Diurnal changes in pineal melatonin content in four rodent species: relationship to photoperiodism. Biol Reprod 24: 778-783 Goldman BD, Darrow JM, Yogev L (1984) Effects of timed melatonin infusions on reproductive development in the Djungarian hamster (Phodopus sungorus). Endocrinology 114: 2074-2083 Heldmaier G, Steinlechner S, Rafael J, Vsiansky P (1981) Photoperiodic control and effects of melatonin on nonshivering thermogenesis and brown adipose tissue. Science 212:917-919 Hoffmann K (1973) The influence of photoperiod and melatonin on the testis size, body weight, and pelage colour in the Djungarian hamster (Phodopussungorus). J Comp Physiol 85 : 26%282 Hoffmann K (1981) The role of the pineal gland in the photoperiodic control of seasonal cycles in hamsters. In: Follett BK, Follett DE (eds) Biological clocks in seasonal reproductive cycles. Wright, Bristol, pp 237-250 Hoffmann K, Illnerovfi H, Van~6ek J (1981) Effect of photoperiod and of one minute light at night-time on the pineal rhythm on N-acetyltransferase activity in the Djungarian hamster Phodopus sungorus. Biol Reprod 24:551-556 Hoffmann K, Illnerovfi H, Van6~ek J (1985) Comparison of pineal melatonin rhythms in young adult and old Djungarian hamsters (Phodopussungorus) under long and short photoperiods. Neurosci Lett 56: 39-43 Hoffmann K, Illnerov/l H, Van~ek J (1986) Change in duration of the nighttime melatonin peak may be a signal driving photoperiodic responses in the Djungarian hamster (Phodopus sungorus). Neurosci Lett 67 : 68-72 lllnerov~t H, Van~ek J (1980) Pineal rhythm in N-acetyltransferase activity in rats under different artificial photoperiods and in natural daylight in the course of a year. Neuroendocrinology 31 : 321-326 Illnerov~ H, Van~ek J (1982) Two-oscillator structure of the pacemaker controlling the circadian rhythm of N-acetyltransferase in the rat pineal gland. J Comp Physiol 145:539-548 Illnerovh H, Van6~ek J (1984) Circadian rhythm in inducibility of rat pineal N-acetyltransferase after brief light pulses at night: control by a morning oscillator. J Comp Physiol A 154: 739-744 Illnerovfi H, Van66ek J, Kr~6ek J, Wetterberg L, S/iiif J (1979) Effect of one minute exposure to light at night on rat pineal serotonin N-acetyltransferase and melatonin. J Neurochem 32: 673-675
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