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Decreased level of light-induced Fos expression in the suprachiasmatic nucleus of diabetic rats
Neuroscience Letters 227 (1997) 103–106
Decreased level of light-induced Fos expression in the suprachiasmatic nucleus of diabetic rats Shiro Yamanouchi a, Takao Shimazoe a ,*, Seiko Nagata a, Takahiro Moriya a, Miyuki Maetani a, Shigenobu Shibata b, Shigenori Watanabe a, Kyoko Miyasaka c, Akira Kono d, Akihiro Funakoshi d a
Department of Pharmacology, Faculty of Pharmaceutical Science, Kyushu University 62, Fukuoka 812-82, Japan b Department of Pharmacology, School of Human Sciences, Waseda University, Tokorozawa, Saitama 359, Japan c Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan d National Kyushu Cancer Center, Fukuoka 815, Japan Received 3 March 1997; revised version received 22 April 1997; accepted 24 April 1997
Abstract We assessed light-induced Fos-immunoreactive cells in the suprachiasmatic nucleus of diabetic rats. The number of Fos-immunoreactive cells significantly decreased in diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats as compared with control Long–Evans Tokushima Otsuka (LETO) rats. In contrast there was no decrease in the number of Fos-immunoreactive cells in young OLETF rats which have not yet developed diabetes. Two months after the administration of streptozotocin (STZ) to Wistar rats, the number of Fosimmunoreactive cells significantly decreased, although 1 week after the administration of STZ, the number had not yet changed in these STZ-induced diabetic rats. These results suggest that chronic diabetic (hyperglycemic) conditions may affect the light entraining responses in the suprachiasmatic nucleus (SCN). 1997 Elsevier Science Ireland Ltd. Keywords: Diabetes; Circadian rhythm; Suprachiasmatic nucleus; Streptozotocin; Otsuka Long–Evans Tokushima Fatty rats; Fos
Several reports have demonstrated that diabetic animals have abnormalities in the circadian rhythms of plasma corticosterone [12,22], locomotor activity [19,22], eating and drinking [15] under the light-dark cycle. Velasco et al. reported that diabetic rats showed similar activity in both phases of the light-dark cycle, and differences of phase angle in plasma corticosterone level [22]. The mammalian suprachiasmatic nucleus (SCN) is known as the center of the circadian timing system, such as eating, drinking, locomotor activity, sleep-wakefulness, plasma corticosterone level and body temperature [6,16]. In addition to its endogenous time-keeping function, the SCN has an important function to mediate the entrainment or synchronization of circadian rhythms to the daily lightdark cycle. The photic entrainment of circadian rhythms may be caused by its modulatory action on the SCN. Fos, one of the immediate early gene products, is involved in regulating the gene transcription cascade coupling external * Corresponding author. Fax: +81 92 6426632.
stimuli to the long-term responses of neuronal and non-neuronal cells [8,18]. The Fos protein is known to increase in the ventrolateral part of the SCN in response to the photic stimulation [13,14]. Moreover, a block of Fos expression by the use of antisense oligonucleotides inhibits the lightinduced phase-shift of the rat free-running activity rhythm [23]. These reports suggest that quantitative analysis of Fos expression is useful for monitoring the photic entrainment in the SCN. However, no reports have discussed the relationship between diabetes-mellitus (DM) induced abnormalities of circadian rhythms and the mechanisms in the SCN. Otsuka Long–Evans Tokushima Fatty (OLETF) rats are a newly developed model of non-insulin dependent diabetes mellitus (NIDDM). The characteristic features of OLETF rats are the late onset of hyperglycemia (after 18 weeks of age), mild obesity, and after 65 weeks of age an insulin deficiency [7]. Therefore, we studied light-induced Fos expression to determine the changes of photic entraining responses in the SCN of this NIDDM model rats. For comparison we also used
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0324-8
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S. Yamanouchi et al. / Neuroscience Letters 277 (1997) 103–106
streptozotocin (STZ)-induced diabetic rats. Male OLETF rats and control male Long–Evans Tokushima Otsuka (LETO) rats were obtained from Otsuka Tokushima Research Institute, Tokushima, Japan. Fos expression was studied in 6–8 and 25–30 week old rats. The body weights of the rats at 6–8 weeks were between 183 and 206 g for LETO and 222–252 g for OLETF. At 25–30 weeks the weights were between 470 and 542 g for LETO and 577– 735 g for OLETF. The plasma glucose levels were 101–119 mg/dl for LETO and 108–125 mg/dl for OLETF at 6–8 weeks, while at 25–30 weeks the levels were 124–139 mg/dl for LETO and 162–229 mg/dl for OLETF. Male Wistar rats (Seac Yoshitomi Labs., Charles Liver; 190–220 g) were also used in this study. STZ was administered (60 mg/kg in 0.2 ml citrate buffer; Sigma Chemical Co., St. Louis, MO, USA) 1 week after adaptation to the light-dark cycle. Citrate buffer was injected as a control. We then assessed the Fos-immunoreactive cells using hyperglycemic rats 1 week and 2 months after the STZ injection. One week after the STZ injection, the body weights were not different from those of control rats (225–260 g). Two months after the vehicle injection, the body weights were 430–470 g. On the other hand, two months after the STZ injection, the body weights were 240–330 g. Then, each plasma glucose level was 244–367 mg/dl 1 week after the injection and more than 400 mg/dl 2 months after the injection. In contrast, plasma glucose level of control rats did not change during the present experiment (92–161 mg/dl). Rats were housed under a 12:12 light-dark cycle (light period 0700–1900 h) with free access to food and water, After 2 days of the constant darkness, rats were exposed to 1 h of light (300 lx of white light for 1 h) at a projected circadian time of 13.5. The rats were then returned to darkness. Two hours after the onset of light stimulation the rats were anesthetized deeply with pentobarbital, and perfused transcardially with 200 ml of saline, followed by 200 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The brains were removed and fixed with 50 ml of 4% paraformaldehyde before being transferred to 30% sucrose solution in 0.1 M phosphate buffer. Hypothalamic blocks were cut into sections at a thickness of 30 mm from rostral to caudal SCN on a freezing microtome. The sections were processed for immunohistochemistry using the avidinbiotin-peroxidase complex technique as reported previously [5,11]. The primary antibody (anti-Fos, Genosys Biotechnologies Inc., USA; 1:1000) was diluted in 0.1 M phosphate buffer containing 1% normal rabbit serum in 0.3% Triton X-100. To determine the rostral-caudal distribution of Fosimmunoreactive neurons through the SCN, sections at 30 mm intervals were selected for analysis from each animal. The number of cells which expressed Fos-immunoreactivity were counted on selected sections from rostral to caudal SCN. The average number of cells per unilateral SCN were calculated and shown as number of cells/SCN. Results are expressed as means ± SEM. A significant
difference between groups was determined by Student’s ttest. Exposure of rats to light at circadian time 13.5 resulted in Fos expression within the SCN region. The majority of cells that displayed Fos-like immunoreactivity were located in the ventrolateral region of the caudal SCN, which corresponds with the lateral field of the retinohypothalamic tract. In 6–8 week old OLETF and LETO rats, the same Fos-like immunoreactivity was observed(55.03 ± 1.39 cells/SCN for LETO rats, 52.82 ± 1.65 cells/SCN for OLETF rats). However, the number of Fos-immunoreactive cells in the SCN of 25–30 week old OLETF rats was significantly lower than that of age-matched LETO rats (44.36 ± 3.38 cells/SCN for 25–30 week old LETO rats; 13.58 ± 3.4 cells/SCN for 25–30 week old OLETF rats; P , 0.001, student’s t-test; Fig. 1). Two months after STZ administration, the number of Fos-immunoreactive cells decreased significantly (41.75 ± 3.75 cells/SCN for control rats; 9.75 ± 0.85 cells/SCN for STZ treated rats; P , 0.001, student’s ttest; Fig. 2). However, 1 week after the administration of STZ no significant difference between STZ and control was observed (52.75 ± 3.99 cells/SCN for control rats; 42.25 ± 6.33 cells/SCN for STZ treated rats). OLETF rats show hyperglycemia and diabetic symptoms after about 18 weeks [7]. In contrast STZ-treated rats show hyperglycemia only 1 day after the injection. These results suggest that continuous hyperglycemia affects circadian rhythms, especially photic entraining responses. Lightinduced Fos expression is a marker for the photic entrainment. Therefore, the decrease of Fos-immunoreactivity in diabetic OLETF rats might result in abnormalities in photic entraining responses, such as entrainment to the light-dark cycle, the light-induced phase-shift of the locomotor activity rhythm, splitting of circadian locomotor activity rhythms under constant condition of illuminations, and re-entrain-
Fig. 1. Light-induced Fos expression in the SCN of OLETF and LETO rats. Rats were exposed to lights for 1 h at circadian time of 13.5 (300 lx, white light). Data represents the mean ± SEM of the number of determinations indicated in parentheses. ###P , 0.001 vs. LETO rats (Student’s ttest).
S. Yamanouchi et al. / Neuroscience Letters 277 (1997) 103–106
Fig. 2. Light-induced Fos expression in the SCN of STZ-induced diabetic and vehicle-treated rats. Rats were exposed to light for 1 h at circadian time of 13.5 (300 lx). Data represents the mean ± SEM of the number of determinations indicated in parentheses. ###P , 0.001 vs. vehicle-treated rats (Student’s t-test).
ment to the light-dark cycle after phase-shift. Locomotor activity of LETO rats and OLETF rats entrained 12:12 light-dark cycle. OLETF rats, however, needed much more days for re-entrainment to new light-dark cycle (light period 1500–0300 h), especially on the onset of the activity (data not shown). On the other hand, cataracts are induced in diabetic rats and DM patients. The cataract may cause the decrease of photic information from the retina to the SCN. Giancarlo et al. showed that STZ-treated rats did not lose photic information after 12 weeks of the injection in spite of progression of cataract [21]. It is also reported that light pulse of less than 30 lx can induce Fos expression [17]. So, for STZ-induced diabetic rats, the decrease of Fos expression may be caused by the decrease of photic information to some extent. However, in 25–30 week old OLETF rats, cataracts were not induced at all. Therefore, more complicated factors should be involved in the decrease of Fos immunoreactive cells. In previous reports, it has been suggested that abnormalities of circadian rhythms in diabetic animals depended on the loss of a circadian feeding schedule, polyphagia and polydipsia [22]. The daily intake of food and the body weight of OLETF rats were much greater than those of LETO rats although the animals were the same age [10]. Some reports suggested that feeding schedule is involved in the expression of circadian rhythms [1,2]. Locomotor activity of OLETF rats entrained to 12:12 light-dark cycle. This result suggests that considerable change of feeding schedule did not occur. So, this factor may not be main cause of the decrease of Fos immunoreactive cells. We have reported previously that due to a genetic abnormality OLETF rats showed no cholecystokinin (CCK)-A receptor gene expression [4]. Thus, OLETF rats represent a naturally occurring CCK-A receptor gene ‘knockout’ rat. One function of CCK-A receptor in the hypothalamus is to regulate food intake. Thus, a dysfunc-
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tional CCK-A receptor may cause some disorders such as hyperglycemia, hyperphagia, and obesity, which are characteristic of NIDDM. Della-Fera et al. reported that CCK is present in the mammalian SCN [3]. Then, CCK mRNA is reported to increase in the hypothalamus of STZ-treated diabetic rats [20]. CCK is also reported to have a circadian regulatory action related to vasopressin and oxytocin release [9]. In our experiments with young OLETF rats, the number of Fos-immunoreactive cells was not different from young LETO rats. These results show that CCK-A receptor is less important for Fos expression, but we still cannot neglect the role of CCK on the circadian system. Neuropeptide Y (NPY) mRNA is also reported to increase in the hypothalamus of STZ-treated diabetic rats [20]. NPY is thought to be one of the transmitters for geniculo-hypothalamic tract. Therefore, the increase of NPY may affect the circadian system. However, we cannot define the cause of the abnormality on the circadian system of diabetic rats at present. In summary, we demonstrated that diabetic rats such as OLETF rats (25–30 weeks old) and STZ-treated rats (2 months after the administration) showed decreased number of Fos-immunoreactive cells in the SCN. These findings suggest that diabetic rats have lower photic responses in the SCN. [1] Aschoff, J., von Goetz, C. and Honma, K., Restricted feeding in rats: effects of varying feeding cycles, Z. Tierpsychol., 63 (1983) 91– 111. [2] Boulos, Z., Rosenwasser, A.M. and Terman, M., Feeding schedules and the circadian organization of behavior in the rat, Behav. Brain Res., 1 (1980) 639–653. [3] Della-fera, M.A. and Koch, J., Gingerrich, R.L. and Baile, C.A., Intestinal infusion of a liquid diet alters CCK and NPY concentrations in specific brain areas of rats, Physiol. Behav., 48 (1990) 423– 428. [4] Funakoshi, A., Miyasaka, K., Shinozaki, H., Masuda, M., Kawanami, T., Takata, Y. and Kono, A., An animal model of congenital defect of gene expression of cholecystokinin (CCK)-A receptor, Biochem. Biophys. Res. Commun., 210 (1995) 787–796. [5] Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577–580. [6] Inoue, S.T. and Shibata, S., Neurochemical organization of circadian rhythm in the suprachiasmatic nucleus, Neurosci. Res., 20 (1994) 109–130. [7] Kawano, K., Hirashima, T., Mori, S., Saitoh, Y., Kurosumi, M. and Natori, T., Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain, Diabetes, 41 (1992) 1422–1428. [8] Marx, J.L., The fos gene as ‘master switch’, Science, 237 (1987) 854–856. [9] Morawska-Barrszceewska, J., Guzek, J.W. and KaczorowssskaSkora, J., Cholecystokinin octapeptide and the daily rhythm of vasopressin and oxytocin release, Exp. Clin. Endocrinol. Diabetes, 104 (1996) 164–171. [10] Miyasaka, K., Kanai, S., Ohta, M., Kawanami, T., Kono, A. and Funakoshi, A., Lack of satiety effect of cholecystokinin (CCK) in a new rat model not expressing the CCK A receptor gene, Neurosci. Lett., 180 (1994) 143–146. [11] Ono, M., Watanabe, A., Matsumoto, Y., Fukushima, T., Nishikawa,
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S. Yamanouchi et al. / Neuroscience Letters 277 (1997) 103–106 Y., Moriya, T., Shibata, S. and Watanabe, S., Methamphetamine modifies the photic entraining responses in the rodent suprachiasmatic nucleus, Neuroscience, 72 (1996) 213–224. Oster, M.H., Castonguay, T.W., Keen, C.L. and Stern, J.S., Circadian rhythm of corticosterone in diabetic rats, Life Sci., 43 (1988) 1643– 1645. Rea, M.A., Light increases Fos-related protein immunoreactivity in the rat suprachiasmatic nuclei, Brain Res. Bull., 23 (1989) 577– 581. Rea, M.A., Different populations of cells in the suprachiasmatic nuclei express c-fos in association with light-induced phase delays and advances of the free-running activity rhythm in hamsters, Brain Res., 579 (1992) 107–112. Reuterving, C.-O. and Hagg, E., Circadian eating and drinking habits in alloxan diabetic rats, Diabetes Metab., 13 (1987) 99–105. Rusak, B. and Binak, G., Neurotransmitters in the mammalian circadian system, Annu. Rev. Neurosci., 13 (1990) 387–401. Rusak, B., Robertson, H.A., Wisden, W. and Hunt, S.P., Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science, 248 (1990) 1237–1240. Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477–485.
[19] Shimomura, Y., Shimizu, H., Takahashi, M., Sato, N., Uehara, Y., Negishi, M., Kobayashi, I. and Kobayashi, S., Ambulatory activity in streptozotocin-induced diabetic rats, Physiol., Behav., 47 (1990) 1153–1155. [20] Sipols, A.J., Baskin, D.G. and Schwartz, M.W., Effect of intracerebroventricular insulin infusion on diabetic hyperphagia and hypothalamic neuropeptide gene expression, Diabetes, 44 (1995) 147–151. [21] Ugazio, G., Bosia, S., Burdino, E. and Gringnolo, F., Amelioration of diabetes and cataract by Na3VO4 plus U-83836E in streptozotocin treated rats, Res. Commun. Mol. Pathol. Pharmacol., 85 (1994) 313– 328. [22] Velasco, A., Huerta, I. and Marin, B., Plasma corticosterone, motor activity and metabolic circadian patterns in streptozotocin-induced diabetes rats, Chronobiol. Int., 5 (1988) 127–135. [23] Wollinik, F., Brysch, W., Uhlmann, E., Gillardon, F., Bravo, R., Zimmermann, M., Schlingensiepen, K.H. and Herdegen, T., Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock, Eur. J. Neurosci., 7 (1995) 388–393.
Decreased level of light-induced Fos expression in the suprachiasmatic nucleus of diabetic rats Shiro Yamanouchi a, Takao Shimazoe a ,*, Seiko Nagata a, Takahiro Moriya a, Miyuki Maetani a, Shigenobu Shibata b, Shigenori Watanabe a, Kyoko Miyasaka c, Akira Kono d, Akihiro Funakoshi d a
Department of Pharmacology, Faculty of Pharmaceutical Science, Kyushu University 62, Fukuoka 812-82, Japan b Department of Pharmacology, School of Human Sciences, Waseda University, Tokorozawa, Saitama 359, Japan c Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan d National Kyushu Cancer Center, Fukuoka 815, Japan Received 3 March 1997; revised version received 22 April 1997; accepted 24 April 1997
Abstract We assessed light-induced Fos-immunoreactive cells in the suprachiasmatic nucleus of diabetic rats. The number of Fos-immunoreactive cells significantly decreased in diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats as compared with control Long–Evans Tokushima Otsuka (LETO) rats. In contrast there was no decrease in the number of Fos-immunoreactive cells in young OLETF rats which have not yet developed diabetes. Two months after the administration of streptozotocin (STZ) to Wistar rats, the number of Fosimmunoreactive cells significantly decreased, although 1 week after the administration of STZ, the number had not yet changed in these STZ-induced diabetic rats. These results suggest that chronic diabetic (hyperglycemic) conditions may affect the light entraining responses in the suprachiasmatic nucleus (SCN). 1997 Elsevier Science Ireland Ltd. Keywords: Diabetes; Circadian rhythm; Suprachiasmatic nucleus; Streptozotocin; Otsuka Long–Evans Tokushima Fatty rats; Fos
Several reports have demonstrated that diabetic animals have abnormalities in the circadian rhythms of plasma corticosterone [12,22], locomotor activity [19,22], eating and drinking [15] under the light-dark cycle. Velasco et al. reported that diabetic rats showed similar activity in both phases of the light-dark cycle, and differences of phase angle in plasma corticosterone level [22]. The mammalian suprachiasmatic nucleus (SCN) is known as the center of the circadian timing system, such as eating, drinking, locomotor activity, sleep-wakefulness, plasma corticosterone level and body temperature [6,16]. In addition to its endogenous time-keeping function, the SCN has an important function to mediate the entrainment or synchronization of circadian rhythms to the daily lightdark cycle. The photic entrainment of circadian rhythms may be caused by its modulatory action on the SCN. Fos, one of the immediate early gene products, is involved in regulating the gene transcription cascade coupling external * Corresponding author. Fax: +81 92 6426632.
stimuli to the long-term responses of neuronal and non-neuronal cells [8,18]. The Fos protein is known to increase in the ventrolateral part of the SCN in response to the photic stimulation [13,14]. Moreover, a block of Fos expression by the use of antisense oligonucleotides inhibits the lightinduced phase-shift of the rat free-running activity rhythm [23]. These reports suggest that quantitative analysis of Fos expression is useful for monitoring the photic entrainment in the SCN. However, no reports have discussed the relationship between diabetes-mellitus (DM) induced abnormalities of circadian rhythms and the mechanisms in the SCN. Otsuka Long–Evans Tokushima Fatty (OLETF) rats are a newly developed model of non-insulin dependent diabetes mellitus (NIDDM). The characteristic features of OLETF rats are the late onset of hyperglycemia (after 18 weeks of age), mild obesity, and after 65 weeks of age an insulin deficiency [7]. Therefore, we studied light-induced Fos expression to determine the changes of photic entraining responses in the SCN of this NIDDM model rats. For comparison we also used
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0324-8
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S. Yamanouchi et al. / Neuroscience Letters 277 (1997) 103–106
streptozotocin (STZ)-induced diabetic rats. Male OLETF rats and control male Long–Evans Tokushima Otsuka (LETO) rats were obtained from Otsuka Tokushima Research Institute, Tokushima, Japan. Fos expression was studied in 6–8 and 25–30 week old rats. The body weights of the rats at 6–8 weeks were between 183 and 206 g for LETO and 222–252 g for OLETF. At 25–30 weeks the weights were between 470 and 542 g for LETO and 577– 735 g for OLETF. The plasma glucose levels were 101–119 mg/dl for LETO and 108–125 mg/dl for OLETF at 6–8 weeks, while at 25–30 weeks the levels were 124–139 mg/dl for LETO and 162–229 mg/dl for OLETF. Male Wistar rats (Seac Yoshitomi Labs., Charles Liver; 190–220 g) were also used in this study. STZ was administered (60 mg/kg in 0.2 ml citrate buffer; Sigma Chemical Co., St. Louis, MO, USA) 1 week after adaptation to the light-dark cycle. Citrate buffer was injected as a control. We then assessed the Fos-immunoreactive cells using hyperglycemic rats 1 week and 2 months after the STZ injection. One week after the STZ injection, the body weights were not different from those of control rats (225–260 g). Two months after the vehicle injection, the body weights were 430–470 g. On the other hand, two months after the STZ injection, the body weights were 240–330 g. Then, each plasma glucose level was 244–367 mg/dl 1 week after the injection and more than 400 mg/dl 2 months after the injection. In contrast, plasma glucose level of control rats did not change during the present experiment (92–161 mg/dl). Rats were housed under a 12:12 light-dark cycle (light period 0700–1900 h) with free access to food and water, After 2 days of the constant darkness, rats were exposed to 1 h of light (300 lx of white light for 1 h) at a projected circadian time of 13.5. The rats were then returned to darkness. Two hours after the onset of light stimulation the rats were anesthetized deeply with pentobarbital, and perfused transcardially with 200 ml of saline, followed by 200 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The brains were removed and fixed with 50 ml of 4% paraformaldehyde before being transferred to 30% sucrose solution in 0.1 M phosphate buffer. Hypothalamic blocks were cut into sections at a thickness of 30 mm from rostral to caudal SCN on a freezing microtome. The sections were processed for immunohistochemistry using the avidinbiotin-peroxidase complex technique as reported previously [5,11]. The primary antibody (anti-Fos, Genosys Biotechnologies Inc., USA; 1:1000) was diluted in 0.1 M phosphate buffer containing 1% normal rabbit serum in 0.3% Triton X-100. To determine the rostral-caudal distribution of Fosimmunoreactive neurons through the SCN, sections at 30 mm intervals were selected for analysis from each animal. The number of cells which expressed Fos-immunoreactivity were counted on selected sections from rostral to caudal SCN. The average number of cells per unilateral SCN were calculated and shown as number of cells/SCN. Results are expressed as means ± SEM. A significant
difference between groups was determined by Student’s ttest. Exposure of rats to light at circadian time 13.5 resulted in Fos expression within the SCN region. The majority of cells that displayed Fos-like immunoreactivity were located in the ventrolateral region of the caudal SCN, which corresponds with the lateral field of the retinohypothalamic tract. In 6–8 week old OLETF and LETO rats, the same Fos-like immunoreactivity was observed(55.03 ± 1.39 cells/SCN for LETO rats, 52.82 ± 1.65 cells/SCN for OLETF rats). However, the number of Fos-immunoreactive cells in the SCN of 25–30 week old OLETF rats was significantly lower than that of age-matched LETO rats (44.36 ± 3.38 cells/SCN for 25–30 week old LETO rats; 13.58 ± 3.4 cells/SCN for 25–30 week old OLETF rats; P , 0.001, student’s t-test; Fig. 1). Two months after STZ administration, the number of Fos-immunoreactive cells decreased significantly (41.75 ± 3.75 cells/SCN for control rats; 9.75 ± 0.85 cells/SCN for STZ treated rats; P , 0.001, student’s ttest; Fig. 2). However, 1 week after the administration of STZ no significant difference between STZ and control was observed (52.75 ± 3.99 cells/SCN for control rats; 42.25 ± 6.33 cells/SCN for STZ treated rats). OLETF rats show hyperglycemia and diabetic symptoms after about 18 weeks [7]. In contrast STZ-treated rats show hyperglycemia only 1 day after the injection. These results suggest that continuous hyperglycemia affects circadian rhythms, especially photic entraining responses. Lightinduced Fos expression is a marker for the photic entrainment. Therefore, the decrease of Fos-immunoreactivity in diabetic OLETF rats might result in abnormalities in photic entraining responses, such as entrainment to the light-dark cycle, the light-induced phase-shift of the locomotor activity rhythm, splitting of circadian locomotor activity rhythms under constant condition of illuminations, and re-entrain-
Fig. 1. Light-induced Fos expression in the SCN of OLETF and LETO rats. Rats were exposed to lights for 1 h at circadian time of 13.5 (300 lx, white light). Data represents the mean ± SEM of the number of determinations indicated in parentheses. ###P , 0.001 vs. LETO rats (Student’s ttest).
S. Yamanouchi et al. / Neuroscience Letters 277 (1997) 103–106
Fig. 2. Light-induced Fos expression in the SCN of STZ-induced diabetic and vehicle-treated rats. Rats were exposed to light for 1 h at circadian time of 13.5 (300 lx). Data represents the mean ± SEM of the number of determinations indicated in parentheses. ###P , 0.001 vs. vehicle-treated rats (Student’s t-test).
ment to the light-dark cycle after phase-shift. Locomotor activity of LETO rats and OLETF rats entrained 12:12 light-dark cycle. OLETF rats, however, needed much more days for re-entrainment to new light-dark cycle (light period 1500–0300 h), especially on the onset of the activity (data not shown). On the other hand, cataracts are induced in diabetic rats and DM patients. The cataract may cause the decrease of photic information from the retina to the SCN. Giancarlo et al. showed that STZ-treated rats did not lose photic information after 12 weeks of the injection in spite of progression of cataract [21]. It is also reported that light pulse of less than 30 lx can induce Fos expression [17]. So, for STZ-induced diabetic rats, the decrease of Fos expression may be caused by the decrease of photic information to some extent. However, in 25–30 week old OLETF rats, cataracts were not induced at all. Therefore, more complicated factors should be involved in the decrease of Fos immunoreactive cells. In previous reports, it has been suggested that abnormalities of circadian rhythms in diabetic animals depended on the loss of a circadian feeding schedule, polyphagia and polydipsia [22]. The daily intake of food and the body weight of OLETF rats were much greater than those of LETO rats although the animals were the same age [10]. Some reports suggested that feeding schedule is involved in the expression of circadian rhythms [1,2]. Locomotor activity of OLETF rats entrained to 12:12 light-dark cycle. This result suggests that considerable change of feeding schedule did not occur. So, this factor may not be main cause of the decrease of Fos immunoreactive cells. We have reported previously that due to a genetic abnormality OLETF rats showed no cholecystokinin (CCK)-A receptor gene expression [4]. Thus, OLETF rats represent a naturally occurring CCK-A receptor gene ‘knockout’ rat. One function of CCK-A receptor in the hypothalamus is to regulate food intake. Thus, a dysfunc-
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tional CCK-A receptor may cause some disorders such as hyperglycemia, hyperphagia, and obesity, which are characteristic of NIDDM. Della-Fera et al. reported that CCK is present in the mammalian SCN [3]. Then, CCK mRNA is reported to increase in the hypothalamus of STZ-treated diabetic rats [20]. CCK is also reported to have a circadian regulatory action related to vasopressin and oxytocin release [9]. In our experiments with young OLETF rats, the number of Fos-immunoreactive cells was not different from young LETO rats. These results show that CCK-A receptor is less important for Fos expression, but we still cannot neglect the role of CCK on the circadian system. Neuropeptide Y (NPY) mRNA is also reported to increase in the hypothalamus of STZ-treated diabetic rats [20]. NPY is thought to be one of the transmitters for geniculo-hypothalamic tract. Therefore, the increase of NPY may affect the circadian system. However, we cannot define the cause of the abnormality on the circadian system of diabetic rats at present. In summary, we demonstrated that diabetic rats such as OLETF rats (25–30 weeks old) and STZ-treated rats (2 months after the administration) showed decreased number of Fos-immunoreactive cells in the SCN. These findings suggest that diabetic rats have lower photic responses in the SCN. [1] Aschoff, J., von Goetz, C. and Honma, K., Restricted feeding in rats: effects of varying feeding cycles, Z. Tierpsychol., 63 (1983) 91– 111. [2] Boulos, Z., Rosenwasser, A.M. and Terman, M., Feeding schedules and the circadian organization of behavior in the rat, Behav. Brain Res., 1 (1980) 639–653. [3] Della-fera, M.A. and Koch, J., Gingerrich, R.L. and Baile, C.A., Intestinal infusion of a liquid diet alters CCK and NPY concentrations in specific brain areas of rats, Physiol. Behav., 48 (1990) 423– 428. [4] Funakoshi, A., Miyasaka, K., Shinozaki, H., Masuda, M., Kawanami, T., Takata, Y. and Kono, A., An animal model of congenital defect of gene expression of cholecystokinin (CCK)-A receptor, Biochem. Biophys. Res. Commun., 210 (1995) 787–796. [5] Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577–580. [6] Inoue, S.T. and Shibata, S., Neurochemical organization of circadian rhythm in the suprachiasmatic nucleus, Neurosci. Res., 20 (1994) 109–130. [7] Kawano, K., Hirashima, T., Mori, S., Saitoh, Y., Kurosumi, M. and Natori, T., Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain, Diabetes, 41 (1992) 1422–1428. [8] Marx, J.L., The fos gene as ‘master switch’, Science, 237 (1987) 854–856. [9] Morawska-Barrszceewska, J., Guzek, J.W. and KaczorowssskaSkora, J., Cholecystokinin octapeptide and the daily rhythm of vasopressin and oxytocin release, Exp. Clin. Endocrinol. Diabetes, 104 (1996) 164–171. [10] Miyasaka, K., Kanai, S., Ohta, M., Kawanami, T., Kono, A. and Funakoshi, A., Lack of satiety effect of cholecystokinin (CCK) in a new rat model not expressing the CCK A receptor gene, Neurosci. Lett., 180 (1994) 143–146. [11] Ono, M., Watanabe, A., Matsumoto, Y., Fukushima, T., Nishikawa,
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