- Email: [email protected]
Circadian rhythm and effects of light on cAMP content of the dwarf hamster suprachiasmatic nucleus
Neuroscience Letters 241 (1998) 131–134
Circadian rhythm and effects of light on cAMP content of the dwarf hamster suprachiasmatic nucleus Stefan Reuss*, Stefan Rimoldi Department of Anatomy, School of Medicine, Johannes Gutenberg-University, Saarstrasse 19–21, D-55099 Mainz, Germany Received 17 November 1997; received in revised form 19 December 1997; accepted 19 December 1997
Abstract The present study was conducted in the dwarf hamster (Phodopus sungorus) to investigate whether a circadian rhythm is present in the content of the second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in the suprachiasmatic nucleus (SCN), the endogenous clock in mammals. In animals held under light/dark conditions (LD), we observed high levels at the end of the light phase and low levels during the night in frozen SCN punches. In animals held in continuous dark, a similar rhythm was seen although a second peak was present in the subjective day. In senile hamsters under LD, the decrease of cAMP levels at the light transition was not seen. These data, obtained for the first time from hamsters, support the view that cAMP is involved in time-keeping mechanisms within the SCN. 1998 Elsevier Science Ireland Ltd.
Keywords: Phodopus sungorus; Siberian hamster; Age; Cyclic adenosine monophosphate; Suprachiasmatic nucleus
It is generally agreed upon that the hypothalamic suprachiasmatic nucleus (SCN) works as the endogenous pacemaker responsible for the generation and entrainment of circadian rhythms in mammals [13,17,22]. Various studies showed that the intrinsic properties of the SCN are responsible for its function. Since single SCN cells exhibit rhythms of their electrical dicharge rate with periods in the circadian range [24], clock function is probably located within the SCN neurons where the transduction cascade involves, inter alia, the second messenger systems. The involvement of the intracellular second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in circadian clock function within the SCN [7] is revealed by two lines of evidence obtained from studies in rats. Firstly, the SCN exhibits a circadian rhythm in the content of cAMP in [15]. Secondly, the application of cAMP during the subjective day advances the endogenous clock by 4–5 h [8]. These observations suggest that the cyclic changes of cAMP are part of the pacemaker mechanisms residing within the SCN. It is, however, not known whether the rhythm of cAMP concentration in the SCN is common for mammals. Further* Corresponding author. Tel.: +49 6131 393207; fax: +49 6131 393719; e-mail: [email protected]
more, the previous data were obtained from albino or hypopigmented rat strains which are known to exhibit neural defects in several parts of the brain [5]. The present study, therefore, was carried out in a pigmented, highly photoperiodic rodent species, the Djungarian hamster Phodopus sungorus. We investigated the contents of cAMP in the SCN during the 24 h cycle in animals held under long photoperiods and in animals that were transferred to continuous darkness for 3 days prior to sampling. We also studied cAMP contents in SCN from hamsters of approximately 2 years of age at selected time-points (2 h before and 2 h after light transition). Djungarian hamsters (Phodopus sungorus) of both sexes with body weights of 46–54 g were used in this study. The animals stem from our own breeding colony (original stock provided by Dr. K. Hoffmann, Clinical Research Unit for Reproductive Medicine, Mu¨nster, Germany) and were held under constant conditions (food and water ad libitum, room temperature 21 ± 1°C). Illumination was provided by bright white light (approximately 500 lux at cage level) for 16 h and safe dim red light for 8 h (long photoperiods; LD 16:8). Lights were switched on at Zeitgeber time 0 (ZT 0). In the first experimental set, animals aged 2–3 months were divided into groups of 5–8 hamsters each and decapi-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00013- 5
132
S. Reuss, S. Rimoldi / Neuroscience Letters 241 (1998) 131–134
tated under light ether anesthesia at seven time points across the 24 h cycle (ZT: 2, 6, 10, 14, 18, 20, 22). Additionally, two groups of five hamsters each with ages of 24–26 months (held under LD 16:8) were killed at ZT 14 or ZT 18, respectively. For the second set, hamsters were transferred from long photoperiod to continuous dark (DD) 3 days prior to experimentation at the beginning of the dark period. In groups of five to eight animals, decapitation was carried out at circadian time (CT) 0, 3, 6, 9, 12, 15, 18 and 21. All darktime samples were collected under dim red light. Brains were dissected out and immediately frozen in liquid nitrogen. Tissue specimens containing the hypothalamus were blocked on a freezing microtome in the frontal plane. According to a stereotaxic atlas of the Phodopus brain based on Nissl-stained sections (S. Reuss, unpublished data), sections of frozen tissue were cut from the anterior part of the brain to the rostral border of the SCN. The bilateral SCN were then collected by use of an NIH-style neuro punch (24 gauge, inner diameter 0.31 mm; Fine Science Tools, Heidelberg, Germany). Samples were individually frozen in Eppendorf tubes in liquid nitrogen and assayed within 2 days. Samples were sonicated in 0.1 ml 0.05 M Tris–HCl buffer (pH 8.0), 1 mM theophylline, 0.1 mM isobutylmethylxanthine and 0.05 ml 0.9 M sodium acetate buffer (pH 4.0). An aliquot was taken for protein determination according to Bradford [2], the remainder heated at 90°C for 3 min and centrifuged. Cyclic AMP was determined from the supernatant by means of a radioimmunoassay kit (Amersham Buchler) with [125I]cAMP as tracer. The tissue punch location was examined in brains from which one SCN was collected. Frozen sections were then cut from the tissue block, fixed in ethanol and stained with cresyl violet. Statistical analysis was performed using a one-way analysis of variance followed by Duncan’s multiple range test for comparing differences between means. A P , 0.05 was regarded as statistically significant. The examination of cresyl violet-stained slices from which one SCN was punched out showed successful
Fig. 1. Cresyl violet-stained frontal section of a hamster brain showing removal of the right SCN. The borders of the left SCN are depicted by arrows. Abbreviations: oc, optic chiasm, V, third ventricle. Scale bar, 400 mm.
Fig. 2. Cyclic AMP levels in the SCN of Phodopus sungorus held under long-day conditions (LD 16:8). Data are the mean ± SEM at seven Zeitgeber time (ZT) points.
removal of the nucleus leaving the contralateral SCN intact (Fig. 1). The protein levels per tissue punch were relatively constant (1.9 ± 0.1 mg). The radioimmunological determination of cAMP from SCN punches showed levels in the range of 10–60 pmol/ mg protein. In animals held under long photoperiods (lights on at ZT 0–16; see Fig. 2), we observed a peak at ZT 14 which was significantly different from levels at ZT 2, 6, 18, 20 and 22. A small peak was seen at CT 2 which did not differ significantly from neighboring timepoints. A nadir was found at ZT 20 which differed statistically from ZT 2, 6, 10, 14, 18 and 22. In aged animals held under LD 16:8, the cAMP levels were 51.9 ± 2.4 pmol/mg protein at ZT 14 and 53.7 ± 6.9 pmol/mg protein at ZT 18 (difference not statistically significant). Under DD conditions (Fig. 3), the rhythm of cAMP content was of similar shape as seen under LD, whereas the range of data at a given timepoint was larger. The peak levels seen at CT 15 and 18 showed a statistically significant difference to nadir levels obtained at CT 9 and CT 0. The second peak (CT 6) as well differed from nadir levels. The present results demonstrate that the cAMP content in the SCN of the dwarf hamster Phodopus sungorus changes during the 24 h cycle under both LD and DD conditions. These first data obtained from a species other than rat reveal that the rhythm of cAMP in the SCN is endogenous (since it continues in DD) and that it is modulated by environmental lighting conditions. Under DD conditions, cAMP concentrations exhibited a biphasic rhythm. Peaks were observed at CT 6 and CT 18 and nadirs at CT 9 and CT 0. Similar results, demonstrating the endogenous nature of cAMP oscillations in the SCN, were previously obtained from rat studies showing a biphasic circadian rhythm of cAMP concentration in the SCN of
S. Reuss, S. Rimoldi / Neuroscience Letters 241 (1998) 131–134
Fig. 3. Cyclic AMP levels in the SCN of Phodopus sungorus held under continuous dark (DD) conditions. Data are the mean ± SEM at eight circadian time (CT) points.
blinded animals [14], of animals in constant darkness [25] and in the SCN in vitro [15]. With regard to the question of which mechanisms are responsible for the fluctuations of cAMP, it is conceivable that the activities of either the cAMP-forming enzyme, adenylate cyclase (AC), or the cAMP-degradative enzymes, phosphodiesterases (PDE), are regulated in a rhythmic manner. Since in rat the AC activity was relatively constant throughout the circadian cycle, it may be speculated that the rhythm of cAMP metabolism in the SCN is due to changes in the activity of the PDE which in the rat SCN showed changes inverse to those of cAMP [15]. In hamsters held under LD, we found a peak of cAMP concentration at the end of the light phase and a nadir in the early dark. Comparing the DD rhythm with the LD rhythm, two observations are conspicuous. First, the biphasic shape of the rhythm changed to monophasic although a second smaller nadir at ZT 6 was still present (not significantly different from ZT 2 and ZT 10). Second, the extreme levels seem to be shifted. Since the free-running periods in Djungarian hamsters are relatively close to 24 h [12], the apparent shift most probably is an artifact caused by the relative uncertainty on the true peak time due to sample intervals. The neuronal pathways by which environmental lighting may modulate the cAMP rhythm include the retinohypothalamic tract which provides strong retino-afferent projection to the Phodopus SCN [6,19,21]. A second photic input to the SCN stems from the metathalamic intergeniculate leaflet (IGL). The major transmitter of the IGL-SCN projection, neuropeptide Y. increased in concentration in the Phodopus SCN at night [20] which may exert inhibitory influence on cAMP metabolism. The shaping of the cAMP rhythm under LD also may involve serotonin which, as part of the rapheSCN projection [10], is augmented during the light period in
133
the rat SCN [4]. It was suggested that serotonin at the 5-HT7 receptor activates adenylate cyclase resulting in augmented cAMP levels [16]. We observed that, in aged hamsters held under LD, cAMP content was similar at ZT 14 and ZT 18, i.e. 2 h before and after lights off, while in young adult animals a dramatic decrease of cAMP was found between these timepoints. Although our investigation did not cover the whole 24 h cycle due to the limited availability of senile animals, we would like to offer two possible explanations for this difference. First, it seems possible that senile animals are less sensitive to light and exhibit a pattern similar to that seen under DD. Second, it is conceivable that the circadian rhythm of PDE activity [15] is abolished with advancing age of the animal, in line with age-related changes of the circadian system [3,18,23]. This point, however, requires further clarification. Our hamster data and those obtained in rat reveal that cAMP is involved in time-keeping mechanisms of the mammalian endogenous clock residing in the SCN. This view is supported by the findings that cAMP may inhibit SCN electrical activity [11], possibly by modulating the inward rectifier current in SCN neurons [1], advance the phase of neuronal firing [8], and stimulate gene expression via the phosphorylation of the cAMP response element binding protein [9]. We thank HSD Dr. R. Spessert for helpful discussion, J. Haase for technical help, Dr. J. Olcese (IHF, Hamburg, Germany) for critically reading the manuscript, and the NMFZ (Rheinland-Pfalz) and DFG (Re 644/2-2) for financial support of our research. Results of this study were presented as part of a thesis by S. Rimoldi in partial fulfillment of his doctoral degree at the Johannes Gutenberg-University, Mainz. [1] Akasu, T. and Shoji, S., cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus, Pflu¨gers Arch. Eur. J. Physiol., 429 (1994) 117–125. [2] Bradford, M.M., A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248–254. [3] Brock, M.A., Biological clocks and aging, Rev. Biol. Res. Aging, 2 (1985) 445–462. [4] Cagampang, F.R.A. and Inouye, S.I.T., Diurnal and circadian changes of serotonin in the suprachiasmatic nuclei: regulation by light and an endogenous pacemaker, Brain Res., 639 (1994) 175–179. [5] Creel, D., Inappropriate use of albino animals as models in research, Pharmacol. Biochem. Behav., 12 (1980) 969–977. [6] Decker, K. and Reuss, S., Nitric oxide-synthesizing neurons in the hamster suprachiasmatic nucleus: a combined NOS- and NADPH-staining and retinohypothalamic tract tracing study, Brain Res., 666 (1994) 284–288. [7] Edmunds, L.N., Carre, I.A., Tamponnet, C. and Tong, J., The role of ions and second messengers in circadian clock function, Chronobiol. Int., 9 (1992) 180–200. [8] Gillette, M.U. and Prosser, R.A., Circadian rhythm of the rat suprachiasmatic brain slice is rapidly reset by daytime application of cAMP analogs, Brain Res., 474 (1988) 348–352.
134
S. Reuss, S. Rimoldi / Neuroscience Letters 241 (1998) 131–134
[9] Ginty, D.D., Kornhauser, J.M., Thompson, M.A., Bading, H., Mayo, K.E., Takahashi, J.S. and Greenberg, M.E., Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock, Science, 260 (1993) 238–241. [10] Kawano, H., Decker, K. and Reuss, S., Is there a direct retinaraphe-suprachiasmatic nucleus pathway in the rat?, Neurosci. Lett., 212 (1996) 143–146. [11] Liou, S.Y., Shibata, S., Shiratsuchi, A. and Ueki, S., Effects of dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparations, Neurosci. Lett., 67 (1986) 339–343. [12] Milette, J.J. and Turek, F.W., Circadian and photoperiodic effects of brief light pulses in male Djungarian hamsters, Biol. Reprod., 35 (1994) 327–335. [13] Moore, R.Y., Organisation and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783–2788. [14] Murakami, N. and Takahashi, K., Circadian rhythm of adenosine-3′,5′monophosphate content in suprachiasmatic nucleus (SCN) and ventromedial hypothalamus (VMH) in the rat, Brain Res., 276 (1983) 297–304. [15] Prosser, R.A. and Gillette, M.U., Cyclic changes in cAMP concentration and phosphodiesterase activity in a mammalian circadian clock studied in vitro, Brain Res., 568 (1991) 185–192. [16] Prosser, R.A., Heller, H.C. and Miller, J.D., Serotonergic phase advances of the mammalian circadian clock involve protein kinase A and K + channel opening, Brain Res., 644 (1994) 67–73. [17] Reuss, S., Components and connections of the circadian timing system in mammals, Cell Tissue Res., 285 (1996) 353–378.
[18] Reuss, S. and Bu¨rger, K., Substance P-like immunoreactivity in the hypothalamic suprachiasmatic nucleus of Phodopus sungorus relation to daytime, photoperiod, sex and age, Brain Res., 638 (1994) 189–195. [19] Reuss, S. and Decker, K., Anterograde tracing of retinohypothalamic afferents with Fluoro-Gold, Brain Res., 745 (1997) 197–204. [20] Reuss, S. and Olcese, J., Neuropeptide Y: distribution of immunoreactivity and quantitative analysis in diencephalic structures and cerebral cortex of dwarf hamsters under different photoperiods, Neuroendocrinology, 61 (1995) 337–347. [21] Reuss, S., Decker, K., Ho¨dl, P. and Sraka, S., Anterograde neuronal tracing of retinohypothalamic projections in the hamster – evidence for the innervation of substance P-containing neurons in the suprachiasmatic nucleus, Neurosci. Lett., 174 (1994) 51–54. [22] Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449–526. [23] Weiland, N.G. and Wise, P.M., Aging progressively decreases the densities and alters the diurnal rhythms of alpha 1-adrenergic receptors in selected hypothalamic regions, Endocrinology, 126 (1990) 2392–2397. [24] Welsh, D.K., Logothetis, D.E., Meister, M. and Reppert, S.M., Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms, Neuron, 14 (1995) 697–706. [25] Yamazaki, S., Maruyama, M., Cagampang, F.R.A. and Inouye, S.T., Circadian fluctuations of cAMP content in the suprachiasmatic nucleus and the anterior hypothalamus of the rat, Brain Res., 651 (1994) 329–331.
Circadian rhythm and effects of light on cAMP content of the dwarf hamster suprachiasmatic nucleus Stefan Reuss*, Stefan Rimoldi Department of Anatomy, School of Medicine, Johannes Gutenberg-University, Saarstrasse 19–21, D-55099 Mainz, Germany Received 17 November 1997; received in revised form 19 December 1997; accepted 19 December 1997
Abstract The present study was conducted in the dwarf hamster (Phodopus sungorus) to investigate whether a circadian rhythm is present in the content of the second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in the suprachiasmatic nucleus (SCN), the endogenous clock in mammals. In animals held under light/dark conditions (LD), we observed high levels at the end of the light phase and low levels during the night in frozen SCN punches. In animals held in continuous dark, a similar rhythm was seen although a second peak was present in the subjective day. In senile hamsters under LD, the decrease of cAMP levels at the light transition was not seen. These data, obtained for the first time from hamsters, support the view that cAMP is involved in time-keeping mechanisms within the SCN. 1998 Elsevier Science Ireland Ltd.
Keywords: Phodopus sungorus; Siberian hamster; Age; Cyclic adenosine monophosphate; Suprachiasmatic nucleus
It is generally agreed upon that the hypothalamic suprachiasmatic nucleus (SCN) works as the endogenous pacemaker responsible for the generation and entrainment of circadian rhythms in mammals [13,17,22]. Various studies showed that the intrinsic properties of the SCN are responsible for its function. Since single SCN cells exhibit rhythms of their electrical dicharge rate with periods in the circadian range [24], clock function is probably located within the SCN neurons where the transduction cascade involves, inter alia, the second messenger systems. The involvement of the intracellular second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) in circadian clock function within the SCN [7] is revealed by two lines of evidence obtained from studies in rats. Firstly, the SCN exhibits a circadian rhythm in the content of cAMP in [15]. Secondly, the application of cAMP during the subjective day advances the endogenous clock by 4–5 h [8]. These observations suggest that the cyclic changes of cAMP are part of the pacemaker mechanisms residing within the SCN. It is, however, not known whether the rhythm of cAMP concentration in the SCN is common for mammals. Further* Corresponding author. Tel.: +49 6131 393207; fax: +49 6131 393719; e-mail: [email protected]
more, the previous data were obtained from albino or hypopigmented rat strains which are known to exhibit neural defects in several parts of the brain [5]. The present study, therefore, was carried out in a pigmented, highly photoperiodic rodent species, the Djungarian hamster Phodopus sungorus. We investigated the contents of cAMP in the SCN during the 24 h cycle in animals held under long photoperiods and in animals that were transferred to continuous darkness for 3 days prior to sampling. We also studied cAMP contents in SCN from hamsters of approximately 2 years of age at selected time-points (2 h before and 2 h after light transition). Djungarian hamsters (Phodopus sungorus) of both sexes with body weights of 46–54 g were used in this study. The animals stem from our own breeding colony (original stock provided by Dr. K. Hoffmann, Clinical Research Unit for Reproductive Medicine, Mu¨nster, Germany) and were held under constant conditions (food and water ad libitum, room temperature 21 ± 1°C). Illumination was provided by bright white light (approximately 500 lux at cage level) for 16 h and safe dim red light for 8 h (long photoperiods; LD 16:8). Lights were switched on at Zeitgeber time 0 (ZT 0). In the first experimental set, animals aged 2–3 months were divided into groups of 5–8 hamsters each and decapi-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00013- 5
132
S. Reuss, S. Rimoldi / Neuroscience Letters 241 (1998) 131–134
tated under light ether anesthesia at seven time points across the 24 h cycle (ZT: 2, 6, 10, 14, 18, 20, 22). Additionally, two groups of five hamsters each with ages of 24–26 months (held under LD 16:8) were killed at ZT 14 or ZT 18, respectively. For the second set, hamsters were transferred from long photoperiod to continuous dark (DD) 3 days prior to experimentation at the beginning of the dark period. In groups of five to eight animals, decapitation was carried out at circadian time (CT) 0, 3, 6, 9, 12, 15, 18 and 21. All darktime samples were collected under dim red light. Brains were dissected out and immediately frozen in liquid nitrogen. Tissue specimens containing the hypothalamus were blocked on a freezing microtome in the frontal plane. According to a stereotaxic atlas of the Phodopus brain based on Nissl-stained sections (S. Reuss, unpublished data), sections of frozen tissue were cut from the anterior part of the brain to the rostral border of the SCN. The bilateral SCN were then collected by use of an NIH-style neuro punch (24 gauge, inner diameter 0.31 mm; Fine Science Tools, Heidelberg, Germany). Samples were individually frozen in Eppendorf tubes in liquid nitrogen and assayed within 2 days. Samples were sonicated in 0.1 ml 0.05 M Tris–HCl buffer (pH 8.0), 1 mM theophylline, 0.1 mM isobutylmethylxanthine and 0.05 ml 0.9 M sodium acetate buffer (pH 4.0). An aliquot was taken for protein determination according to Bradford [2], the remainder heated at 90°C for 3 min and centrifuged. Cyclic AMP was determined from the supernatant by means of a radioimmunoassay kit (Amersham Buchler) with [125I]cAMP as tracer. The tissue punch location was examined in brains from which one SCN was collected. Frozen sections were then cut from the tissue block, fixed in ethanol and stained with cresyl violet. Statistical analysis was performed using a one-way analysis of variance followed by Duncan’s multiple range test for comparing differences between means. A P , 0.05 was regarded as statistically significant. The examination of cresyl violet-stained slices from which one SCN was punched out showed successful
Fig. 1. Cresyl violet-stained frontal section of a hamster brain showing removal of the right SCN. The borders of the left SCN are depicted by arrows. Abbreviations: oc, optic chiasm, V, third ventricle. Scale bar, 400 mm.
Fig. 2. Cyclic AMP levels in the SCN of Phodopus sungorus held under long-day conditions (LD 16:8). Data are the mean ± SEM at seven Zeitgeber time (ZT) points.
removal of the nucleus leaving the contralateral SCN intact (Fig. 1). The protein levels per tissue punch were relatively constant (1.9 ± 0.1 mg). The radioimmunological determination of cAMP from SCN punches showed levels in the range of 10–60 pmol/ mg protein. In animals held under long photoperiods (lights on at ZT 0–16; see Fig. 2), we observed a peak at ZT 14 which was significantly different from levels at ZT 2, 6, 18, 20 and 22. A small peak was seen at CT 2 which did not differ significantly from neighboring timepoints. A nadir was found at ZT 20 which differed statistically from ZT 2, 6, 10, 14, 18 and 22. In aged animals held under LD 16:8, the cAMP levels were 51.9 ± 2.4 pmol/mg protein at ZT 14 and 53.7 ± 6.9 pmol/mg protein at ZT 18 (difference not statistically significant). Under DD conditions (Fig. 3), the rhythm of cAMP content was of similar shape as seen under LD, whereas the range of data at a given timepoint was larger. The peak levels seen at CT 15 and 18 showed a statistically significant difference to nadir levels obtained at CT 9 and CT 0. The second peak (CT 6) as well differed from nadir levels. The present results demonstrate that the cAMP content in the SCN of the dwarf hamster Phodopus sungorus changes during the 24 h cycle under both LD and DD conditions. These first data obtained from a species other than rat reveal that the rhythm of cAMP in the SCN is endogenous (since it continues in DD) and that it is modulated by environmental lighting conditions. Under DD conditions, cAMP concentrations exhibited a biphasic rhythm. Peaks were observed at CT 6 and CT 18 and nadirs at CT 9 and CT 0. Similar results, demonstrating the endogenous nature of cAMP oscillations in the SCN, were previously obtained from rat studies showing a biphasic circadian rhythm of cAMP concentration in the SCN of
S. Reuss, S. Rimoldi / Neuroscience Letters 241 (1998) 131–134
Fig. 3. Cyclic AMP levels in the SCN of Phodopus sungorus held under continuous dark (DD) conditions. Data are the mean ± SEM at eight circadian time (CT) points.
blinded animals [14], of animals in constant darkness [25] and in the SCN in vitro [15]. With regard to the question of which mechanisms are responsible for the fluctuations of cAMP, it is conceivable that the activities of either the cAMP-forming enzyme, adenylate cyclase (AC), or the cAMP-degradative enzymes, phosphodiesterases (PDE), are regulated in a rhythmic manner. Since in rat the AC activity was relatively constant throughout the circadian cycle, it may be speculated that the rhythm of cAMP metabolism in the SCN is due to changes in the activity of the PDE which in the rat SCN showed changes inverse to those of cAMP [15]. In hamsters held under LD, we found a peak of cAMP concentration at the end of the light phase and a nadir in the early dark. Comparing the DD rhythm with the LD rhythm, two observations are conspicuous. First, the biphasic shape of the rhythm changed to monophasic although a second smaller nadir at ZT 6 was still present (not significantly different from ZT 2 and ZT 10). Second, the extreme levels seem to be shifted. Since the free-running periods in Djungarian hamsters are relatively close to 24 h [12], the apparent shift most probably is an artifact caused by the relative uncertainty on the true peak time due to sample intervals. The neuronal pathways by which environmental lighting may modulate the cAMP rhythm include the retinohypothalamic tract which provides strong retino-afferent projection to the Phodopus SCN [6,19,21]. A second photic input to the SCN stems from the metathalamic intergeniculate leaflet (IGL). The major transmitter of the IGL-SCN projection, neuropeptide Y. increased in concentration in the Phodopus SCN at night [20] which may exert inhibitory influence on cAMP metabolism. The shaping of the cAMP rhythm under LD also may involve serotonin which, as part of the rapheSCN projection [10], is augmented during the light period in
133
the rat SCN [4]. It was suggested that serotonin at the 5-HT7 receptor activates adenylate cyclase resulting in augmented cAMP levels [16]. We observed that, in aged hamsters held under LD, cAMP content was similar at ZT 14 and ZT 18, i.e. 2 h before and after lights off, while in young adult animals a dramatic decrease of cAMP was found between these timepoints. Although our investigation did not cover the whole 24 h cycle due to the limited availability of senile animals, we would like to offer two possible explanations for this difference. First, it seems possible that senile animals are less sensitive to light and exhibit a pattern similar to that seen under DD. Second, it is conceivable that the circadian rhythm of PDE activity [15] is abolished with advancing age of the animal, in line with age-related changes of the circadian system [3,18,23]. This point, however, requires further clarification. Our hamster data and those obtained in rat reveal that cAMP is involved in time-keeping mechanisms of the mammalian endogenous clock residing in the SCN. This view is supported by the findings that cAMP may inhibit SCN electrical activity [11], possibly by modulating the inward rectifier current in SCN neurons [1], advance the phase of neuronal firing [8], and stimulate gene expression via the phosphorylation of the cAMP response element binding protein [9]. We thank HSD Dr. R. Spessert for helpful discussion, J. Haase for technical help, Dr. J. Olcese (IHF, Hamburg, Germany) for critically reading the manuscript, and the NMFZ (Rheinland-Pfalz) and DFG (Re 644/2-2) for financial support of our research. Results of this study were presented as part of a thesis by S. Rimoldi in partial fulfillment of his doctoral degree at the Johannes Gutenberg-University, Mainz. [1] Akasu, T. and Shoji, S., cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus, Pflu¨gers Arch. Eur. J. Physiol., 429 (1994) 117–125. [2] Bradford, M.M., A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248–254. [3] Brock, M.A., Biological clocks and aging, Rev. Biol. Res. Aging, 2 (1985) 445–462. [4] Cagampang, F.R.A. and Inouye, S.I.T., Diurnal and circadian changes of serotonin in the suprachiasmatic nuclei: regulation by light and an endogenous pacemaker, Brain Res., 639 (1994) 175–179. [5] Creel, D., Inappropriate use of albino animals as models in research, Pharmacol. Biochem. Behav., 12 (1980) 969–977. [6] Decker, K. and Reuss, S., Nitric oxide-synthesizing neurons in the hamster suprachiasmatic nucleus: a combined NOS- and NADPH-staining and retinohypothalamic tract tracing study, Brain Res., 666 (1994) 284–288. [7] Edmunds, L.N., Carre, I.A., Tamponnet, C. and Tong, J., The role of ions and second messengers in circadian clock function, Chronobiol. Int., 9 (1992) 180–200. [8] Gillette, M.U. and Prosser, R.A., Circadian rhythm of the rat suprachiasmatic brain slice is rapidly reset by daytime application of cAMP analogs, Brain Res., 474 (1988) 348–352.
134
S. Reuss, S. Rimoldi / Neuroscience Letters 241 (1998) 131–134
[9] Ginty, D.D., Kornhauser, J.M., Thompson, M.A., Bading, H., Mayo, K.E., Takahashi, J.S. and Greenberg, M.E., Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock, Science, 260 (1993) 238–241. [10] Kawano, H., Decker, K. and Reuss, S., Is there a direct retinaraphe-suprachiasmatic nucleus pathway in the rat?, Neurosci. Lett., 212 (1996) 143–146. [11] Liou, S.Y., Shibata, S., Shiratsuchi, A. and Ueki, S., Effects of dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparations, Neurosci. Lett., 67 (1986) 339–343. [12] Milette, J.J. and Turek, F.W., Circadian and photoperiodic effects of brief light pulses in male Djungarian hamsters, Biol. Reprod., 35 (1994) 327–335. [13] Moore, R.Y., Organisation and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783–2788. [14] Murakami, N. and Takahashi, K., Circadian rhythm of adenosine-3′,5′monophosphate content in suprachiasmatic nucleus (SCN) and ventromedial hypothalamus (VMH) in the rat, Brain Res., 276 (1983) 297–304. [15] Prosser, R.A. and Gillette, M.U., Cyclic changes in cAMP concentration and phosphodiesterase activity in a mammalian circadian clock studied in vitro, Brain Res., 568 (1991) 185–192. [16] Prosser, R.A., Heller, H.C. and Miller, J.D., Serotonergic phase advances of the mammalian circadian clock involve protein kinase A and K + channel opening, Brain Res., 644 (1994) 67–73. [17] Reuss, S., Components and connections of the circadian timing system in mammals, Cell Tissue Res., 285 (1996) 353–378.
[18] Reuss, S. and Bu¨rger, K., Substance P-like immunoreactivity in the hypothalamic suprachiasmatic nucleus of Phodopus sungorus relation to daytime, photoperiod, sex and age, Brain Res., 638 (1994) 189–195. [19] Reuss, S. and Decker, K., Anterograde tracing of retinohypothalamic afferents with Fluoro-Gold, Brain Res., 745 (1997) 197–204. [20] Reuss, S. and Olcese, J., Neuropeptide Y: distribution of immunoreactivity and quantitative analysis in diencephalic structures and cerebral cortex of dwarf hamsters under different photoperiods, Neuroendocrinology, 61 (1995) 337–347. [21] Reuss, S., Decker, K., Ho¨dl, P. and Sraka, S., Anterograde neuronal tracing of retinohypothalamic projections in the hamster – evidence for the innervation of substance P-containing neurons in the suprachiasmatic nucleus, Neurosci. Lett., 174 (1994) 51–54. [22] Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449–526. [23] Weiland, N.G. and Wise, P.M., Aging progressively decreases the densities and alters the diurnal rhythms of alpha 1-adrenergic receptors in selected hypothalamic regions, Endocrinology, 126 (1990) 2392–2397. [24] Welsh, D.K., Logothetis, D.E., Meister, M. and Reppert, S.M., Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms, Neuron, 14 (1995) 697–706. [25] Yamazaki, S., Maruyama, M., Cagampang, F.R.A. and Inouye, S.T., Circadian fluctuations of cAMP content in the suprachiasmatic nucleus and the anterior hypothalamus of the rat, Brain Res., 651 (1994) 329–331.