Abstract 

 Section 1 

 Section 2 

 Section 3 

 Section 4 

 Section 5 
 
 
 

Discussion 
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 In the rat, the rhythmic pineal melatonin production is driven by the circadian rhythm in N-acetyltransferase (NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87)(18,21). The NAT rhythm, similarly as other overt rhythms, e.g. in locomotor activity, is controlled by a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus (20). The SCN receives photic input directly from the retina, via the retinohypothalamic tract, and indirectly via the intergeniculate leaflet of the lateral geniculate body (4,25). Following a light stimulus, in addition to other processes, immediate early genes c-fos and jun-B are transcriptionaly activated, mostly in the ventrolateral (VL) SCN (22,27). These genes are believed to function in coupling short-term signals elicited by extracellular events to long-term changes in cellular phenotype by mediating subsequent changes in gene expression (5). Importantly, light induces c-fos and jun-B mRNA expression and elevates c-Fos and Jun-B proteins in the mammalian SCN only during the subjective night, when it also phase shifts circadian rhythmicity (22,26,27,30,33). The rhythm in the light-induced c-fos and jun-B mRNA and c-Fos and Jun-B protein, thus represents an endogenous rhythm in SCN sensitivity to light. Recently, with a highly sensitive antibody, we described a circadian rhythm in the SCN c-Fos immunoreactivity in darkness, with the maximum in the morning and through during the subjective night (32). The spontaneous rhythmic c-Fos induction occurred mostly in the dorsomedial (DM) SCN and might indicate an elevated dorsomedial neuronal activity in the early subjective day. 

Rhythmic processes in the pineal, though they are controlled by the SCN, might be also partly independent of the SCN. It has been suggested that the pineal itself may have residual clock properties and affect via the cyclic AMP-responsive element modulator (CREM) its rhythmic melatonin production (3). In addition, independent of the SCN, the pineal melatonin might be directly affected by light (10) and via its highly sensitive SCN receptors (35) might itself modulate the SCN rhythmicity. 

The present study was undertaken to find out whether and how the rhythmic melatonin production reflects the intrinsic SCN rhythmicity or whether it is partly independent of the rhythmicity. To elucidate this question, the pineal NAT rhythm under various conditions and following various stimuli was compared with the rhythm in the light-induced c-fos mRNA and c-Fos protein which was present mostly in the VL-SCN and with the spontaneous rhythmic c-Fos induction in darkness which occurred predominantly in the DM-SCN. 

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 2.1. Rhythms in the pineal NAT and in the SCN c-fos photoinduction  

The pineal NAT activity was determined by a radiometric method as described elsewhere (11-17).Induction of c-Fos protein by a 30-min light pulse was followed by the immunocytochemical method, with the primary antiserum generated against the amino acids 2-17 of the N-terminal peptide sequence of c-Fos; the antiserum was kindly provided by D. Hancock, Imperial Cancer Research Fund, London, and generously supplied by M. Hastings, University of Cambridge, U.K.) (30,31,33). Eventually, c-fos mRNA was measured by the in situ hybridization method (33). 

Rats were maintained under a light-dark regime with 12 h of light (06 h to 18 h) and 12 h of darkness (18 h to 06 h) per day (LD 12:12), unless indicated otherwise, then they were released into darkness and the next night the NAT activity and c-Fos photoinduction were followed. The SCN rhythm in the light-induced c-Fos immunoreactivity had two well defined phase markers, namely the time of the evening rise and the time of the morning decline (30). Similarly, the pineal NAT rhythm had also two markers, namely the time of the evening NAT rise and the time of the morning decline (9,12,16). Whereas the pineal NAT rise laged by about 2 h the SCN rise in c-Fos photoinduction, the morning decline in both the SCN and pineal variables occurred at about the same time. The gradual evening rise in the number of the light-induced c-Fos immunopositive cells indicates that in the course of the early night more and more SCN neurons begin to be in such a phase that they respond to light. 

2.2. Photic resetting of the SCN rhythm in c-Fos photoinduction and of the pineal NAT rhythm 

When rats were exposed to a light stimulus in the early night and then released into darkness, the next day both the SCN rhythm in c-Fos photoinduction and the pineal NAT rhythms were phase-delayed as compared with the rhythm profiles in control, unexposed animals; in both rhythms, the evening marker was delayed to a larger extent than the morning one (9,16,17,30). However, when such delays of both rhythms were studied under an extremely long photoperiod, LD 18:6, the morning markers were phase delayed more than the evening ones, due apparently to the state of the underlying pacemaking system (7,30,34). When rats were exposed to a light stimulus in the late night and then released to darkness, the next day, during the first transient cycle, only the morning decline in the SCN c-Fos photoinduction and in the pineal NAT, respectively, was phase advanced, as compared with the decline in control rats, but not the evening rise (16,17,30). Apparently, the evening and the morning SCN and pineal markers do not necessarily phase shift in parallel. The finding suggests a complex nature of the underlying SCN pacemaker where groups of neurons might be first reset together and via coupling might entrain other groups (23,37). The evening NAT rise started to be phase advanced only within four days following a late night light stimulus, and even then to a still lesser extent than the morning decline(16). 

2.3. Non-photic resetting of the SCN rhythm in c-Fos photo- induction and of the pineal NAT rhythm by melatonin  

A single evening melatonin administration phase-advanced instantaneously the evening rise in the light-induced SCN c-Fos immunoreactivity (34). Similarly, a single melatonin administration before the evening dark onset phase-advanced instantaneously the evening pineal NAT rise (6). The magnitude of phase shifts of the intrinsic SCN rhythmicity induced by melatonin administration in vivo was similar to the magnitude of phase-shifts of the pineal NAT rhythm and was less than half of the magnitude attained in in vitro experiments following melatonin application to the rat SCN slices during late subjective day (6,24,28). 

Importantly, melatonin administration in the late day phase-advanced just the evening NAT rise, but not the morning decline (6). Recently, it has been reported that daily melatonin administration at the time of the former dark onset keeps the rhythm in the pineal melatonin production entrained to the 24-h day just for few weeks after a release of rats from a LD cycle to constant darkness; thereafter, the whole rhythm begins to free-run (1). But at first, shortly after the release from the LD cycle, it is the morning melatonin decline which starts to free-run, with the ensuing extension of the melatonin signal duration, and only then the entrainment of the whole rhythm breaks. Altogether, the data suggest that melatonin administered in late day entrains primarily an evening component of circadian rhythmicity. 

2.4. Effect of photoperiod 

The finding that an early night light stimulus phase delays primarily the evening marker of the SCN and pineal rhythms and a late night light stimulus phase advances primarily the morning marker of both rhythms suggests that on long days light perturbing into the late evening and early morning hours may compress the waveform of the SCN and pineal rhythms; on short days, the waveform may decompress. This actually happens. The interval between the evening rise in c-fos photoinduction and the morning decline under unmasked conditions in darkness as well as that between the evening NAT rise and the morning decline were by about 5 h longer under a short, LD 8:16 photoperiod, than under a long, LD 16:8 photoperiod (8,9,12,33). In both rhythms, the interval under the short photoperiod was extended assymetricaly, into the morning hours. This indicates a more important role of the morning than of the evening light in entrainment of the rat circadian pacemaking system (9,15). Similarly, in Syrian and European hamsters, the interval between the evening rise and the morning decline in the SCN c-fos photoinduction is also longer on short than on long days (36). Importantly, the photoperiod affected the waveform of the SCN rhythm in the light-induced c-Fos immunoreactivity directly, and not via the pineal melatonin (32). 

A long light stimulus encompassing the middle of the night compressed the waveform of the SCN rhythm in c-Fos photoinduction and of the pineal NAT rhythm in a manner similar to the effect of a long photoperiod, i.e., by phase delaying the evening marker of both rhythms and phase advancing the morning one (9,17,30). When rats were maintained under an extremely long, LD 18:6 photoperiod, even a 5-min or a shorter pulse had such an effect, i.e., it phase-delayed the evening marker, phase-advanced the morning one and further compressed the SCN and the pineal rhythm waveform (15,30). 

When rats were transferred from a long, LD 16:8, photoperiod to a short, LD 8:16, photoperiod, the waveform of the SCN rhythm in c-Fos photoinduction as well as that of the pineal NAT rhythm extended just gradually and it took two weeks before the full extension was achieved (11,31). However, when rats were transferred from a short to a long photoperiod, compression of the interval enabling high SCN c-Fos photoinduction occurred within three days (34). It appears that the memory on long but not on short days is stored in the SCN itself. 

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3.1. Endogenous rhythm of c-Fos immunoreactivity  

The c-Fos immunoreactivity was determined with a highly sensitive antiserum generated against the amino acids 2-17 of the N-terminal peptide of c-Fos and characterized elsewhere (38); the antiserum was kindly provided by J.D. Mikkelsen, H. Lundbeck, Copenhagen. In rats maintained in LD 12:12 and released to darkness, c-Fos immunoreactivity in the DM-SCN peaked in the early subjective day and then slowly declined; the decrease to low nighttime levels occurred after the expected dark onset( 32). Before the expected light onset, c-Fos started spontaneously to increase. 

3.2. Effect of photoperiod  

The interval of the spontaneous c-Fos induction in the SCN, namely in the DM-SCN, was longer in rats released into darkness from a long photoperiod than in those released into darkness from a short photoperiod (Sumová, Trávníčková, Jáč and Illnerová, in preparation). In other words, low nighttime c-Fos immunoreactivity lasted for a shorter time under the long than under the short photoperiod. Hence even the spontaneous rhythm of c-Fos in the SCN was photoperiod dependent. Under the long photoperiod as well as under the short one, the morning rise in c-Fos immunoreactivity occurred before the expected light onset and was locked to the morning light whereas the evening decline occurred at about the same time under both photoperiods. Hence it appears that not just the rhythm in the pineal NAT and in the light-induced c-Fos immunoreactivity, but also the rhythm in the spontaneous c-Fos immunoreactivity, are entrained mostly by the morning light. 

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 4.1. Discussion  

A striking similarity exists between resetting of the SCN rhythm in c-Fos photoinduction and resetting of the pineal NAT rhythm. Following photic stimuli in the early, middle and late night, respectively, both the SCN and the pineal rhythms phase shift in a similar way during the first transient cycle. Importantly, the evening markers of both rhythms do not necessarily phase shift in parallel with the morning ones, suggesting a complex nature of the underlying pacemaking system (9,13). Following melatonin administration in late day, the evening rise in the SCN c-Fos photoinduction as well as the pineal NAT rise are phase advanced instantaneously, by about the same amount. 

A striking similarity exists also in the response of the SCN rhythm in c-Fos photoinduction and in the response of the pineal NAT rhythm to the photoperiod. Both rhythms are photoperiod-dependent: under a long photoperiod, the interval enabling high c-Fos photoinduction as well as the interval of elevated NAT activity are short and under a short photoperiod they are long. Under LD 16:8, LD 12:12 and LD 8:16, respectively, the morning decline in the light-induced c-Fos occurs at about the same time as that in the NAT activity, whereas the evening NAT rise occurs by 1 to 2 h later than the rise in c-Fos photoinduction. The 1 to 2 h delay in the NAT rise may be explained by the time interval necessary for the NAT mRNA and protein formation (19): following administration of isoproterenol, a beta adrenergic agonist, it takes 1 to 2 h before the NAT activity markedly increases (14). The gradual evening rise in the number of the light-induced c-Fos immunopositive cells suggests that more and more SCN neurons start to be in a light-responsive phase, and towards the middle of the night all cells capable of c-Fos photoinduction may respond. Similarly, strength of the signal coming from the SCN into the pineal may gradually increase; under an extremely long, LD 20:4 photoperiod, NAT activity rises as rapidly as after isoproterenol administration (14). 

After transition of rats from a long to a short photoperiod, decompression of the waveform of the SCN rhythm in c-Fos photoinduction as well as of the pineal NAT rhythm waveform proceeds just gradually and is roughly completed within two weeks whereas compression of the SCN rhythms after a change from a short to a long photoperiod is roughly achieved already within three days. Memory on long days stored in the SCN pacemaking system may explain "the carry-over phenomenon" when infrequent long day treatment induces long day responses (2). 

A similarity exits also in the response of the DM-SCN rhythm in the spontaneous c-Fos immunoreactivity and in the responsse of the pineal NAT rhythm to the photoperiod.The interval of low c-Fos immunoreactivity which indicates low neuronal activity and at the same time the subjective night, is shorter on long than on short days, similarly as the interval of elevated NAT activity. 

4.2. Conclusions  

The aforementioned data indicate that changes of the rat pineal NAT and hence also of the melatonin rhythm reflect mostly changes of the intrinsic SCN rhythmicity. If there is an intrinsic time-keeping mechanism in the rat pineal, it is only a marginal one. 

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