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Nicotine phase shifts the 6-sulphatoxymelatonin rhythm and induces c-Fos in the SCN of rats
Brain Research Bulletin, Vol. 48, No. 5, pp. 527–538, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(99)00033-7
Nicotine phase shifts the 6-sulphatoxymelatonin rhythm and induces c-Fos in the SCN of rats Sally A. Ferguson,* David J. Kennaway and Robert W. Moyer Department of Obstetrics and Gynaecology, University of Adelaide, Medical School, Adelaide, South Australia, Australia [Received 3 September 1998; Revised 5 January 1999; Accepted 5 January 1999] ABSTRACT: The neurotransmitter acetylcholine is not found in the major suprachiasmatic nuclei afferents reported to mediate light effects on entrainment and phase shifts in mammals; however it clearly has some role in the control of circadian rhythmicity. This study examined the effect of the cholinergic agonists nicotine and oxotremorine on (1) the rhythmic production of melatonin using the metabolite, 6-sulphatoxymelatonin as a marker, and (2) the expression of c-Fos protein in the suprachiasmatic nuclei (SCN) of the rat. Nicotine administration (1 mg/ kg, s.c.) caused phase delays in the timing of the onset of 6-sulphatoxymelatonin excretion (compared to the pre-treatment night), when administered at circadian time (CT)16 (1.7 6 0.3 h delay) and CT18 (1.7 6 0.2 h delay) but not at CT14 (0.8 6 0.3 h delay), whereas oxotremorine and saline administration had no effect on the timing of the melatonin rhythm. Nicotine administration also caused the induction of c-Fos-like immunoreactivity in the SCN in a dose- and time-dependent manner. Further, pre-treatment with the nicotinic antagonist mecamylamine reduced the number of nicotine-induced c-Fospositive cells in the SCN by 65%. These data indicate that cholinergic neurons may alter the timing of the onset of melatonin excretion by a direct or indirect effect on the SCN possibly mediated by the nicotinic receptor. © 1999 Elsevier Science Inc.
which are important in the R-GHT [29,36], and serotonin is the major neurotransmitter of the raphe projection [1]. In addition to the neurotransmitters in the major afferents, many other neuroactive substances have been identified in the SCN [50], one of which is acetylcholine (ACh). The role of ACh in the control of SCN rhythmicity is not well defined. Although ACh is not present in the RHT, R-GHT or raphe projections to the SCN, cholinergic projections from the brainstem and basal forebrain have been shown to terminate in the SCN [5]. The muscarinic and nicotinic ACh receptor subtypes have been localised to rat SCN cells using monoclonal antibodies [47,51], and the synthetic enzyme of ACh, choline acetyltransferase, was also found in the SCN [6]. A three-fold rise in ACh content was demonstrated in the rat SCN after a light pulse administered 3 h after subjective dark onset [33], implicating ACh in light-induced effects on SCN function. In addition, the non-specific cholinergic agonist carbachol, when microinjected into the SCN of rats, caused phase shifts in the rhythmic production of the enzyme responsible for melatonin synthesis, N-acetyltransferase (NAT), similar to those caused by light [55]. Taken together this information suggests that the cholinergic system may have a role in the mediation of light effects on the SCN of rats, although the receptor mechanisms and neural pathways involved are yet to be characterised. The aim of this study was to examine the effect of two cholinergic agonists, nicotine and oxotremorine, on (1) the melatonin rhythm and (2) the expression of c-Fos protein in the SCN. Our group has shown previously that the rhythmic excretion of the melatonin metabolite, 6-sulphatoxymelatonin (aMT.6S) is an accurate marker of the timing of SCN output [18]. Light pulses during the early–mid night delayed the aMT.6S excretion rhythm consistent with the typical phase response curves (PRC) published for activity rhythms [9]. Drugs mimicking the neurotransmitters involved in the mediation of light effects are expected to cause similar time-gated phase changes as light pulses. As additional support that the drugs were acting through the SCN, we used the induction of the immediate early gene c-fos in the SCN as a marker for stimulus reception. Further, the nicotinic antagonist mecamylamine was administered in an attempt to block the effects produced with nicotine treatment and to further characterise the receptor mechanisms involved.
KEY WORDS: Circadian, Melatonin, Acetylcholine, Suprachiasmatic nucleus.
INTRODUCTION In mammals, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus are primarily responsible for the generation and maintenance of a wide range of circadian rhythms. The neurochemistry of the circadian timing system is the subject of much research to determine the mechanisms by which the SCN is entrained to the environment via light. Three main SCN afferents from the retina have been reported: the retino-hypothalamic tract (RHT), directly from the retina to the SCN [28]; the retino-geniculo-hypothalamic tract (R-GHT), from the retina via the intergeniculate leaflet to the SCN [13]; and third, the retino-raphe-hypothalamic tract via the raphe nucleus [16,46]. The excitatory amino acids (EAA) are thought to be the primary transmitters in the RHT [8,24], GABA and neuropeptide Y,
* Address for correspondence: Sally Ferguson, Department of Obstetrics and Gynaecology, University of Adelaide, Medical School, Frome Road, Adelaide, South Australia 5005, Australia. Fax: 61-8-83034099; E-mail: [email protected]
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528 MATERIALS AND METHODS Melatonin Studies In all experiments male Wistar albino rats weighing 80 –100 g were obtained from the Central Animal House, University of Adelaide, where they had been maintained on a 12L:12D lighting cycle with lights off at 1900 h. Four days before the experiments commenced animals were transferred to metabolism cages in lightcontrolled chambers and fed a liquid diet (Osmolite HN, Ross Laboratories, Columbus, OH, USA) to promote high urine output. Experiments were carried out over 3 or 4 consecutive nights, with lights remaining off from 1900 h on night 1 for the duration of the study. Throughout this report the term CT12 represents the time of subjective lights off and ZT12 the actual time of lights off. Urine was collected hourly each subjective night using an automatic collection system [18], and the vials were weighed to determine the urine volume and stored for radioimmunoassay (RIA). Night 1 was used as a control collection with no intervention. Night 2 was the treatment night, and animals received: 1. Nicotine (0.3 mg/kg, 1 mg/kg or 3 mg/kg, s.c.), oxotremorine (0.8 mg/kg, 2 mg/kg or 4 mg/kg, s.c.), a light pulse (1 min/2 lux) or saline at CT16. 2. Nicotine (1 mg/kg, s.c.) or saline at CT14, CT16 or CT18. 3. Mecamylamine (5 mg/kg) at CT15.5, 30 min prior to nicotine (1 mg/kg) or saline. Nights 3 and 4 (where applicable) were the post-treatment nights and served to detect phase shifts from the control night. All drugs were obtained from either Sigma Chemical Company (St. Louis, MO, USA) or Research Biochemicals International (Natick, MA, USA) and were administered in complete darkness using infrared vision equipment and infrared torches. The 6-sulphatoxymelatonin was assayed in 50 ml of a 1:50 dilution of urine by radioimmunoassay [2] using reagents purchased from Stockgrand Ltd. (Guildford, Surrey, UK). Samples from individual animals were assayed together and control and experimental animals alternately in each assay. The onset time of aMT.6S excretion was used as a phase marker and was defined as the time at which the excretion rate rose above 20 pmol/h. This approach has been used in previous studies from our group, and the timing of aMT.6S excretion is highly correlated with the secretion of melatonin into blood [21]. The onset times for individual animals on each experimental night were analysed by one-way analysis of variance (ANOVA) with repeated measures using the SPSS for Windows package. Significance was taken as p , 0.05, and post-hoc test was Student’s t-test, with significance set at p , 0.03 (Bonferroni correction). c-Fos Immunocytochemical Studies Male albino Wistar rats weighing 80 –100 g were maintained in home cages in groups of five in a 12L:12D photoperiod, with lights off at 1900 h. On the day of the experiment, animals were treated at: 1. ZT16 after 4 h of darkness with nicotine (0.3 mg/kg, 1 mg/kg or 3 mg/kg, s.c.), oxotremorine (4 mg/kg), a light pulse (1 min/2 lux) or saline. 2. CT6 after 18 h of darkness with nicotine (1 mg/kg, s.c.), a light pulse (1 min/2 lux) or saline. 3. ZT15.5 with mecamylamine (0.3 mg/kg) 30 min prior to ZT16 administration of nicotine (1 mg/kg) or saline, or ZT15.5 with mecamylamine (5 mg/kg) 30 min prior to ZT16 administration of a light pulse (1 min/2 lux). Two hours after treatment, animals were decapitated and brains rapidly removed and fixed by immersion in 4% paraformaldehyde
FERGUSON, KENNAWAY AND MOYER in 0.1-M phosphate buffer (pH 7.4). Four 70-mm coronal sections encompassing the SCN were cut from each brain on a Vibroslicet microtome and processed for c-Fos protein using a protocol reported previously [30]. Results are expressed as the number of immunopositive cells per animal (calculated by averaging the number of positive cells in the left and right SCN). Data were analysed by Kruskal Wallis non-parametric ANOVA and MannWhitney U tests post hoc. RESULTS Melatonin Studies Dose response. Nicotine administration at each dose (0.3 mg/ kg, 1 mg/kg and 3 mg/kg) at CT16 resulted in a small transient decrease in aMT.6S excretion into urine (Fig. 1) whereas light treatment produced a sustained acute suppression of aMT.6S excretion (Fig. 2a). Significant phase delays in the onset of aMT.6S were observed on night 3, compared to the control night at the 3 mg/kg and 1 mg/kg doses of 2.2 6 0.5 h and 1.7 6 0.3 h, respectively; however, at 0.3 mg/kg, no significant delay resulted (0.1 6 0.3 h) (Fig. 1, Table 1). Oxotremorine administered at CT16 did not have any effect acutely or on the following night at any dose used (Table 1). A 1-min/2-lux light pulse at CT16 caused a significant delay in aMT.6S onset on night 3 (2.6 6 0.2 h) (Fig. 2a, Table 1), whereas saline treatment at this time had no acute or phase-delaying effects (Fig. 2b, Table 1). Phase response. Nicotine caused a small transient acute decrease in aMT.6S excretion rate at each treatment time (data not shown). On post-treatment nights 3 and 4, aMT.6S onset was delayed in animals treated with nicotine (1 mg/kg) at CT16 (1.7 6 0.3 h and 1.7 6 0.2 h) and CT18 (1.7 6 0.2 h and 2.1 6 0.3 h) but not at CT14 (0.6 6 0.3 h and 0.2 6 0.1 h) or in animals treated with saline at any time compared with the control night (saline data for CT14 and CT18 not shown) (Fig. 3). Antagonist Studies Mecamylamine treatment (5 mg/kg) at CT15.5 caused a total suppression of the melatonin excretion rate from the time of administration (Fig. 4). When administered prior to saline the nicotinic antagonist did not cause any phase shifts of aMT.6S rhythmicity (Fig. 4b). The antagonist also failed to have an effect on the phase delay in the melatonin excretion rate following nicotine treatment, as animals receiving mecamylamine prior to nicotine exhibited delays of 1.1 6 0.2 h on night 3 and 1.9 6 0.3 h on night 4 (Fig. 4a). c-Fos Immunocytochemical Studies Dose response. At each dose of nicotine (0.3 mg/kg, 1 mg/kg and 3 mg/kg), significantly more c-Fos immunopositive cells were observed in the SCN than following saline administration (p , 0.05) (Fig. 5a). Although there was a dose-dependent increase in the number of labelled cells, even at the maximal dose (3 mg/kg) (Fig. 5a) the response was significantly less (approximately a third) than that observed after a light pulse (Fig. 6a). In both nicotineand light-treated animals the majority of the immunopositive cells were identified in the ventrolateral region of the SCN, with a lower proportion in the ventromedial area. Administration of oxotremorine (4 mg/kg) resulted in significantly more immunopositive cells in the SCN than following saline, but the number was similar to that provoked by the lowest dose of nicotine (Fig. 5a). Phase response. Nicotine (1 mg/kg) administered at CT6 did not result in the appearance of c-Fos-positive neurons in the SCN. In addition, both a light pulse (1 min/2 lux) and a saline injection failed to induce the gene at this time (Fig. 5b).
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FIG. 1. 6-Sulphatoxymelatonin excretion profiles for animals treated with nicotine at doses of (a) 3 mg/kg, (b) 1 mg/kg or (c) 0.3 mg/kg at CT16. Three consecutive nights are shown. Data from night 1, represented by solid symbols, overlay data from nights 2 and 3 (open symbols). Arrows indicate time of treatment on night 2. Each data point represents the mean 6 SEM for five animals.
Antagonist studies. Mecamylamine administration (0.3 mg/kg) prior to saline failed to induce a significant number of c-Fos immunopositive cells in the SCN (Fig. 7). Nicotine (1 mg/kg) at ZT16 resulted in a significant number of immunopositive cells in the SCN (Fig. 8a), and pre-treatment with mecamylamine (0.3 mg/kg) 30 min prior caused a 65% reduction in the number of labelled cells (Figs. 7 and 8b). Mecamylamine (5 mg/kg) administration prior to a 1-min/2-lux light pulse did not reduce the
number of light-induced c-Fos-positive cells in the SCN (data not shown). DISCUSSION The cholinergic agonist nicotine caused phase delays in the timing of the 6-sulphatoxymelatonin excretion rhythm in a dosedependent and time-gated manner. Significant phase delays were
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FIG. 2. 6-Sulphatoxymelatonin excretion profiles for animals treated with (a) a light pulse (1 min/2 lux) or (b) saline at CT16. Four consecutive nights are shown. Data from night 1, represented by solid symbols, overlay data from nights 2, 3 and 4 (open symbols). Arrows indicate time of treatment on night 2. Each data point represents the mean 6 SEM for five animals.
recorded after nicotine (1 mg/kg) treatment at CT16 and CT18 but not CT14. Acting as an agonist of the nicotinic receptor, the drug also stimulated induction of the immediate early gene, c-fos in the suprachiasmatic nucleus at ZT16 but not CT6, again indicating a
TABLE 1 EFFECTS OF CHOLINERGIC DRUGS OR LIGHT ON THE TIMING OF THE ONSET OF MELATONIN PRODUCTION IN THE RAT Treatment
Dose
Time
Delay on Night 3
Significance
Nicotine Nicotine Nicotine Nicotine Nicotine Oxotremorine Oxotremorine Oxotremorine Light Saline
3 mg/kg 1 mg/kg 0.3 mg/kg 1 mg/kg 1 mg/kg 4 mg/kg 2 mg/kg 0.8 mg/kg 1 min/2 lux
CT16 CT16 CT16 CT16 CT16 CT16 CT16 CT16 CT16 CT16
2.2 6 0.5 1.7 6 0.3 0.1 6 0.3 1.7 6 0.2 0.8 6 0.3 0.1 6 0.2 0.2 6 0.1 0.2 6 0.2 2.3 6 0.2 0.3 6 0.1
Yes Yes No Yes No No No No Yes No
The data show the mean 6 SEM (hours, n 5 5) delay in the onset of the 6-sulphatoxymelatonin excretion on the night following administration of the various treatments.
phase dependence of the SCN response. Pre-treatment with mecamylamine (a nicotinic antagonist) reduced the number of nicotine-induced c-Fos immunopositive cells by 65% in the SCN at ZT16 but failed to affect the number of light-induced c-Fospositive cells at this time. Oxotremorine (a muscarinic cholinergic agonist) had no effect on the timing of the aMT.6S excretion rhythm but did induce a small number of immunopositive cells when administered at the highest dose. Together the data suggest that ACh acting through the nicotinic receptor has some role in the control of timing of the melatonin excretion rhythm and that this may be due to action (direct or indirect) at the level of the SCN. Nicotine at the two higher doses (1 mg/kg and 3 mg/kg) caused significant phase delays in the aMT.6S excretion rate rhythm when administered at CT16, whereas the lowest dose was ineffective. The highest dose of oxotremorine used in this experiment (4 mg/kg) was five times larger than the dose used to produce a significant decrease in core body temperature during the day [37], suggesting that the dose was sufficient to invoke central effects. Nevertheless, oxotremorine failed to affect the timing of the aMT.6S rhythm. Using the minimal effective dose of nicotine (1 mg/kg), a partial phase response curve was established. In a previous study a 15-min light pulse given 4 or 6 h after dark onset delayed the timing of the aMT.6S excretion rhythm; however, a pulse at 2 h after dark onset resulted in no delay [18]. Our current results show that the nicotine-induced delays in the melatonin rhythm were phase dependent in this portion of the curve, in a
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FIG. 3. Phase shifts in the timing of the 6-sulphatoxymelatonin (aMT.6S) onset recorded after treatment with nicotine (1 mg/kg) at various circadian times. Delays from night 1 are represented by negative values. (a) Phase shifts on night 3 as compared to the control night and (b) night 4 shifts. CON indicates the response of the aMT.6S excretion rate after treatment with saline at CT16 (data from saline treatment at CT14 and CT18 not shown). Each data point represents the mean 6 SEM for five animals.
similar manner to light-induced delays, with the drug effective only at CT16 and CT18 but not CT14. Although the protocol used in the previous study applied Aschoff’s method IV (phase response in LD) and the present study used method II (acute continuous darkness) [3], the phase delays observed following light treatment 4 h after (subjective) lights off are comparable (2.4 6 0.2 h [18]; cf. 2.6 6 0.2 h in the present study). Further, the lack of c-Fosimmunopositive cells stained after treatment with nicotine at CT6 (light also failed to have an effect at this time), also suggests a phase responsiveness of the SCN to this agonist. Therefore, nicotine elicited similar phase delays in the timing of aMT.6S excretion as light, in an identical time-gated manner. The model employed for assessment of phase shifts in this study has been used successfully in previous studies in our laboratory [19,22,43]. The main advantage of the system is the ability to record phase changes in individual animals by using each as its own control. In addition, this negates the possibility of misinterpretation of results due to individual variation in timing and amplitude of aMT.6S excretion. The dose-response curve in the present study was established using only 1 post-treatment night to
assess the degree of phase shift that occurred after nicotine treatment. These phase delays were analysed both in relation to the individual shifts from the control night and the shifts recorded in another group of animals after treatment with saline. Using the system of automated urine collection and RIA protocol involving 2 post-treatment nights, significant delays on night 3 induced by light or drugs are always followed by similar or greater magnitude delays on night 4 [22]. A report from Mrosovsky explains in some detail why he considers the Aschoff Type II method to be valid and important in this research area. One of the most important points made is that transients resulting from a change in lighting conditions are minimal and often negligible, and thus assessment of the first or second onset after the phase-shifting stimulus is valid [31]. Furthermore, Illnerova and Vanecek [15] showed that transients were not observed in the NAT rhythms in rats when light pulses were administered in the first half of the night. This treatment also produced shifts in both onset and offset that were complete after 1 night. Honma et al. [14] also showed that the activity onset on the day after manipulation was similar to that obtained after a “steadystate” had been achieved, specifically in the delay portion of the
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FIG. 4. 6-Sulphatoxymelatonin excretion profiles for animals treated with mecamylamine (5 mg/kg) prior to (a) nicotine (1 mg/kg) or (b) saline at CT16. Four consecutive nights are shown. Data from night 1, represented by solid symbols, overlay data from nights 2, 3 and 4 (open symbols). Each data point represents the mean 6 SEM for five animals.
PRC. For these reasons, the phase changes recorded on night 3 in this study are considered statistically and physiologically meaningful. A light pulse at CT18 caused both an acute suppression at the time of treatment and a subsequent phase delay of melatonin production rate on the following night [20]. It has been demonstrated that propranolol (adrenergic antagonist) also caused acute suppression of melatonin at CT18 but failed to provoke a phase change on the following night (Kennaway and Rowe, unpublished results). Thus, the acute suppression of melatonin excretion alone does not result in a subsequent delay in onset on the following night. It was also reported that nicotine had no effect on the melatonin level in the pineal of rats when infused at night [10]. In the present study, nicotine caused a small transient suppression of aMT.6S excretion at the time of treatment compared to the robust acute inhibition of production following the light pulse. Because some urine is lost when the animals are briefly handled during the injections, we cannot be sure whether this decrease in excretion rate is of physiological significance or an artefact. However, as melatonin suppression alone cannot cause phase changes of the aMT.6S rhythm, the phase shifts reported in the present study are clearly a result of a response of the SCN to previous nicotinic administration. Much of the work examining the role of ACh in the circadian
timing system has focussed on the non-specific agonist, carbachol. In vivo studies showed that intraventricular administration of carbachol caused phase shifts in the circadian rhythm of locomotor activity of hamsters similar to those seen after a light pulse. Phase advances were demonstrated after treatment during late subjective night/early subjective morning, and phase delays after treatment during early subjective evening [26]. Similarly, when hamsters were maintained in constant darkness for at least 2 weeks and treated with intraventricular or intra-SCN carbachol 2 h (CT14) or 10 h (CT22) after activity onset, identical phase changes were reported [53]. This result was reported in mice also using wheelrunning activity as a marker [57]. Carbachol administered intraventricularly to the rat caused phase delays of the N-acetyltransferase rhythm in the pineal gland similar to those seen with light pulses [55], and continuous infusion of carbachol shortened the free-running period of locomotor activity and drinking behaviour in rats [12,32]. More recently, a similar pattern of carbacholinduced phase shifts in hamster locomotor activity was reported, and the shifts were blocked with the muscarinic antagonist atropine but not the nicotinic antagonist mecamylamine [4]. In a separate study, mecamylamine was shown to block light-induced phase shifts of locomotor activity in the hamster [17]. This discrepancy between studies using the same species may be explained by a possible action of mecamylamine in the glutaminergic system
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FIG. 5. The number of immunopositive cells recorded after treatment with cholinergic or light stimuli. (a) Data from animals treated with nicotine (3 mg/kg, 1 mg/kg or 0.3 mg/kg, s.c.), oxotremorine (4 mg/kg), light (1 min/2 lux) or saline at CT16. (b) Data from animals treated with light (1 min/2 lux) at CT16, light (1 min/2 lux) at CT6, nicotine (1 mg/kg) at CT6 or saline at CT6. Each data set represents the mean 6 SEM for nine animals. Stars indicate significant difference in number of cells counted compared to data from saline-treated animals.
[35], as the EAA are involved in the mediation of light effects on SCN timing in the hamster. There is evidence that the neural control of SCN function may vary between species. The EAA are the main mediators of light effects on SCN timing in the hamster circadian timing system; however, the agonist N-methyl-D-aspartate (NMDA) had no effect on melatonin or running rhythmicity in rats, and the antagonist MK-801 did not have any blocking effect on light-induced changes in timing [43]. Serotonergic agonists on the other hand did cause phase delays in the aMT.6S excretion rhythm and resulted in the expression of c-Fos immunopositive cells in the SCN of rats, suggesting that this transmitter may also be important in mediating the effects of light in the rat [19,22,30]. Further, serotonin has been reported to have opposite effects to light pulses in the hamster circadian system. For example, the serotonergic agonist 8-OHDPAT blocked the phase-shifting effects of light on hamster running rhythms [41], whereas the antagonist NAN-190 potentiated the phase shifts induced by light in that species [40]. Therefore, although it is yet to be established if the importance of ACh in SCN function varies between species, caution must be exercised
when directly comparing studies using different species and protocols with regard to this neurotransmitter. Although the non-specific agonist carbachol has been shown to induce phase shifts in the three species studied (hamster, rat and mouse), data are not conclusive with regard to the receptor subtype mediating these effects. The early work of Miller et al. [27] demonstrated that suprachiasmatic nucleus neurons were responsive to nicotinic stimuli. Both a-bungarotoxin and D-tubocurarine, antagonists of nicotinic receptors, blocked the photic- and carbachol-induced effects on the circadian rhythm of pineal enzyme in rats [56]. A more recent study using the circadian rhythm of neuronal activity in suprachiasmatic nucleus explants has also shown nicotine to be effective at shifting the timing of SCN output in the rat [49], where phase advances were produced by nicotine at all times of the cycle studied. A similar protocol demonstrated carbachol to be effective at causing phase advances whereas nicotine also “caused a small phase advance” [25], and these investigators concluded that a muscarinic mechanism was mediating the cholinergic effects. It is important to give results from such in vitro studies the appropriate emphasis.
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FIG. 6. Representative micrographs of the suprachiasmatic nuclei (SCN) region of rats treated with (a) a 1-min/2-lux light pulse or (b) saline injection at ZT16. Horizontal bars 5 500 mm. Arrows indicate the region of the SCN, OC indicates optic chiasm, and V indicates the third ventricle.
The phase changes reported in in vitro experiments are often of much greater magnitude than those recorded in whole animals using slave rhythms (evident by the .6-h shifts after carbachol treatment recorded by Liu and Gillette [25]). Further, both the direction and phase dependence of the changes are often not comparable to in vivo studies. The Trachsel et al. study [49] reported phase advances with nicotine at all times of the cycle, and Liu and Gillette [25] indicated that the cholinergic agents carbachol and ACh, as well as muscarinic agents, produced phase advances only when applied in the subjective night. These factors suggest that the SCN, when isolated from the rest of the circadian machinery, behaves differently to manipulation from how it be-
haves in the intact animal. Despite the lack of direct correlation between in vitro and in vivo studies with respect to direction, magnitude and phase dependence of shifts, these studies do tell us that the SCN is responsive to cholinergic agents, although it is still not clear which receptor subtype is mediating the responses. The present study has shown that nicotine caused phase delays when administered at a time when light is also effective in the intact animal, whereas oxotremorine had no effect. Our data therefore suggest that a receptor of the nicotinic subtype(s) is mediating the cholinergic effects on the melatonin rhythm in the rat. The nicotinic antagonist mecamylamine was administered in the present study to further define the mechanism of action of the
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FIG. 7. The number of immunopositive cells recorded after treatment with cholinergic stimuli. Animals were treated with either nicotine (1 mg/kg, s.c.) alone at ZT16 or mecamylamine (0.3 mg/kg, s.c.) at ZT15.5 prior to either nicotine (1 mg/kg, s.c.) or saline at ZT16.
nicotine-induced delays in aMT.6S excretion rate and appearance of c-Fos-positive cells in the SCN. Mecamylamine caused a total suppression of the melatonin excretion rate that lasted for the entire subjective night. The subsequent lack of phase change on the post-treatment nights suggests, however, that this suppression occurred via an action of mecamylamine at the level of the input to the pineal gland from the superior cervical ganglion. Mecamylamine pre-treatment prior to nicotine did not have any effect on the phase delay of the aMT.6S rhythm caused by the agonist; however, as drug administration interfered with the actual measurement of the aMT.6S excretion rate, mecamylamine is not an appropriate tool in this model for the identification of the receptor involved. Further work will need to focus on an alternate rhythm such as temperature or locomotor activity. The neural mechanisms through which nicotine affects the timing of the circadian rhythm of melatonin excretion were examined further using the induction of the immediate early gene c-fos in the SCN. Rusak et al. [44] first illustrated the phase-dependent response of c-fos induction in the rat SCN, showing that light pulses during the subjective night caused expression of the c-Fos protein, whereas pulses during the subjective day did not. Studies in our laboratory have recently shown that a light pulse as short as 1 min and at an intensity of only 2 lux given 3– 6 h after lights off resulted in a large number of c-Fos immunopositive cells appearing in the rat SCN and confirmed a lack of c-Fos induction when light was administered during the subjective day [30]. The present results show that nicotine caused the induction of the c-Fos protein at the same circadian time as a light pulse (CT16) and also failed to induce c-Fos at CT6, as did light. To our knowledge, only one other study has reported the effect of nicotine on c-fos induction in the rat. Clegg et al. [7] showed that the drug resulted in c-fos mRNA production in the fetal SCN but not the maternal SCN. The treatment time reported by the authors (CT6 –7) is in the nonresponsive portion of the intact adult SCN response curve, and, as mentioned in a number of studies, in addition to the present one, it has been shown that light cannot induce the gene at this time [30,39]. In the hamster, carbachol was incapable of inducing Fos-like immunoreactivity in the SCN 8 h after activity onset [8]. This result differs from those reported in the current study; how-
ever, the possibility exists that the role of the cholinergic system in light stimulation of the SCN differs between rats and hamsters in a similar manner to the EAA and serotonergic systems. The only other Fos-related study reported that mecamylamine blocked the CT19 light-induced c-fos induction in hamsters [58], which may be due to an interaction of mecamylamine with the glutaminergic system, a fact that makes direct comparison between studies difficult. The present study is the first to demonstrate cholinergic stimulation of c-fos in the SCN of adult rats and demonstrates distinct day–night differences in the response of the clock to nicotine. There was no significant induction of c-Fos immunopositive cells in the SCN after treatment with the cholinergic antagonist mecamylamine at a dose identical to the minimum effective dose of nicotine in this model. Mecamylamine did, however, antagonise the nicotine-induced expression of c-Fos. A 65% reduction of immunopositive cells in the SCN after pre-treatment with mecamylamine suggests that the action of nicotine on the SCN is mediated by the nicotinic receptor. The antagonist had no effect on the light-induced c-Fos response in the SCN, suggesting that the cholinergic component of the circadian timing system may involve minor neural pathways distinct from the major SCN afferents. As previously stated, mecamylamine has antagonist actions at the NMDA receptor [35]. Although we acknowledge that mecamylamine may not be an appropriate tool for analysing melatonin rhythmicity, several arguments exist for its valid use in our c-Fos protocol. First, it is a widely used agent in this field of study, used in several species and is the best antagonist available at present for nicotinic receptors. Second, although it does antagonise NMDA receptors, we believe that the time of the circadian cycle mecamylamine was administered in this study precludes interaction with the EAA system. Rowe and Kennaway [43] showed that during subjective darkness, NMDA did not stimulate and MK801 did not block light-induced phase shifts in melatonin or running activity. Thus, the blocking action of mecamylamine on nicotine in this study indicates action at a nicotinic receptor. Oxotremorine also induced a small but significant number of immunopositive cells in the SCN, a response similar to that seen with the lowest dose of nicotine (0.3 mg/kg), but neither treatment
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FIG. 8. Representative micrographs of the suprachiasmatic nuclei (SCN) region of rats treated with (a) nicotine (1 mg/kg, s.c.) at ZT16 or (b) mecamylamine (0.3 mg/kg, s.c.) at ZT15.5 prior to nicotine (1 mg/kg, s.c.). Horizontal bars 5 500 mm. Arrows indicate the region of the SCN, OC indicates optic chiasm, and V indicates the third ventricle.
caused delays in the aMT.6S rhythm. It has been suggested that induction of the c-fos gene does not necessarily result in subsequent phase shifts and indeed that phase shifts do not require the induction of the gene [42]. Because these data do not supply information about the neurochemistry of the specific cells being activated by the stimuli, it is difficult to make definitive conclusions about the relationship between c-fos induction and phase changes in these studies. Rea et al. [42] suggested that c-Fos induction may be a parallel process to phase shifts rather than an event in a cascade that leads to phase shifts, but Wollnik et al. [54] suggested c-Fos and Jun-B to be fundamental parts of light-
induced phase shifts. The results from the present study indicate that nicotine and oxotremorine can have effects on the SCN of rats either directly or indirectly via brain centres innervating the SCN. The mechanisms of action of ACh in the functioning of the SCN are becoming clearer. The presence of the receptors [47,51] and the synthetic enzyme [6] for ACh in the SCN along with the report that ACh levels rise in the nucleus after a light pulse [33] supply functional evidence for a role for ACh in the circadian timing system. Excitation of SCN neurons by iontophoresis of ACh [34] or systemic injection of cholinergic agonists [27] provides electrophysiological evidence, and the pharmacological stud-
NICOTINE AND CIRCADIAN RHYTHMICITY ies reporting phase shifts with carbachol and other cholinergic agonists [11,25,53] confirm that the cholinergic neurotransmitter system is involved in the regulation of SCN function. A recent study provided us with morphological evidence that cholinergic afferents of the SCN may act directly on neurons in the nucleus. Kiss and Halasz [23] demonstrated the existence of separate contacts between choline acetyltransferase-immunopositive elements and SCN neurons. There are no cholinergic somata in the SCN [38,48], and it is reported that cholinergic neurons whose terminals are located in the SCN may originate from brainstem and forebrain areas [5], basal forebrain and mesopontine tegmentum [23] as well as several other hypothalamic areas [38,48]. It may be that the cholinergic pathways from the forebrain/brainstem regions play a modulatory role in light information transfer to the SCN. The lower magnitude phase shifts and smaller number of Fos-immunoreactive cells induced by nicotine treatment as compared to a light pulse are possibly due to activation of a different population and/or a smaller number of cells in the SCN. Characterisation of the structure and neurochemistry of the specific cells activated by nicotine and light will further elucidate the precise role of ACh in the circadian timing system of rats. It is also important to remember that activation of nicotinic ACh receptors can facilitate the release of other neurotransmitters including noradrenaline [45] and norepinephrine [52]. It is a limitation of this work that we are unable to draw any conclusions about the pre- or post-synaptic localisation of nicotine or its specific action, other than to suggest that it may play some role in the control of circadian timing in rats. In conclusion, this study has shown that nicotine administered peripherally can induce phase-dependent delays in an identical pattern as that seen with light pulses. Further, treatment with the cholinergic agonist caused the induction of the immediate-early gene c-fos in the suprachiasmatic nucleus, also in a phase-dependent manner, suggesting a response from the SCN, possibly as a result of stimulation via minor cholinergic afferents. Our data show therefore that ACh acting through a nicotinic receptor may have a modulatory role in the functioning of the SCN of rats. ACKNOWLEDEGMENTS
The authors wish to acknowledge the contribution of Shawn Rowe in some of the reported experiments. This work was supported in part by a grant from the National Health and Medical Research Council of Australia to Dr. D. J. Kennaway. Sally A. Ferguson was supported by the Benjamin Poulton scholarship.
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46. Shen, H.; Semba, K. A direct retinal projection to the dorsal raphe nucleus in the rat. Brain Res. 635:159 –168; 1994. 47. Swanson, L.; Simmons, D. H.; Whiting, P. J.; Lindstrom, J. Immunohistochemical localization of neuronal nicotinic receptors in rodent central nervous system. J. Neurosci. 7:3334 –3342; 1987. 48. Tago, H.; McGeer, P. L.; McGeer, E. G.; Akiyama, H.; Hersh, L. B. Distribution of choline acetyltransferase immunopositive structures in the rat brainstem. Brain Res. 495:271–297; 1989. 49. Trachsel, L.; Heller, H. C.; Miller, J. D. Nicotine phase-advances the circadian neuronal activity rhythm in rat suprachiasmatic nuclei explants. Neuroscience 65:797– 803; 1995. 50. van den Pol, A. N.; Tsujimoto, T. Neurotransmitters of the hypothalamic suprachiasmatic nucleus: Immunocytochemical analysis of 25 antigens. Neuroscience 15:1049 –1086; 1985. 51. van der Zee, E. A.; Streefland, C.; Strosberg, A. D.; Schroder, H.; Luiten, P. G. Colocalization of muscarinic and nicotinic receptors in cholinoceptive neurons of the suprachiasmatic region in young and aged rats. Brain Res. 542:348 –352; 1991. 52. Vizi, E. S.; Sershen, H.; Balla, A.; Mike, A.; Windisch, K.; Juranyi, Z.; Lajtha, A. Neurochemical evidence of heterogeneity of presynaptic and somatodendritic nicotinic acetylcholine receptors. Ann. N. Y. Acad. Sci. 757:84 –99; 1995. 53. Wee, B. E.; Anderson, K. D.; Kouchis, N. S.; Turek, F. W. Administration of carbachol into the lateral ventricle and suprachiasmatic nucleus (SCN) produces dose-dependent phase shifts in the circadian rhythm of locomotor activity. Neurosci. Lett. 137:211–215; 1992. 54. Wollnik, F.; Brysch, W.; Uhlmann, E.; Gillardon, F.; Bravo, R.; Zimmermann, M.; Schlingensiepen, K. H.; Herdegen, T. Block of c-Fos and Jun-B expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock. Eur. J. Neurosci. 7:388 –393; 1995. 55. Zatz, M.; Brownstein, M. J. Intraventricular carbachol mimics the effects of light on the circadian rhythm in the rat pineal gland. Science 203:358 –361; 1979. 56. Zatz, M.; Brownstein, M. J. Injection of a-bungarotoxin near the suprachiasmatic nucleus blocks the effects of light on nocturnal pineal enzyme activity. Brain Res. 213:438 – 442; 1981. 57. Zatz, M.; Herkenham, M. A. Intraventricular carbachol mimics the phase-shifting effect of light on the circadian rhythm of wheel-running activity. Brain Res. 212:234 –238; 1981. 58. Zhang, Y.; Zee, P. C.; Kirby, J. D.; Takahashi, J. S.; Turek, F. W. A cholinergic antagonist, mecamylamine, blocks light-induced Fos immunoreactivity in specific regions of the hamster suprachiasmatic nucleus. Brain Res. 615:107–112; 1993.
PII S0361-9230(99)00033-7
Nicotine phase shifts the 6-sulphatoxymelatonin rhythm and induces c-Fos in the SCN of rats Sally A. Ferguson,* David J. Kennaway and Robert W. Moyer Department of Obstetrics and Gynaecology, University of Adelaide, Medical School, Adelaide, South Australia, Australia [Received 3 September 1998; Revised 5 January 1999; Accepted 5 January 1999] ABSTRACT: The neurotransmitter acetylcholine is not found in the major suprachiasmatic nuclei afferents reported to mediate light effects on entrainment and phase shifts in mammals; however it clearly has some role in the control of circadian rhythmicity. This study examined the effect of the cholinergic agonists nicotine and oxotremorine on (1) the rhythmic production of melatonin using the metabolite, 6-sulphatoxymelatonin as a marker, and (2) the expression of c-Fos protein in the suprachiasmatic nuclei (SCN) of the rat. Nicotine administration (1 mg/ kg, s.c.) caused phase delays in the timing of the onset of 6-sulphatoxymelatonin excretion (compared to the pre-treatment night), when administered at circadian time (CT)16 (1.7 6 0.3 h delay) and CT18 (1.7 6 0.2 h delay) but not at CT14 (0.8 6 0.3 h delay), whereas oxotremorine and saline administration had no effect on the timing of the melatonin rhythm. Nicotine administration also caused the induction of c-Fos-like immunoreactivity in the SCN in a dose- and time-dependent manner. Further, pre-treatment with the nicotinic antagonist mecamylamine reduced the number of nicotine-induced c-Fospositive cells in the SCN by 65%. These data indicate that cholinergic neurons may alter the timing of the onset of melatonin excretion by a direct or indirect effect on the SCN possibly mediated by the nicotinic receptor. © 1999 Elsevier Science Inc.
which are important in the R-GHT [29,36], and serotonin is the major neurotransmitter of the raphe projection [1]. In addition to the neurotransmitters in the major afferents, many other neuroactive substances have been identified in the SCN [50], one of which is acetylcholine (ACh). The role of ACh in the control of SCN rhythmicity is not well defined. Although ACh is not present in the RHT, R-GHT or raphe projections to the SCN, cholinergic projections from the brainstem and basal forebrain have been shown to terminate in the SCN [5]. The muscarinic and nicotinic ACh receptor subtypes have been localised to rat SCN cells using monoclonal antibodies [47,51], and the synthetic enzyme of ACh, choline acetyltransferase, was also found in the SCN [6]. A three-fold rise in ACh content was demonstrated in the rat SCN after a light pulse administered 3 h after subjective dark onset [33], implicating ACh in light-induced effects on SCN function. In addition, the non-specific cholinergic agonist carbachol, when microinjected into the SCN of rats, caused phase shifts in the rhythmic production of the enzyme responsible for melatonin synthesis, N-acetyltransferase (NAT), similar to those caused by light [55]. Taken together this information suggests that the cholinergic system may have a role in the mediation of light effects on the SCN of rats, although the receptor mechanisms and neural pathways involved are yet to be characterised. The aim of this study was to examine the effect of two cholinergic agonists, nicotine and oxotremorine, on (1) the melatonin rhythm and (2) the expression of c-Fos protein in the SCN. Our group has shown previously that the rhythmic excretion of the melatonin metabolite, 6-sulphatoxymelatonin (aMT.6S) is an accurate marker of the timing of SCN output [18]. Light pulses during the early–mid night delayed the aMT.6S excretion rhythm consistent with the typical phase response curves (PRC) published for activity rhythms [9]. Drugs mimicking the neurotransmitters involved in the mediation of light effects are expected to cause similar time-gated phase changes as light pulses. As additional support that the drugs were acting through the SCN, we used the induction of the immediate early gene c-fos in the SCN as a marker for stimulus reception. Further, the nicotinic antagonist mecamylamine was administered in an attempt to block the effects produced with nicotine treatment and to further characterise the receptor mechanisms involved.
KEY WORDS: Circadian, Melatonin, Acetylcholine, Suprachiasmatic nucleus.
INTRODUCTION In mammals, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus are primarily responsible for the generation and maintenance of a wide range of circadian rhythms. The neurochemistry of the circadian timing system is the subject of much research to determine the mechanisms by which the SCN is entrained to the environment via light. Three main SCN afferents from the retina have been reported: the retino-hypothalamic tract (RHT), directly from the retina to the SCN [28]; the retino-geniculo-hypothalamic tract (R-GHT), from the retina via the intergeniculate leaflet to the SCN [13]; and third, the retino-raphe-hypothalamic tract via the raphe nucleus [16,46]. The excitatory amino acids (EAA) are thought to be the primary transmitters in the RHT [8,24], GABA and neuropeptide Y,
* Address for correspondence: Sally Ferguson, Department of Obstetrics and Gynaecology, University of Adelaide, Medical School, Frome Road, Adelaide, South Australia 5005, Australia. Fax: 61-8-83034099; E-mail: [email protected]
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528 MATERIALS AND METHODS Melatonin Studies In all experiments male Wistar albino rats weighing 80 –100 g were obtained from the Central Animal House, University of Adelaide, where they had been maintained on a 12L:12D lighting cycle with lights off at 1900 h. Four days before the experiments commenced animals were transferred to metabolism cages in lightcontrolled chambers and fed a liquid diet (Osmolite HN, Ross Laboratories, Columbus, OH, USA) to promote high urine output. Experiments were carried out over 3 or 4 consecutive nights, with lights remaining off from 1900 h on night 1 for the duration of the study. Throughout this report the term CT12 represents the time of subjective lights off and ZT12 the actual time of lights off. Urine was collected hourly each subjective night using an automatic collection system [18], and the vials were weighed to determine the urine volume and stored for radioimmunoassay (RIA). Night 1 was used as a control collection with no intervention. Night 2 was the treatment night, and animals received: 1. Nicotine (0.3 mg/kg, 1 mg/kg or 3 mg/kg, s.c.), oxotremorine (0.8 mg/kg, 2 mg/kg or 4 mg/kg, s.c.), a light pulse (1 min/2 lux) or saline at CT16. 2. Nicotine (1 mg/kg, s.c.) or saline at CT14, CT16 or CT18. 3. Mecamylamine (5 mg/kg) at CT15.5, 30 min prior to nicotine (1 mg/kg) or saline. Nights 3 and 4 (where applicable) were the post-treatment nights and served to detect phase shifts from the control night. All drugs were obtained from either Sigma Chemical Company (St. Louis, MO, USA) or Research Biochemicals International (Natick, MA, USA) and were administered in complete darkness using infrared vision equipment and infrared torches. The 6-sulphatoxymelatonin was assayed in 50 ml of a 1:50 dilution of urine by radioimmunoassay [2] using reagents purchased from Stockgrand Ltd. (Guildford, Surrey, UK). Samples from individual animals were assayed together and control and experimental animals alternately in each assay. The onset time of aMT.6S excretion was used as a phase marker and was defined as the time at which the excretion rate rose above 20 pmol/h. This approach has been used in previous studies from our group, and the timing of aMT.6S excretion is highly correlated with the secretion of melatonin into blood [21]. The onset times for individual animals on each experimental night were analysed by one-way analysis of variance (ANOVA) with repeated measures using the SPSS for Windows package. Significance was taken as p , 0.05, and post-hoc test was Student’s t-test, with significance set at p , 0.03 (Bonferroni correction). c-Fos Immunocytochemical Studies Male albino Wistar rats weighing 80 –100 g were maintained in home cages in groups of five in a 12L:12D photoperiod, with lights off at 1900 h. On the day of the experiment, animals were treated at: 1. ZT16 after 4 h of darkness with nicotine (0.3 mg/kg, 1 mg/kg or 3 mg/kg, s.c.), oxotremorine (4 mg/kg), a light pulse (1 min/2 lux) or saline. 2. CT6 after 18 h of darkness with nicotine (1 mg/kg, s.c.), a light pulse (1 min/2 lux) or saline. 3. ZT15.5 with mecamylamine (0.3 mg/kg) 30 min prior to ZT16 administration of nicotine (1 mg/kg) or saline, or ZT15.5 with mecamylamine (5 mg/kg) 30 min prior to ZT16 administration of a light pulse (1 min/2 lux). Two hours after treatment, animals were decapitated and brains rapidly removed and fixed by immersion in 4% paraformaldehyde
FERGUSON, KENNAWAY AND MOYER in 0.1-M phosphate buffer (pH 7.4). Four 70-mm coronal sections encompassing the SCN were cut from each brain on a Vibroslicet microtome and processed for c-Fos protein using a protocol reported previously [30]. Results are expressed as the number of immunopositive cells per animal (calculated by averaging the number of positive cells in the left and right SCN). Data were analysed by Kruskal Wallis non-parametric ANOVA and MannWhitney U tests post hoc. RESULTS Melatonin Studies Dose response. Nicotine administration at each dose (0.3 mg/ kg, 1 mg/kg and 3 mg/kg) at CT16 resulted in a small transient decrease in aMT.6S excretion into urine (Fig. 1) whereas light treatment produced a sustained acute suppression of aMT.6S excretion (Fig. 2a). Significant phase delays in the onset of aMT.6S were observed on night 3, compared to the control night at the 3 mg/kg and 1 mg/kg doses of 2.2 6 0.5 h and 1.7 6 0.3 h, respectively; however, at 0.3 mg/kg, no significant delay resulted (0.1 6 0.3 h) (Fig. 1, Table 1). Oxotremorine administered at CT16 did not have any effect acutely or on the following night at any dose used (Table 1). A 1-min/2-lux light pulse at CT16 caused a significant delay in aMT.6S onset on night 3 (2.6 6 0.2 h) (Fig. 2a, Table 1), whereas saline treatment at this time had no acute or phase-delaying effects (Fig. 2b, Table 1). Phase response. Nicotine caused a small transient acute decrease in aMT.6S excretion rate at each treatment time (data not shown). On post-treatment nights 3 and 4, aMT.6S onset was delayed in animals treated with nicotine (1 mg/kg) at CT16 (1.7 6 0.3 h and 1.7 6 0.2 h) and CT18 (1.7 6 0.2 h and 2.1 6 0.3 h) but not at CT14 (0.6 6 0.3 h and 0.2 6 0.1 h) or in animals treated with saline at any time compared with the control night (saline data for CT14 and CT18 not shown) (Fig. 3). Antagonist Studies Mecamylamine treatment (5 mg/kg) at CT15.5 caused a total suppression of the melatonin excretion rate from the time of administration (Fig. 4). When administered prior to saline the nicotinic antagonist did not cause any phase shifts of aMT.6S rhythmicity (Fig. 4b). The antagonist also failed to have an effect on the phase delay in the melatonin excretion rate following nicotine treatment, as animals receiving mecamylamine prior to nicotine exhibited delays of 1.1 6 0.2 h on night 3 and 1.9 6 0.3 h on night 4 (Fig. 4a). c-Fos Immunocytochemical Studies Dose response. At each dose of nicotine (0.3 mg/kg, 1 mg/kg and 3 mg/kg), significantly more c-Fos immunopositive cells were observed in the SCN than following saline administration (p , 0.05) (Fig. 5a). Although there was a dose-dependent increase in the number of labelled cells, even at the maximal dose (3 mg/kg) (Fig. 5a) the response was significantly less (approximately a third) than that observed after a light pulse (Fig. 6a). In both nicotineand light-treated animals the majority of the immunopositive cells were identified in the ventrolateral region of the SCN, with a lower proportion in the ventromedial area. Administration of oxotremorine (4 mg/kg) resulted in significantly more immunopositive cells in the SCN than following saline, but the number was similar to that provoked by the lowest dose of nicotine (Fig. 5a). Phase response. Nicotine (1 mg/kg) administered at CT6 did not result in the appearance of c-Fos-positive neurons in the SCN. In addition, both a light pulse (1 min/2 lux) and a saline injection failed to induce the gene at this time (Fig. 5b).
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FIG. 1. 6-Sulphatoxymelatonin excretion profiles for animals treated with nicotine at doses of (a) 3 mg/kg, (b) 1 mg/kg or (c) 0.3 mg/kg at CT16. Three consecutive nights are shown. Data from night 1, represented by solid symbols, overlay data from nights 2 and 3 (open symbols). Arrows indicate time of treatment on night 2. Each data point represents the mean 6 SEM for five animals.
Antagonist studies. Mecamylamine administration (0.3 mg/kg) prior to saline failed to induce a significant number of c-Fos immunopositive cells in the SCN (Fig. 7). Nicotine (1 mg/kg) at ZT16 resulted in a significant number of immunopositive cells in the SCN (Fig. 8a), and pre-treatment with mecamylamine (0.3 mg/kg) 30 min prior caused a 65% reduction in the number of labelled cells (Figs. 7 and 8b). Mecamylamine (5 mg/kg) administration prior to a 1-min/2-lux light pulse did not reduce the
number of light-induced c-Fos-positive cells in the SCN (data not shown). DISCUSSION The cholinergic agonist nicotine caused phase delays in the timing of the 6-sulphatoxymelatonin excretion rhythm in a dosedependent and time-gated manner. Significant phase delays were
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FIG. 2. 6-Sulphatoxymelatonin excretion profiles for animals treated with (a) a light pulse (1 min/2 lux) or (b) saline at CT16. Four consecutive nights are shown. Data from night 1, represented by solid symbols, overlay data from nights 2, 3 and 4 (open symbols). Arrows indicate time of treatment on night 2. Each data point represents the mean 6 SEM for five animals.
recorded after nicotine (1 mg/kg) treatment at CT16 and CT18 but not CT14. Acting as an agonist of the nicotinic receptor, the drug also stimulated induction of the immediate early gene, c-fos in the suprachiasmatic nucleus at ZT16 but not CT6, again indicating a
TABLE 1 EFFECTS OF CHOLINERGIC DRUGS OR LIGHT ON THE TIMING OF THE ONSET OF MELATONIN PRODUCTION IN THE RAT Treatment
Dose
Time
Delay on Night 3
Significance
Nicotine Nicotine Nicotine Nicotine Nicotine Oxotremorine Oxotremorine Oxotremorine Light Saline
3 mg/kg 1 mg/kg 0.3 mg/kg 1 mg/kg 1 mg/kg 4 mg/kg 2 mg/kg 0.8 mg/kg 1 min/2 lux
CT16 CT16 CT16 CT16 CT16 CT16 CT16 CT16 CT16 CT16
2.2 6 0.5 1.7 6 0.3 0.1 6 0.3 1.7 6 0.2 0.8 6 0.3 0.1 6 0.2 0.2 6 0.1 0.2 6 0.2 2.3 6 0.2 0.3 6 0.1
Yes Yes No Yes No No No No Yes No
The data show the mean 6 SEM (hours, n 5 5) delay in the onset of the 6-sulphatoxymelatonin excretion on the night following administration of the various treatments.
phase dependence of the SCN response. Pre-treatment with mecamylamine (a nicotinic antagonist) reduced the number of nicotine-induced c-Fos immunopositive cells by 65% in the SCN at ZT16 but failed to affect the number of light-induced c-Fospositive cells at this time. Oxotremorine (a muscarinic cholinergic agonist) had no effect on the timing of the aMT.6S excretion rhythm but did induce a small number of immunopositive cells when administered at the highest dose. Together the data suggest that ACh acting through the nicotinic receptor has some role in the control of timing of the melatonin excretion rhythm and that this may be due to action (direct or indirect) at the level of the SCN. Nicotine at the two higher doses (1 mg/kg and 3 mg/kg) caused significant phase delays in the aMT.6S excretion rate rhythm when administered at CT16, whereas the lowest dose was ineffective. The highest dose of oxotremorine used in this experiment (4 mg/kg) was five times larger than the dose used to produce a significant decrease in core body temperature during the day [37], suggesting that the dose was sufficient to invoke central effects. Nevertheless, oxotremorine failed to affect the timing of the aMT.6S rhythm. Using the minimal effective dose of nicotine (1 mg/kg), a partial phase response curve was established. In a previous study a 15-min light pulse given 4 or 6 h after dark onset delayed the timing of the aMT.6S excretion rhythm; however, a pulse at 2 h after dark onset resulted in no delay [18]. Our current results show that the nicotine-induced delays in the melatonin rhythm were phase dependent in this portion of the curve, in a
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FIG. 3. Phase shifts in the timing of the 6-sulphatoxymelatonin (aMT.6S) onset recorded after treatment with nicotine (1 mg/kg) at various circadian times. Delays from night 1 are represented by negative values. (a) Phase shifts on night 3 as compared to the control night and (b) night 4 shifts. CON indicates the response of the aMT.6S excretion rate after treatment with saline at CT16 (data from saline treatment at CT14 and CT18 not shown). Each data point represents the mean 6 SEM for five animals.
similar manner to light-induced delays, with the drug effective only at CT16 and CT18 but not CT14. Although the protocol used in the previous study applied Aschoff’s method IV (phase response in LD) and the present study used method II (acute continuous darkness) [3], the phase delays observed following light treatment 4 h after (subjective) lights off are comparable (2.4 6 0.2 h [18]; cf. 2.6 6 0.2 h in the present study). Further, the lack of c-Fosimmunopositive cells stained after treatment with nicotine at CT6 (light also failed to have an effect at this time), also suggests a phase responsiveness of the SCN to this agonist. Therefore, nicotine elicited similar phase delays in the timing of aMT.6S excretion as light, in an identical time-gated manner. The model employed for assessment of phase shifts in this study has been used successfully in previous studies in our laboratory [19,22,43]. The main advantage of the system is the ability to record phase changes in individual animals by using each as its own control. In addition, this negates the possibility of misinterpretation of results due to individual variation in timing and amplitude of aMT.6S excretion. The dose-response curve in the present study was established using only 1 post-treatment night to
assess the degree of phase shift that occurred after nicotine treatment. These phase delays were analysed both in relation to the individual shifts from the control night and the shifts recorded in another group of animals after treatment with saline. Using the system of automated urine collection and RIA protocol involving 2 post-treatment nights, significant delays on night 3 induced by light or drugs are always followed by similar or greater magnitude delays on night 4 [22]. A report from Mrosovsky explains in some detail why he considers the Aschoff Type II method to be valid and important in this research area. One of the most important points made is that transients resulting from a change in lighting conditions are minimal and often negligible, and thus assessment of the first or second onset after the phase-shifting stimulus is valid [31]. Furthermore, Illnerova and Vanecek [15] showed that transients were not observed in the NAT rhythms in rats when light pulses were administered in the first half of the night. This treatment also produced shifts in both onset and offset that were complete after 1 night. Honma et al. [14] also showed that the activity onset on the day after manipulation was similar to that obtained after a “steadystate” had been achieved, specifically in the delay portion of the
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FIG. 4. 6-Sulphatoxymelatonin excretion profiles for animals treated with mecamylamine (5 mg/kg) prior to (a) nicotine (1 mg/kg) or (b) saline at CT16. Four consecutive nights are shown. Data from night 1, represented by solid symbols, overlay data from nights 2, 3 and 4 (open symbols). Each data point represents the mean 6 SEM for five animals.
PRC. For these reasons, the phase changes recorded on night 3 in this study are considered statistically and physiologically meaningful. A light pulse at CT18 caused both an acute suppression at the time of treatment and a subsequent phase delay of melatonin production rate on the following night [20]. It has been demonstrated that propranolol (adrenergic antagonist) also caused acute suppression of melatonin at CT18 but failed to provoke a phase change on the following night (Kennaway and Rowe, unpublished results). Thus, the acute suppression of melatonin excretion alone does not result in a subsequent delay in onset on the following night. It was also reported that nicotine had no effect on the melatonin level in the pineal of rats when infused at night [10]. In the present study, nicotine caused a small transient suppression of aMT.6S excretion at the time of treatment compared to the robust acute inhibition of production following the light pulse. Because some urine is lost when the animals are briefly handled during the injections, we cannot be sure whether this decrease in excretion rate is of physiological significance or an artefact. However, as melatonin suppression alone cannot cause phase changes of the aMT.6S rhythm, the phase shifts reported in the present study are clearly a result of a response of the SCN to previous nicotinic administration. Much of the work examining the role of ACh in the circadian
timing system has focussed on the non-specific agonist, carbachol. In vivo studies showed that intraventricular administration of carbachol caused phase shifts in the circadian rhythm of locomotor activity of hamsters similar to those seen after a light pulse. Phase advances were demonstrated after treatment during late subjective night/early subjective morning, and phase delays after treatment during early subjective evening [26]. Similarly, when hamsters were maintained in constant darkness for at least 2 weeks and treated with intraventricular or intra-SCN carbachol 2 h (CT14) or 10 h (CT22) after activity onset, identical phase changes were reported [53]. This result was reported in mice also using wheelrunning activity as a marker [57]. Carbachol administered intraventricularly to the rat caused phase delays of the N-acetyltransferase rhythm in the pineal gland similar to those seen with light pulses [55], and continuous infusion of carbachol shortened the free-running period of locomotor activity and drinking behaviour in rats [12,32]. More recently, a similar pattern of carbacholinduced phase shifts in hamster locomotor activity was reported, and the shifts were blocked with the muscarinic antagonist atropine but not the nicotinic antagonist mecamylamine [4]. In a separate study, mecamylamine was shown to block light-induced phase shifts of locomotor activity in the hamster [17]. This discrepancy between studies using the same species may be explained by a possible action of mecamylamine in the glutaminergic system
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FIG. 5. The number of immunopositive cells recorded after treatment with cholinergic or light stimuli. (a) Data from animals treated with nicotine (3 mg/kg, 1 mg/kg or 0.3 mg/kg, s.c.), oxotremorine (4 mg/kg), light (1 min/2 lux) or saline at CT16. (b) Data from animals treated with light (1 min/2 lux) at CT16, light (1 min/2 lux) at CT6, nicotine (1 mg/kg) at CT6 or saline at CT6. Each data set represents the mean 6 SEM for nine animals. Stars indicate significant difference in number of cells counted compared to data from saline-treated animals.
[35], as the EAA are involved in the mediation of light effects on SCN timing in the hamster. There is evidence that the neural control of SCN function may vary between species. The EAA are the main mediators of light effects on SCN timing in the hamster circadian timing system; however, the agonist N-methyl-D-aspartate (NMDA) had no effect on melatonin or running rhythmicity in rats, and the antagonist MK-801 did not have any blocking effect on light-induced changes in timing [43]. Serotonergic agonists on the other hand did cause phase delays in the aMT.6S excretion rhythm and resulted in the expression of c-Fos immunopositive cells in the SCN of rats, suggesting that this transmitter may also be important in mediating the effects of light in the rat [19,22,30]. Further, serotonin has been reported to have opposite effects to light pulses in the hamster circadian system. For example, the serotonergic agonist 8-OHDPAT blocked the phase-shifting effects of light on hamster running rhythms [41], whereas the antagonist NAN-190 potentiated the phase shifts induced by light in that species [40]. Therefore, although it is yet to be established if the importance of ACh in SCN function varies between species, caution must be exercised
when directly comparing studies using different species and protocols with regard to this neurotransmitter. Although the non-specific agonist carbachol has been shown to induce phase shifts in the three species studied (hamster, rat and mouse), data are not conclusive with regard to the receptor subtype mediating these effects. The early work of Miller et al. [27] demonstrated that suprachiasmatic nucleus neurons were responsive to nicotinic stimuli. Both a-bungarotoxin and D-tubocurarine, antagonists of nicotinic receptors, blocked the photic- and carbachol-induced effects on the circadian rhythm of pineal enzyme in rats [56]. A more recent study using the circadian rhythm of neuronal activity in suprachiasmatic nucleus explants has also shown nicotine to be effective at shifting the timing of SCN output in the rat [49], where phase advances were produced by nicotine at all times of the cycle studied. A similar protocol demonstrated carbachol to be effective at causing phase advances whereas nicotine also “caused a small phase advance” [25], and these investigators concluded that a muscarinic mechanism was mediating the cholinergic effects. It is important to give results from such in vitro studies the appropriate emphasis.
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FIG. 6. Representative micrographs of the suprachiasmatic nuclei (SCN) region of rats treated with (a) a 1-min/2-lux light pulse or (b) saline injection at ZT16. Horizontal bars 5 500 mm. Arrows indicate the region of the SCN, OC indicates optic chiasm, and V indicates the third ventricle.
The phase changes reported in in vitro experiments are often of much greater magnitude than those recorded in whole animals using slave rhythms (evident by the .6-h shifts after carbachol treatment recorded by Liu and Gillette [25]). Further, both the direction and phase dependence of the changes are often not comparable to in vivo studies. The Trachsel et al. study [49] reported phase advances with nicotine at all times of the cycle, and Liu and Gillette [25] indicated that the cholinergic agents carbachol and ACh, as well as muscarinic agents, produced phase advances only when applied in the subjective night. These factors suggest that the SCN, when isolated from the rest of the circadian machinery, behaves differently to manipulation from how it be-
haves in the intact animal. Despite the lack of direct correlation between in vitro and in vivo studies with respect to direction, magnitude and phase dependence of shifts, these studies do tell us that the SCN is responsive to cholinergic agents, although it is still not clear which receptor subtype is mediating the responses. The present study has shown that nicotine caused phase delays when administered at a time when light is also effective in the intact animal, whereas oxotremorine had no effect. Our data therefore suggest that a receptor of the nicotinic subtype(s) is mediating the cholinergic effects on the melatonin rhythm in the rat. The nicotinic antagonist mecamylamine was administered in the present study to further define the mechanism of action of the
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FIG. 7. The number of immunopositive cells recorded after treatment with cholinergic stimuli. Animals were treated with either nicotine (1 mg/kg, s.c.) alone at ZT16 or mecamylamine (0.3 mg/kg, s.c.) at ZT15.5 prior to either nicotine (1 mg/kg, s.c.) or saline at ZT16.
nicotine-induced delays in aMT.6S excretion rate and appearance of c-Fos-positive cells in the SCN. Mecamylamine caused a total suppression of the melatonin excretion rate that lasted for the entire subjective night. The subsequent lack of phase change on the post-treatment nights suggests, however, that this suppression occurred via an action of mecamylamine at the level of the input to the pineal gland from the superior cervical ganglion. Mecamylamine pre-treatment prior to nicotine did not have any effect on the phase delay of the aMT.6S rhythm caused by the agonist; however, as drug administration interfered with the actual measurement of the aMT.6S excretion rate, mecamylamine is not an appropriate tool in this model for the identification of the receptor involved. Further work will need to focus on an alternate rhythm such as temperature or locomotor activity. The neural mechanisms through which nicotine affects the timing of the circadian rhythm of melatonin excretion were examined further using the induction of the immediate early gene c-fos in the SCN. Rusak et al. [44] first illustrated the phase-dependent response of c-fos induction in the rat SCN, showing that light pulses during the subjective night caused expression of the c-Fos protein, whereas pulses during the subjective day did not. Studies in our laboratory have recently shown that a light pulse as short as 1 min and at an intensity of only 2 lux given 3– 6 h after lights off resulted in a large number of c-Fos immunopositive cells appearing in the rat SCN and confirmed a lack of c-Fos induction when light was administered during the subjective day [30]. The present results show that nicotine caused the induction of the c-Fos protein at the same circadian time as a light pulse (CT16) and also failed to induce c-Fos at CT6, as did light. To our knowledge, only one other study has reported the effect of nicotine on c-fos induction in the rat. Clegg et al. [7] showed that the drug resulted in c-fos mRNA production in the fetal SCN but not the maternal SCN. The treatment time reported by the authors (CT6 –7) is in the nonresponsive portion of the intact adult SCN response curve, and, as mentioned in a number of studies, in addition to the present one, it has been shown that light cannot induce the gene at this time [30,39]. In the hamster, carbachol was incapable of inducing Fos-like immunoreactivity in the SCN 8 h after activity onset [8]. This result differs from those reported in the current study; how-
ever, the possibility exists that the role of the cholinergic system in light stimulation of the SCN differs between rats and hamsters in a similar manner to the EAA and serotonergic systems. The only other Fos-related study reported that mecamylamine blocked the CT19 light-induced c-fos induction in hamsters [58], which may be due to an interaction of mecamylamine with the glutaminergic system, a fact that makes direct comparison between studies difficult. The present study is the first to demonstrate cholinergic stimulation of c-fos in the SCN of adult rats and demonstrates distinct day–night differences in the response of the clock to nicotine. There was no significant induction of c-Fos immunopositive cells in the SCN after treatment with the cholinergic antagonist mecamylamine at a dose identical to the minimum effective dose of nicotine in this model. Mecamylamine did, however, antagonise the nicotine-induced expression of c-Fos. A 65% reduction of immunopositive cells in the SCN after pre-treatment with mecamylamine suggests that the action of nicotine on the SCN is mediated by the nicotinic receptor. The antagonist had no effect on the light-induced c-Fos response in the SCN, suggesting that the cholinergic component of the circadian timing system may involve minor neural pathways distinct from the major SCN afferents. As previously stated, mecamylamine has antagonist actions at the NMDA receptor [35]. Although we acknowledge that mecamylamine may not be an appropriate tool for analysing melatonin rhythmicity, several arguments exist for its valid use in our c-Fos protocol. First, it is a widely used agent in this field of study, used in several species and is the best antagonist available at present for nicotinic receptors. Second, although it does antagonise NMDA receptors, we believe that the time of the circadian cycle mecamylamine was administered in this study precludes interaction with the EAA system. Rowe and Kennaway [43] showed that during subjective darkness, NMDA did not stimulate and MK801 did not block light-induced phase shifts in melatonin or running activity. Thus, the blocking action of mecamylamine on nicotine in this study indicates action at a nicotinic receptor. Oxotremorine also induced a small but significant number of immunopositive cells in the SCN, a response similar to that seen with the lowest dose of nicotine (0.3 mg/kg), but neither treatment
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FIG. 8. Representative micrographs of the suprachiasmatic nuclei (SCN) region of rats treated with (a) nicotine (1 mg/kg, s.c.) at ZT16 or (b) mecamylamine (0.3 mg/kg, s.c.) at ZT15.5 prior to nicotine (1 mg/kg, s.c.). Horizontal bars 5 500 mm. Arrows indicate the region of the SCN, OC indicates optic chiasm, and V indicates the third ventricle.
caused delays in the aMT.6S rhythm. It has been suggested that induction of the c-fos gene does not necessarily result in subsequent phase shifts and indeed that phase shifts do not require the induction of the gene [42]. Because these data do not supply information about the neurochemistry of the specific cells being activated by the stimuli, it is difficult to make definitive conclusions about the relationship between c-fos induction and phase changes in these studies. Rea et al. [42] suggested that c-Fos induction may be a parallel process to phase shifts rather than an event in a cascade that leads to phase shifts, but Wollnik et al. [54] suggested c-Fos and Jun-B to be fundamental parts of light-
induced phase shifts. The results from the present study indicate that nicotine and oxotremorine can have effects on the SCN of rats either directly or indirectly via brain centres innervating the SCN. The mechanisms of action of ACh in the functioning of the SCN are becoming clearer. The presence of the receptors [47,51] and the synthetic enzyme [6] for ACh in the SCN along with the report that ACh levels rise in the nucleus after a light pulse [33] supply functional evidence for a role for ACh in the circadian timing system. Excitation of SCN neurons by iontophoresis of ACh [34] or systemic injection of cholinergic agonists [27] provides electrophysiological evidence, and the pharmacological stud-
NICOTINE AND CIRCADIAN RHYTHMICITY ies reporting phase shifts with carbachol and other cholinergic agonists [11,25,53] confirm that the cholinergic neurotransmitter system is involved in the regulation of SCN function. A recent study provided us with morphological evidence that cholinergic afferents of the SCN may act directly on neurons in the nucleus. Kiss and Halasz [23] demonstrated the existence of separate contacts between choline acetyltransferase-immunopositive elements and SCN neurons. There are no cholinergic somata in the SCN [38,48], and it is reported that cholinergic neurons whose terminals are located in the SCN may originate from brainstem and forebrain areas [5], basal forebrain and mesopontine tegmentum [23] as well as several other hypothalamic areas [38,48]. It may be that the cholinergic pathways from the forebrain/brainstem regions play a modulatory role in light information transfer to the SCN. The lower magnitude phase shifts and smaller number of Fos-immunoreactive cells induced by nicotine treatment as compared to a light pulse are possibly due to activation of a different population and/or a smaller number of cells in the SCN. Characterisation of the structure and neurochemistry of the specific cells activated by nicotine and light will further elucidate the precise role of ACh in the circadian timing system of rats. It is also important to remember that activation of nicotinic ACh receptors can facilitate the release of other neurotransmitters including noradrenaline [45] and norepinephrine [52]. It is a limitation of this work that we are unable to draw any conclusions about the pre- or post-synaptic localisation of nicotine or its specific action, other than to suggest that it may play some role in the control of circadian timing in rats. In conclusion, this study has shown that nicotine administered peripherally can induce phase-dependent delays in an identical pattern as that seen with light pulses. Further, treatment with the cholinergic agonist caused the induction of the immediate-early gene c-fos in the suprachiasmatic nucleus, also in a phase-dependent manner, suggesting a response from the SCN, possibly as a result of stimulation via minor cholinergic afferents. Our data show therefore that ACh acting through a nicotinic receptor may have a modulatory role in the functioning of the SCN of rats. ACKNOWLEDEGMENTS
The authors wish to acknowledge the contribution of Shawn Rowe in some of the reported experiments. This work was supported in part by a grant from the National Health and Medical Research Council of Australia to Dr. D. J. Kennaway. Sally A. Ferguson was supported by the Benjamin Poulton scholarship.
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