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Calbindin-D28k immunoreactivity in the suprachiasmatic nucleus and the circadian response to constant light in the rat
Photic modulation of calbindin in the circadian clock
Pergamon PII: S0306-4522(00)00327-4
Neuroscience Vol. 99, No. 3, pp. 397–401, 2000 397 䉷 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
Letter to Neuroscience CALBINDIN-D28K IMMUNOREACTIVITY IN THE SUPRACHIASMATIC NUCLEUS AND THE CIRCADIAN RESPONSE TO CONSTANT LIGHT IN THE RAT A. ARVANITOGIANNIS, B. ROBINSON, C. BEAULE´ and S. AMIR* Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 deMaisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8 Key words: circadian activity rhythms, immunocytochemistry.
subnucleus reminiscent of the compact CaB subnucleus present in the core region of the SCN in hamsters. 18,19,32 CaB immunostaining in the dorsal region of the SCN was sparse. This pattern of differential regional expression coincides with retinal ganglion cell axons innervation of the SCN in rats 15 and closely resembles the distribution of several inducible transcription factors, transmitters and peptides implicated in photic signaling. 13,17,26 As previously described, 4 immunostaining for CaB marked a population of neurons in the intergeniculate leaflet (IGL, see Fig. 1), a retinorecipient structure implicated in both photic and non-photic regulation of circadian rhythms. 3,8,12,21,25,28 Statistical analysis of cell counts revealed that significantly fewer CaB-labeled neurons were present in the ventral SCN of rats perfused during the light phase (65.5 ^ 5.33, n 4) than in the dark phase (90.25 ^ 6.78, n 4) of the LD cycle (F1,6 8.225, P ⬍ 0.028). The mean number of CaB-labeled neurons seen in the IGL during the light phase (31.9 ^ 1.97, n 4) equaled that noted in the dark phase (35.56 ^ 1.54, n 4), indicating that the effect of light on CaB expression within the circadian system is specific to the SCN. This finding of differential sensitivity to light is consistent with other evidence that photic responses in the SCN and IGL in rats are mediated by different mechanisms. 1,6,9 Next, we examined CaB immunoreactivity in the SCN of intact rats that were housed in either constant light (LL, 300 lux) or constant darkness (DD) for four weeks. All rats housed in DD (n 11) displayed free running rhythms with periods close to 24 h, whereas, as was expected, 7 LL-housed rats (n 7) displayed disrupted rhythms (see representative actograms in Fig. 2a). To determine the influence of circadian phase on CaB immunoreactivity, rats from the DD group were perfused either during the subjective night (SN) or during the subjective day (SD), 4–6 h into the active or inactive phase. Rats in the LL group were all perfused during the day, between 10 a.m. and 4 p.m. Significantly fewer CaBlabeled neurons were seen in the ventral SCN of LL-housed rats than were seen in that of DD-housed rats, regardless of whether the latter were perfused in the SN or the SD (F2,15 9.47, P ⬍ 0.002; Fig. 2b). Importantly, in the SCN of DD-housed rats there were equal numbers of labeled neurons in the SN and SD (Fig. 2b). These findings are consistent with previous evidence from hamsters, 19 showing that
Recent studies in the hamster have led to the discovery that the expression of the calcium binding protein, calbindinD28k, is a defining feature of neurons in the suprachiasmatic nucleus involved in the regulation of circadian rhythms by environmental light. 2,18,19,32 To study further the involvement of calbindin-D28k, we examined the effect of exposure to constant light on calbindin-D28k immunoreactivity in the suprachiasmatic nucleus of intact rats and of rats treated neonatally with the retinal neurotoxin, monosodium glutamate. Exposure to constant light is known to disrupt circadian rhythms in rodents and we found previously that treatment with monosodium glutamate selectively prevents the disruptive effect of constant light on circadian rhythms in rats. 7,9 In the present study we found that exposure to light suppresses calbindin-D28k expression in the ventrolateral retinorecipient region of the suprachiasmatic nucleus of rats and that neonatal treatment with monosodium glutamate blocks the suppressive effect of constant light on calbindinD28k expression. These findings are consistent with the proposed role of calbindin-D28k in photic signaling in the suprachiasmatic nucleus, 32 and point to the possibility that suppression of calbindin-D28k expression is linked to the mechanism by which constant light disrupts circadian rhythms. 䉷 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.
We first examined the distribution of calbindin-D28k (CaB) immunoreactivity in the suprachiasmatic nucleus (SCN) of intact rats housed under a 12:12-h light–dark cycle (LD, 300 lux). Rats were perfused either during the day or during the night, 4–6 h after lights on (n 4) or off (n 4). As previously described, 30 immunostaining for CaB was evident throughout the rostro-caudal extent of the SCN, but was particularly dense in the ventrolateral region of the nucleus (Fig. 1); in this area labeled neurons appeared as a circumscribed *To whom correspondence should be addressed: Tel.: ⫹1-514-848-2188; Fax: ⫹1-514-848-2817. E-mail address: [email protected] (S. Amir). Abbreviations: ANOVA, analysis of variance; CaB, calbindin-D28k; DD, constant dark; IGL, intergeniculate leaflet; LD cycle, light–dark cycle; LL, constant light; MSG, monosodium glutamate; SCN, suprachiasmatic nucleus; SN, subjective night; SD, subjective day; TBS, Tris-buffered saline. 397
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Fig. 1. Photomicrograph of coronal brain sections showing examples of CaB immunostaining in the median SCN, (top) and rostral IGL (bottom).
CaB expression was independent of circadian phase. Interestingly, the mean number of CaB-labeled neurons seen in the SCN of DD-housed rats perfused in the SN (90.3 ^ 5.1, n 7) or SD (91.7 ^ 1.4, n 4) equaled that noted in the SCN of LD-housed rats perfused during the dark phase (90.25 ^ 6.78, n 4), and the number of CaB neurons seen in the SCN of LL-housed rats (69.28 ^ 3.34, n 7) equaled that noted in the SCN of LD-housed rats perfused during the day (65.5 ^ 5.33, n 4). This indicates that, in rats, housing in DD for four weeks is not sufficient to increase CaB expression in the SCN, as was previously shown in hamsters after seven weeks in DD, and, furthermore, that housing in constant light for four weeks does not lead to a further decrease in CaB expression in the SCN. Finally, lighting conditions did not affect CaB immunoreactivity in the IGL (Fig. 2b), further demonstrating that the expression of CaB in the SCN and IGL is differentially influenced by light. In a third experiment, we assessed CaB expression in the SCN of rats that were treated with monosodium glutamate (MSG) during the neonatal period and then housed in LL for three months starting at 21 days of age. Neonatal MSG treatment prevents the disruptive effect of LL housing on circadian rhythms in adult rats, 7,9 without affecting photic entrainment, 5,23,24 light-induced phase shifts and Fos protein expression in the SCN, 9,29 and light-induced suppression of pineal N-acetyltransferase activity. 27 As shown in the
actograms in Fig. 3a, the saline-treated control rats (n 6) displayed disrupted activity rhythms typically associated with extended LL housing, whereas as was expected, rats treated with MSG (n 6) displayed free running rhythms. Counts of SCN neurons immunoreactive to CaB revealed a significantly greater number of labeled neurons in MSGtreated rats compared to saline-treated control rats (F1,10 73.016, P ⬍ 0.0001; Fig. 3b). Treatment with MSG had no effect on CaB immunoreactivity in the IGL (Fig. 3b). Importantly, the mean number of CaB neurons seen in the SCN of saline-treated rats, which, in the present experiment, were housed in LL for three months (48.04 ^ 3.03, n 6), was significantly lower than that seen in rats perfused in the light phase of the LD cycle (65.5 ^ 5.33, n 4) or after four weeks in LL (69.28 ^ 3.34, n 7) (F2,14 10.1, P ⬍ .002; Tukey Compromise, P ⬍ 0.05). In contrast, the number of CaB neurons seen in the ventral SCN of MSGtreated rats (80.54 ^ 2.29, n 6) equaled that seen in intact rats perfused in the dark phase of the LD cycle (90.25 ^ 6.78, n 4) or in rats perfused after four weeks in DD (90.84 ^ 3.18, n 11) (F2,18 2.18, P 0.14). Thus, prolonged LL housing leads to a progressive, albeit slow, decrease in CaB expression in the SCN, and treatment with MSG, which blocks the disruptive effect of LL housing on circadian rhythms, prevents this effect. This latter finding suggests that the suppression of CaB expression in the SCN
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Fig. 2. (a) Double-plotted actograms showing examples of the last 15 days of wheel-running activity rhythms of animals housed in DD or LL for four weeks. The vertical marks indicate periods of activity of at least 10 wheel revolutions/10 min. Horizontal scale denotes time of day in hours. Successive days are plotted from top to bottom. (b) Number (mean ^ S.E.M.) of cells immunoreactive for CaB counted in the SCN and IGL of DD-housed rats perfused in the SN (n 7) or SD (n 4) and in LL-housed rats (n 7).*Significant difference from SN and SD (Tukey Compromise, P ⬍ 0.05).
Fig. 3. Double-plotted actograms showing examples of the last 15 days of wheel-running activity rhythms of animals treated neonatally with saline or MSG and housed in LL for three months starting at 21 days of age. (b) Number (mean ^ S.E.M.) of cells immunoreactive for CaB counted in the SCN and IGL of saline- and MSG-treated rats housed in LL for three months (n 6/group).*Significant difference from Saline (Tukey Compromise, P ⬍ 0.05).
and the disruption of circadian rhythms might be functionally related. Our findings support the idea that CaB, presumably by regulating intracellular calcium concentrations in retinorecipient neurons, influences photic signaling within the SCN. 19 Furthermore, they are consistent with evidence that the effect of light within the SCN involves activation of calcium-requiring transduction mechanisms. 10,11,16,31,35 It is worth noting that a high proportion of CaB-positive neurons in the SCN express the transcriptional regulatory protein, Fos,
in response to light, 32 and there is evidence to suggest that Fos is involved in the mechanism underlying clock resetting by light. 17,37 Further investigation of a possible interaction between the photically-regulated Fos and CaB expression should provide valuable insight into the light-responsive clock-resetting mechanism. Finally, it is interesting to consider the possibility that suppression of CaB expression in the SCN is part of the mechanism by which exposure to constant light disrupts circadian rhythms. The normal expression of circadian
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rhythms is presumed to require coupling among multiple SCN pacemaker neurons, 14,22,36 and it is conceivable that prolonged exposure to constant light disrupts circadian rhythms by altering processes responsible for coupling among these neurons. 20,38 In this context it is noteworthy that a role for calcium in the coupling of pacemaker neurons has been proposed 22,33,34 and further, that CaB, via an effect on intracellular calcium, could influence this coupling process. 19 Consistent with these ideas, we propose that changes in CaB expression produced by exposure to constant light may lead to persistent alterations in calcium dynamics and, in turn, to enduring uncoupling among pacemaker neurons in the SCN. EXPERIMENTAL PROCEDURES
The experimental procedures followed the guidelines of the Canadian Council on Animal Care. The procedures were approved by the Animal Care Committee, Concordia University, and all efforts were made to minimize the number of animals used and their suffering. Experiments were carried out in male Wistar rats (275–325 g, Charles River Canada, St Constant, Quebec). The rats were housed individually in cages equipped with running wheel and had free access to food and water. Circadian activity rhythms were monitored continuously using DataCol data acquisition hardware and software (Mini Mitter Co., Sunriver, OR, USA). Activity data were displayed as actograms using Circadia software. For treatment with MSG, rat pups housed with their mothers under a 12:12-h LD cycle, were treated subcutaneously with 2 mg/g of MSG (in distilled water) or saline (10% NaCl
solution) on postnatal days 1, 3, 5, 7, and 9, as previously described. 9 For CaB immunocytochemistry, the rats were anaesthetized with sodium pentobarbital (100 mg/kg i.p.) and perfused transcardially with 300 ml of cold physiological saline (0.9% NaCl) followed by 300 ml of cold, fresh 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.3). Brains were removed, postfixed in 4% paraformaldehyde (4⬚C) overnight, and cut on a vibratome in 50-mm-thick coronal sections. Immunostaining was carried-out on free-floating sections using a mouse anti-CaB monoclonal antibody (Sigma) diluted 1:20,000 with a solution of 0.3% Triton X-100 in tris-buffered saline (TBS) with 1% normal horse serum. Sections were incubated with the anti-CaB antibody for 48 h at 4⬚C, rinsed in TBS, and then transferred to a solution of 0.3% Triton X-100 in TBS containing biotinylated antimouse secondary antibody (1:200; Vector Labs). Calbindin immunoreactivity was detected with a Vectastain Elite ABC Kit (Vector Labs, ON, Canada) using diaminobenzidine as the chromogen. Counts of CaB-immunoreactive neurons within the SCN and IGL were obtained using a computerized image acquisition and analysis system with NIH Image software. Each region was imaged under high magnification ( × 40) and labeled cells were individually marked and manually counted. For analysis, the mean number of CaB immunoreactive cells was calculated from the counts of six alternate images showing the highest number of labeled cells. Group means were derived from these values and analysed using one-way ANOVA followed by Tukey Compromise test.
Acknowledgements—Supported by grants from the Medical Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, and from the Fonds pour la Formation de Chercheurs et l’Aide a` la Recherche (FCAR, Que´bec).
REFERENCES
1. Amir S. and Edelstein K. (1997) A blocker of nitric oxide synthase, NG-nitro-l-arginine methyl ester, attenuates light-induced Fos protein expression in rat suprachiasmatic nucleus. Neurosci. Lett. 224, 29–32. 2. Bryant D. N., LeSauter J., Silver R. and Romero M. T. (2000) Retinal innervation of calbindin-D28K cells in the hamster suprachiasmatic nucleus: ultrastructural characterization. J. biol. Rhythms 15, 103–111. 3. Card J. P. and Moore R. Y. (1989) Organization of lateral geniculate-hypothalamic connections in the rat. J. comp. Neurol. 284, 135–147. 4. Celio M. R. (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475. 5. Chambille I. and Serviere J. (1993) Neurotoxic effects of neonatal injections of monosodium l-glutamate (l- MSG) on the retinal ganglion cell layer of the golden hamster: anatomical and functional consequences on the circadian system. J. comp. Neurol. 338, 67–82. 6. Edelstein K. and Amir S. (1998) Glutamatergic antagonists do not attenuate light-induced fos protein in rat intergeniculate leaflet. Brain Res. 810, 264–268. 7. Edelstein K. and Amir S. (1999) The intergeniculate leaflet does not mediate the disruptive effects of constant light on circadian rhythms in the rat. Neuroscience 90, 1093–1101. 8. Edelstein K. and Amir S. (1999) The role of the intergeniculate leaflet in entrainment of circadian rhythms to a skeleton photoperiod. J. Neurosci. 19, 372–380. 9. Edelstein K., Pfaus J. G., Rusak B. and Amir S. (1995) Neonatal monosodium glutamate treatment prevents effects of constant light on circadian temperature rhythms of adult rats. Brain Res. 675, 135–142. 10. Golombek D. A. and Ralph M. R. (1994) KN-62, an inhibitor of Ca 2⫹/calmodulin kinase II, attenuates circadian responses to light. NeuroReport 5, 1638–1640. 11. Golombek D. A. and Ralph M. R. (1995) Circadian responses to light: the calmodulin connection. Neurosci. Lett. 192, 101–104. 12. Harrington M. E. (1997) The ventral lateral geniculate nucleus and the intergeniculate leaflet: interrelated structures in the visual and circadian systems. Neurosci. Biobehav. Rev. 21, 705–727. 13. Hastings M. H., Ebling F. J., Grosse J., Herbert J., Maywood E. S., Mikkelsen J. D. and Sumova A. (1995) Immediate-early genes and the neural bases of photic and non-photic entrainment. Ciba Found. Symp. 183, 175–189. 14. Herzog E. D., Takahashi J. S. and Block G. D. (1998) Clock controls circadian period in isolated suprachiasmatic nucleus neurons. Nat. Neurosci. 1, 708–713. 15. Johnson R. F., Morin L. P. and Moore R. Y. (1988) Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin. Brain Res. 462, 301–312. 16. Kako K., Wakamatsu H. and Ishida N. (1996) c-fos CRE-binding activity of CREB/ATF family in the SCN is regulated by light but not a circadian clock. Neurosci. Lett. 216, 159–162. 17. Kornhauser J. M., Mayo K. E. and Takahashi J. S. (1996) Light, immediate-early genes, and circadian rhythms. Behav. Genet. 26, 221–240. 18. LeSauter J. and Silver R. (1999) Localization of a suprachiasmatic nucleus subregion regulating locomotor rhythmicity. J. Neurosci. 19, 5574–5585. 19. LeSauter J., Stevens P., Jansen H., Lehman M. N. and Silver R. (1999) Calbindin expression in the hamster SCN is influenced by circadian genotype and by photic conditions. NeuroReport 10, 3159–3163. 20. Mason R. (1991) The effects of continuous light exposure on Syrian hamster suprachiasmatic (SCN) neuronal discharge activity in vitro. Neurosci. Lett. 123, 160–163. 21. Maywood E. S., Smith E., Hall S. J. and Hastings M. H. (1997) A thalamic contribution to arousal-induced, non-photic entrainment of the circadian clock of the Syrian hamster. Eur. J. Neurosci. 9, 1739–1747. 22. Miller J. D. (1998) The SCN is comprised of a population of coupled oscillators. Chronobiol. Int. 15, 489–511. 23. Miyabo S., Ooya E., Yamamura I. and Hayashi S. (1982) Ontogeny of circadian corticosterone rhythm in rats treated with monosodium glutamate neonatally. Brain Res. 248, 341–345.
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24. Miyabo S., Yamamura I., Ooya E., Aoyagi N., Horikawa Y. and Hayashi S. (1985) Effects of neonatal treatment with monosodium glutamate on circadian locomotor rhythm in the rat. Brain Res. 339, 201–208. 25. Moore R. Y. and Card J. P. (1994) Intergeniculate leaflet: an anatomically and functionally distinct subdivision of the lateral geniculate complex. J. comp. Neurol. 344, 403–430. 26. Moore R. Y., Gustafson E. L. and Card J. P. (1984) Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide, molluscan cardioexcitatory peptide and neuropeptide Y. Cell Tiss. Res. 236, 41–46. 27. Nemeroff C. B., Konkol R. J., Bissette G., Youngblood W., Martin J. B., Brazeau P., Rone M. S., Prange A. J. Jr, Breese G. R. and Kizer J. S. (1977) Analysis of the disruption in hypothalamic-pituitary regulation in rats treated neonatally with monosodium l-glutamate (MSG): evidence for the involvement of tuberoinfundibular cholinergic and dopaminergic systems in neuroendocrine regulation. Endocrinology 101, 613–622. 28. Pickard G. E. (1994) Intergeniculate leaflet ablation alters circadian rhythms in the mouse. NeuroReport 5, 2186–2188. 29. Pickard G. E., Turek F. W., Lamperti A. A. and Silverman A. J. (1982) The effect of neonatally administered monosodium glutamate (MSG) on the development of retinofugal projections and entrainment of circadian locomotor activity. Behav. neural. Biol. 34, 433–444. 30. Rogers J. H. and Resibois A. (1992) Calretinin and calbindin-D28k in rat brain: patterns of partial co- localization. Neuroscience 51, 843–865. 31. Shibata S. and Moore R. Y. (1994) Calmodulin inhibitors produce phase shifts of circadian rhythms in vivo and in vitro. J. Biol. Rhythms 9, 27–41. 32. Silver R., Romero M. T., Besmer H. R., Leak R., Nunez J. M. and LeSauter J. (1996) Calbindin-D28K cells in the hamster SCN express light-induced Fos. NeuroReport 7, 1224–1228. 33. van den Pol A. N. and Dudek F. E. (1993) Cellular communication in the circadian clock, the suprachiasmatic nucleus. Neuroscience 56, 793–811. 34. van den Pol A. N., Finkbeiner S. M. and Cornell-Bell A. H. (1992) Calcium excitability and oscillations in suprachiasmatic nucleus neurons and glia in vitro. J. Neurosci. 12, 2648–2664. 35. von Gall C., Duffield G. E., Hastings M. H., Kopp M. D., Dehghani F., Korf H. W. and Stehle J. H. (1998) CREB in the mouse SCN: a molecular interface coding the phase-adjusting stimuli light, glutamate, PACAP, and melatonin for clockwork access. J. Neurosci. 18, 10,389–10,397. 36. Welsh D. K., Logothetis D. E., Meister M. and Reppert S. M. (1995) Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14, 697–706. 37. Wollnik F., Brysch W., Uhlmann E., Gillardon F., Bravo R., Zimmermann M., Schlingensiepen K. H. and Herdegen T. (1995) Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock. Eur. J. Neurosci. 7, 388–393. 38. Zlomanczuk P., Margraf R. R. and Lynch G. R. (1991) In vitro electrical activity in the suprachiasmatic nucleus following splitting and masking of wheel-running behavior. Brain Res. 559, 94–99. (Accepted 28 June 2000)
Pergamon PII: S0306-4522(00)00327-4
Neuroscience Vol. 99, No. 3, pp. 397–401, 2000 397 䉷 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
Letter to Neuroscience CALBINDIN-D28K IMMUNOREACTIVITY IN THE SUPRACHIASMATIC NUCLEUS AND THE CIRCADIAN RESPONSE TO CONSTANT LIGHT IN THE RAT A. ARVANITOGIANNIS, B. ROBINSON, C. BEAULE´ and S. AMIR* Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 deMaisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8 Key words: circadian activity rhythms, immunocytochemistry.
subnucleus reminiscent of the compact CaB subnucleus present in the core region of the SCN in hamsters. 18,19,32 CaB immunostaining in the dorsal region of the SCN was sparse. This pattern of differential regional expression coincides with retinal ganglion cell axons innervation of the SCN in rats 15 and closely resembles the distribution of several inducible transcription factors, transmitters and peptides implicated in photic signaling. 13,17,26 As previously described, 4 immunostaining for CaB marked a population of neurons in the intergeniculate leaflet (IGL, see Fig. 1), a retinorecipient structure implicated in both photic and non-photic regulation of circadian rhythms. 3,8,12,21,25,28 Statistical analysis of cell counts revealed that significantly fewer CaB-labeled neurons were present in the ventral SCN of rats perfused during the light phase (65.5 ^ 5.33, n 4) than in the dark phase (90.25 ^ 6.78, n 4) of the LD cycle (F1,6 8.225, P ⬍ 0.028). The mean number of CaB-labeled neurons seen in the IGL during the light phase (31.9 ^ 1.97, n 4) equaled that noted in the dark phase (35.56 ^ 1.54, n 4), indicating that the effect of light on CaB expression within the circadian system is specific to the SCN. This finding of differential sensitivity to light is consistent with other evidence that photic responses in the SCN and IGL in rats are mediated by different mechanisms. 1,6,9 Next, we examined CaB immunoreactivity in the SCN of intact rats that were housed in either constant light (LL, 300 lux) or constant darkness (DD) for four weeks. All rats housed in DD (n 11) displayed free running rhythms with periods close to 24 h, whereas, as was expected, 7 LL-housed rats (n 7) displayed disrupted rhythms (see representative actograms in Fig. 2a). To determine the influence of circadian phase on CaB immunoreactivity, rats from the DD group were perfused either during the subjective night (SN) or during the subjective day (SD), 4–6 h into the active or inactive phase. Rats in the LL group were all perfused during the day, between 10 a.m. and 4 p.m. Significantly fewer CaBlabeled neurons were seen in the ventral SCN of LL-housed rats than were seen in that of DD-housed rats, regardless of whether the latter were perfused in the SN or the SD (F2,15 9.47, P ⬍ 0.002; Fig. 2b). Importantly, in the SCN of DD-housed rats there were equal numbers of labeled neurons in the SN and SD (Fig. 2b). These findings are consistent with previous evidence from hamsters, 19 showing that
Recent studies in the hamster have led to the discovery that the expression of the calcium binding protein, calbindinD28k, is a defining feature of neurons in the suprachiasmatic nucleus involved in the regulation of circadian rhythms by environmental light. 2,18,19,32 To study further the involvement of calbindin-D28k, we examined the effect of exposure to constant light on calbindin-D28k immunoreactivity in the suprachiasmatic nucleus of intact rats and of rats treated neonatally with the retinal neurotoxin, monosodium glutamate. Exposure to constant light is known to disrupt circadian rhythms in rodents and we found previously that treatment with monosodium glutamate selectively prevents the disruptive effect of constant light on circadian rhythms in rats. 7,9 In the present study we found that exposure to light suppresses calbindin-D28k expression in the ventrolateral retinorecipient region of the suprachiasmatic nucleus of rats and that neonatal treatment with monosodium glutamate blocks the suppressive effect of constant light on calbindinD28k expression. These findings are consistent with the proposed role of calbindin-D28k in photic signaling in the suprachiasmatic nucleus, 32 and point to the possibility that suppression of calbindin-D28k expression is linked to the mechanism by which constant light disrupts circadian rhythms. 䉷 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved.
We first examined the distribution of calbindin-D28k (CaB) immunoreactivity in the suprachiasmatic nucleus (SCN) of intact rats housed under a 12:12-h light–dark cycle (LD, 300 lux). Rats were perfused either during the day or during the night, 4–6 h after lights on (n 4) or off (n 4). As previously described, 30 immunostaining for CaB was evident throughout the rostro-caudal extent of the SCN, but was particularly dense in the ventrolateral region of the nucleus (Fig. 1); in this area labeled neurons appeared as a circumscribed *To whom correspondence should be addressed: Tel.: ⫹1-514-848-2188; Fax: ⫹1-514-848-2817. E-mail address: [email protected] (S. Amir). Abbreviations: ANOVA, analysis of variance; CaB, calbindin-D28k; DD, constant dark; IGL, intergeniculate leaflet; LD cycle, light–dark cycle; LL, constant light; MSG, monosodium glutamate; SCN, suprachiasmatic nucleus; SN, subjective night; SD, subjective day; TBS, Tris-buffered saline. 397
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Fig. 1. Photomicrograph of coronal brain sections showing examples of CaB immunostaining in the median SCN, (top) and rostral IGL (bottom).
CaB expression was independent of circadian phase. Interestingly, the mean number of CaB-labeled neurons seen in the SCN of DD-housed rats perfused in the SN (90.3 ^ 5.1, n 7) or SD (91.7 ^ 1.4, n 4) equaled that noted in the SCN of LD-housed rats perfused during the dark phase (90.25 ^ 6.78, n 4), and the number of CaB neurons seen in the SCN of LL-housed rats (69.28 ^ 3.34, n 7) equaled that noted in the SCN of LD-housed rats perfused during the day (65.5 ^ 5.33, n 4). This indicates that, in rats, housing in DD for four weeks is not sufficient to increase CaB expression in the SCN, as was previously shown in hamsters after seven weeks in DD, and, furthermore, that housing in constant light for four weeks does not lead to a further decrease in CaB expression in the SCN. Finally, lighting conditions did not affect CaB immunoreactivity in the IGL (Fig. 2b), further demonstrating that the expression of CaB in the SCN and IGL is differentially influenced by light. In a third experiment, we assessed CaB expression in the SCN of rats that were treated with monosodium glutamate (MSG) during the neonatal period and then housed in LL for three months starting at 21 days of age. Neonatal MSG treatment prevents the disruptive effect of LL housing on circadian rhythms in adult rats, 7,9 without affecting photic entrainment, 5,23,24 light-induced phase shifts and Fos protein expression in the SCN, 9,29 and light-induced suppression of pineal N-acetyltransferase activity. 27 As shown in the
actograms in Fig. 3a, the saline-treated control rats (n 6) displayed disrupted activity rhythms typically associated with extended LL housing, whereas as was expected, rats treated with MSG (n 6) displayed free running rhythms. Counts of SCN neurons immunoreactive to CaB revealed a significantly greater number of labeled neurons in MSGtreated rats compared to saline-treated control rats (F1,10 73.016, P ⬍ 0.0001; Fig. 3b). Treatment with MSG had no effect on CaB immunoreactivity in the IGL (Fig. 3b). Importantly, the mean number of CaB neurons seen in the SCN of saline-treated rats, which, in the present experiment, were housed in LL for three months (48.04 ^ 3.03, n 6), was significantly lower than that seen in rats perfused in the light phase of the LD cycle (65.5 ^ 5.33, n 4) or after four weeks in LL (69.28 ^ 3.34, n 7) (F2,14 10.1, P ⬍ .002; Tukey Compromise, P ⬍ 0.05). In contrast, the number of CaB neurons seen in the ventral SCN of MSGtreated rats (80.54 ^ 2.29, n 6) equaled that seen in intact rats perfused in the dark phase of the LD cycle (90.25 ^ 6.78, n 4) or in rats perfused after four weeks in DD (90.84 ^ 3.18, n 11) (F2,18 2.18, P 0.14). Thus, prolonged LL housing leads to a progressive, albeit slow, decrease in CaB expression in the SCN, and treatment with MSG, which blocks the disruptive effect of LL housing on circadian rhythms, prevents this effect. This latter finding suggests that the suppression of CaB expression in the SCN
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Fig. 2. (a) Double-plotted actograms showing examples of the last 15 days of wheel-running activity rhythms of animals housed in DD or LL for four weeks. The vertical marks indicate periods of activity of at least 10 wheel revolutions/10 min. Horizontal scale denotes time of day in hours. Successive days are plotted from top to bottom. (b) Number (mean ^ S.E.M.) of cells immunoreactive for CaB counted in the SCN and IGL of DD-housed rats perfused in the SN (n 7) or SD (n 4) and in LL-housed rats (n 7).*Significant difference from SN and SD (Tukey Compromise, P ⬍ 0.05).
Fig. 3. Double-plotted actograms showing examples of the last 15 days of wheel-running activity rhythms of animals treated neonatally with saline or MSG and housed in LL for three months starting at 21 days of age. (b) Number (mean ^ S.E.M.) of cells immunoreactive for CaB counted in the SCN and IGL of saline- and MSG-treated rats housed in LL for three months (n 6/group).*Significant difference from Saline (Tukey Compromise, P ⬍ 0.05).
and the disruption of circadian rhythms might be functionally related. Our findings support the idea that CaB, presumably by regulating intracellular calcium concentrations in retinorecipient neurons, influences photic signaling within the SCN. 19 Furthermore, they are consistent with evidence that the effect of light within the SCN involves activation of calcium-requiring transduction mechanisms. 10,11,16,31,35 It is worth noting that a high proportion of CaB-positive neurons in the SCN express the transcriptional regulatory protein, Fos,
in response to light, 32 and there is evidence to suggest that Fos is involved in the mechanism underlying clock resetting by light. 17,37 Further investigation of a possible interaction between the photically-regulated Fos and CaB expression should provide valuable insight into the light-responsive clock-resetting mechanism. Finally, it is interesting to consider the possibility that suppression of CaB expression in the SCN is part of the mechanism by which exposure to constant light disrupts circadian rhythms. The normal expression of circadian
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rhythms is presumed to require coupling among multiple SCN pacemaker neurons, 14,22,36 and it is conceivable that prolonged exposure to constant light disrupts circadian rhythms by altering processes responsible for coupling among these neurons. 20,38 In this context it is noteworthy that a role for calcium in the coupling of pacemaker neurons has been proposed 22,33,34 and further, that CaB, via an effect on intracellular calcium, could influence this coupling process. 19 Consistent with these ideas, we propose that changes in CaB expression produced by exposure to constant light may lead to persistent alterations in calcium dynamics and, in turn, to enduring uncoupling among pacemaker neurons in the SCN. EXPERIMENTAL PROCEDURES
The experimental procedures followed the guidelines of the Canadian Council on Animal Care. The procedures were approved by the Animal Care Committee, Concordia University, and all efforts were made to minimize the number of animals used and their suffering. Experiments were carried out in male Wistar rats (275–325 g, Charles River Canada, St Constant, Quebec). The rats were housed individually in cages equipped with running wheel and had free access to food and water. Circadian activity rhythms were monitored continuously using DataCol data acquisition hardware and software (Mini Mitter Co., Sunriver, OR, USA). Activity data were displayed as actograms using Circadia software. For treatment with MSG, rat pups housed with their mothers under a 12:12-h LD cycle, were treated subcutaneously with 2 mg/g of MSG (in distilled water) or saline (10% NaCl
solution) on postnatal days 1, 3, 5, 7, and 9, as previously described. 9 For CaB immunocytochemistry, the rats were anaesthetized with sodium pentobarbital (100 mg/kg i.p.) and perfused transcardially with 300 ml of cold physiological saline (0.9% NaCl) followed by 300 ml of cold, fresh 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.3). Brains were removed, postfixed in 4% paraformaldehyde (4⬚C) overnight, and cut on a vibratome in 50-mm-thick coronal sections. Immunostaining was carried-out on free-floating sections using a mouse anti-CaB monoclonal antibody (Sigma) diluted 1:20,000 with a solution of 0.3% Triton X-100 in tris-buffered saline (TBS) with 1% normal horse serum. Sections were incubated with the anti-CaB antibody for 48 h at 4⬚C, rinsed in TBS, and then transferred to a solution of 0.3% Triton X-100 in TBS containing biotinylated antimouse secondary antibody (1:200; Vector Labs). Calbindin immunoreactivity was detected with a Vectastain Elite ABC Kit (Vector Labs, ON, Canada) using diaminobenzidine as the chromogen. Counts of CaB-immunoreactive neurons within the SCN and IGL were obtained using a computerized image acquisition and analysis system with NIH Image software. Each region was imaged under high magnification ( × 40) and labeled cells were individually marked and manually counted. For analysis, the mean number of CaB immunoreactive cells was calculated from the counts of six alternate images showing the highest number of labeled cells. Group means were derived from these values and analysed using one-way ANOVA followed by Tukey Compromise test.
Acknowledgements—Supported by grants from the Medical Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, and from the Fonds pour la Formation de Chercheurs et l’Aide a` la Recherche (FCAR, Que´bec).
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