Bram Research, 582 (1992) 353-356 © 1992 Elsewer Science Publishers B V All rights reserved. 0006-8993/92/$05.00
353
BRES 25214
Phase-resetting effect of 8-OH-DPAT, a serotoninlA receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro Shigenobu Shibata, Atsuko Tsuneyoshi, Toshiyuki Hamada, Keiko Tominaga and Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceuttcal Scwnces, Kyushu University, Fukuoka (Japan) (Accepted 3 March 1992) Key words 5-Hydroxytryptamine, 8-OH-DPAT, Serotonm 5-HT~A; Suprachmsmatlc nucleus; Circadian rhythm
The 5-HTergic neurons m the mesencephahc raphe nucle~ provide a robust projection to the hypothalam~c suprachlasmat~c nucleus (SCN), the s~te of a putatwe neuronal c~rcadian pacemaker. Although ~t has been suggested that 5-HT neurons may play a role m the circa&an timing system, this role has not yet been specified. Prosser et al. (Bram Res., 534 (1990) 336-339) reported that 1 h treatments with qmpazine reduce robust phase shifts in vitro, and that this effect depends upon the c~rcadmn t~me of treatment However, qmpazine is a nonspecific 5-HT agomst. Besides, it is reported that the 5-HT~A agomst, 8-hydroxy-2-(dl-n-propylamino)tetrahne hydrobromlde (8-OH-DPAT) affected a circadian rhythm of hamster wheel-running activity In the present study we investigated whether the 5-HT~A agomst 8-OH-DPAT can reset the phase of the SCN clock when it is isolated m vitro The present results show that 1 h treatments with 8-OH-DPAT induce robust phase advances m vitro when it was administered during the subjectwe day. This result suggests that 5HTtA receptor functioning may play a role m modulating the phase of SCN clock, especially during the subjective day The mammalian suprachiasmatic nuclei (SCN) have been identified as a circadian pacemaker for behavioral and physiological functions 13'2°. There is a dense serotonergic (5-HT) projection to the SCN from cells located primarily in the dorsal and medial raphe nuclei ~'12. However, the functional significance of the input from the raphe to the circadian system is controversial. To elucidate the role of 5-HT function, many investigators have used the chemical and electrical destruction of the 5-HT system 2'4-6. To study the function of this projection in the mammalian circadian system, Prosser et al. 16 reported that quipazine could reset the phase of the SCN circadian pacemaker. Treatments during subjective daytime induced phase advances, while nighttime treatments induced phase delays. Because quipazine is a non-specific 5-HT agonist, it is uncertain whether the phase change induced by quipazine is due to actions at 5-HT1, at 5-HT 2 or at 5-HT 3 receptor subtypes. Recently we reported that the administration of the 5-HT1A agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone and ipsapirone during subjective day induced a phase advance of wheel-running activity under constant light condition 2s. However, administration of these drugs during subjective night did not induce phase changes. Besides, we found that the high
uptake of 2-deoxyglucose in the SCN during subjective day was inhibited by the perfusion of 8-OH-DPAT, but the low uptake of 2-deoxyglucose during subjective night was unaffected 24. The purpose of the present experiment was therefore to examine whether 8-OH-DPAT, which produces an inhibitory action of the SCN neurons, could reset the phase of the SCN circadian pacemaker. Male Wistar rats were housed in a 12:12 light-dark cycle. Each animal was killed and the brain quickly removed from the skull. Coronal hypothalamic slices (450 /~m thickness) were prepared through the SCN and anterior hypothalamic area ( A H A ) , using a tissue chopper. The slices were placed on disk-type chambers and were continuously perfused with warmed Krebs Ringer solution at 36°C equilibrated with 95% 02/5% CO2. The composition of Krebs Ringer was (in mM): NaC1 129, MgSO 4 1.3, N a H C O 3 22.4, KH2PO 4 1.2, KC1 4.2, glucose 10.0, CaCI 2 1.5, H E P E S 10 and gentamycin (0.5%). The buffer was maintained with the p H at 7.3-7.4. At a specified circadian time (CT; time referenced to the donor colony light cycle since slices were kept in constant light, thus, CT0 refers to light-on and CT12 to light-off in the colony) during the first day in vitro, perfusion was stopped and the medium inside the small
Correspondence. S Shibata, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu Umverslty 62, Fukuoka 812, Japan
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Circadian Time( hr ) Fig. 1 Neuronal electrical actwlty rhythms recorded from SCN m vitro on day 2 Plotted are the 2 h means + S.E.M. of finng rates of single SCN neurons. Each plot represents a single experiment A' control expenment with vehicle treated slices. The electrical activity peaked at CT6 on day 2 B - D shces were treated with 8-OH-DPAT 10, 1 and 0 1 ~M at CT7-8 on day 1 m vitro. The subsequent time of peak occurs
at CT2, 4 and 5, re&eating 4, 2 and 1 h earher than m control shces. Horizontal bar, hghts off mammal colony, vertical bar, Ume of treatment, dotted line means ume of peak m vehicle treated slices (6.14 + 0 14 h, n = 7)
chamber was replaced with m e d i u m s u p p l e m e n t e d with 8 - O H - D P A T (Research Biochemicals Inc.) A f t e r a 1 h exposure, 8 - O H - D P A T containing m e d i u m was replaced by normal m e d i u m and perfusion was reinstated. The spontaneous electrical activity of single SCN cells as recorded extracellularly through glass electrodes filled with 2 M NaC1 ( D C resistance, 2-10 M f~ in Krebs solution) during the second day in wtro. Single unit activity that r e m a i n e d stable was recorded over 5 min intervals. The activities of all cells recorded during a single ex-
p e r i m e n t were averaged into 2 h intervals using 1 h lags. Previous studies have shown that this p r o c e d u r e yields a pattern of electrical activity for the population of SCN neurons that varies little between animals, and that the time of peak electrical activity in particular is a reliable m a r k e r of the phase of the SCN p a c e m a k e r ls'16. Previously, Prosser et al. r e p o r t e d that the mean time of p e a k for untreated slices was CT 6.0 + 0.32 h (n = 5) 16. This indicates that the process of changing the medium does not alter the phase of the clock. The p e a k in electrical activity in control slices occurs around CT6 on
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F~g 2. Phase response curve for 8-OH-DPAT m individual experiments are plotted according to the circadian time of treatment. Treatments during most of the subjective day produce slgmficant phase advances, whereas treatments during most of the subjective mght produce little or no phase changes. Circles, phase shifts determined i d after treatment
In control experiments slices were treated for 1 h on day 1 in vitro between CT6 and CT10 with perfusion medium not supplemented with 8-OH-DPAT. In these experiments, the mean time of peak on the subsequent day occurred at CT6:14 + 0.14 h (n = 7). When slices were treated at CT7-8 on day 1 in vitro with 10 /~M 8-OH-DPAT, the peak in electrical activity was around CT2 on day 2 (Figs. 1B and 2). The mean phaseadvance induced by 10 /~M 8-OH-DPAT for 1 h at CT6-10 was 3.3 ___ 0.4 h (n = 4). The changes induced by 10 f~M 8-OH-DPAT treatment at different times throughout the circadian cycle are summarized in Fig. 2. The overall pattern shows that 8-OH-DPAT advances the time-of-peak when applied during mid-subjective day. A slight delay or no change is seen after 8-OHDPAT treatment during subjective night. In the next experiment, we observed the dose-dependent effect of 8-OH-DPAT on the magnitude of phase shift at the time of maximal phase advance (CT7-8). Treatment with 8-OH-DPAT produced phase advance in a dose-depend a y 216.
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dent manner, as shown in Fig. 3. The concentrations of 1 and 10/~M caused significant effects. In the present study, we demonstrated that 8-OHDPAT, a 5-HT1A agonist, could reset the phase of the circadian pacemaker in the SCN. This suggested that the SCN circadian pacemaker can be modulated by excitation of 5-HTIA receptors in this nucleus. Actually, the importance of 5-HT1A receptors in the SCN in the circadian pacemaker mechanism was recently reported H'lT. Our present result is consistent with these observations. Recently we reported that intraperitoneal administration of the 5-HTIA agonists 8-OH-DPAT, buspirone and ipsapirone during subjective day, especially around CT8, induced a phase advance of wheel-running activity25, while administration of these drugs during subjective night did not induce phase changes. In addition, we reported that the high uptake of 2-deoxyglucose in the SCN during subjective day was inhibited by the perfusion of 8-OH-DPAT, but low uptake of 2-deoxyglucose during subjective night was unaffected24. Moreover, single unit activity in the SCN and SCN field potentials elicited by optic nerve stimulation were inhibited by the application of 5-HT and 8-OH-DPAT 7'9'14'23. Starting from our previous observations and present results, it is suggested that the phase of the SCN clock can be modulated by the excitation of 5-HTIA receptors in the SCN, and that a 5-HTIA receptor mechanism is involved in phase advances during subjective day. Prosser and Gillette ~5 demonstrated that application of cAMP induced only phase advances during subjective day. Our present 8-OH-DPAT result is consistent with the effects of cAMP. A stimulatory effect of 8-OH-DPAT on the activity of adenylate cyclase in the guinea pig hippocampus and rat cerebral cortex have been reported previously8'22. Therefore, the 8-OH-DPAT induced phase advance may be mediated through the activation of adenylate cyclase and a subsequent increase in endogenous cAMP in the SCN. Recently Prosser et a1.16 reported the phase-resetting effect of quipazine on SCN circadian pacemakers. In the case of quipazine, however, the treatment produces both phase advances and phase delays and so does not entirely mimic the effects of cAMP analogs and 8-OH-DPAT. Thus quipazine may affect the pacemaker mechanism in the SCN through 5-HT1, 5-HT 2 or 5-HT 3 receptor subtypes 21. Starting from the similarity of quipazine's effects to those of 8-OH-DPAT and cAMP in inducing phase advances during subjective day, quipazine may affect the clock through a 5TH1A receptor mechanism in the SCN. Recently it was reported that intense running-wheel activity induces phase-shifts ~9 and entrains the circadian rhythms of rodents. However, the treatment produces both big phase advance and small phase delays and so
356 d o e s n o t e n t i r e l y m i m i c t h e effects o f 8 - O H - D P A T
in
receptor
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the
SCN.
In conclusion,
the
vitro. A n i n c r e a s e in 5 - H T e r g i c activity h a s b e e n c o r r e -
present result suggested
l a t e d w i t h i n c r e a s e d a r o u s a l in r a t s 1°, a n d 5 - H T m e t a b -
a g o n i s t , c o u l d r e s e t t h e p h a s e of t h e c i r c a d i a n p a c e -
o l i s m is h i g h d u r i n g t h e l a t e d a y a n d n i g h t 3"~8. T h e r e f o r e
m a k e r m t h e S C N e s p e c i a l l y d u r i n g t h e s u b j e c t i v e daytime.
it is p o s s i b l e t h a t t h e b e h a v i o r a l l y i n d u c e d p h a s e ad-
that 8-OH-DPAT,
a 5-HTtA
v a n c e s d u r i n g d a y t i m e a r e m e d i a t e d t h r o u g h a 5-HT~A
1 Azmlta, E C, and Segal, M , An autora&ographic analysis of differential ascendmg projections of the dorsal and me&an raphe nuclei of the rat, J Comp Neurol, 179 (1978) 641-668 2 Block, M and Zucker, I , Clrcadmn rhythms of rat locomotor activity after lesions of the mldbrain raphe nuclei, J Comp Physwl., 109 (1976) 325-347 3 Faradji, H., Cespugllo, R. and Jouvet, M , Voltammetric measurements of 5-hydroxyIndole compounds m the suprachlasmatlc nuclei; clrcadmn fluctuations, Brain Res , 279 (1983) 111119 4 Honma, K., Watanabe, K. and Hiroshlge, T., Effects of parachlorophenylalanlne and 5,6-&hydroxytryptamlne on the free-running rhythms of locomotor activity and plasma cort~costerone in the rat exposed to continuous hght, Bram Res, 169 (1979) 531-544 5 Kam, L M and Moberg, G.P., Effect of raphe lesmns on the circadian pattern of wheel runnmg in the rat, Phystol Behav , 18 (1977) 213-217 6 Levlne, J.D., Rosenwasser, A . M , Yanovskl, J.A and Adler, N T., Circadian actiwty rhythms m rats wath mldbram raphe lestuns, Brain Res , 384 (1986) 240-249 7 Liou, S.Y., Shlbata, S. and Ueki, S , Effects of monoamines on field potentials in the suprachiasmatxc nucleus of shces of hypothalamus of the rat evoked by stimulation of the optic nerve, Neuropharmacology, 25 (1986) 1009-1014 8 Markstem, R , Hoyer, D and Engel, G., 5-HTiA-receptors mediate stimulation of adenylate cyclase in rat hlppocampus, Naunyn-Schmwdeberg's Arch. Pharmacol., 333 (1986) 335-341 9 Mason, R., Circadian variation xn sensitivity of suprachlasmat~c and lateral geniculate neurones to 5-hydroxytryptamine m the rat, J. Phystol, 377 (1986) 1-13. 10 McGmty, D. and Szymusmk, R , Neuronal umt activity patterns m behaving ammals: bramstem and limblc system, Annu Rev Psychol, 39 (1988) 135-168 i1 Medamc, M. and Gdlette, M , Serotonerglc agomsts advance the c~rcadmn rhythm of neuronal activity m rat SCN in v~tro, Soc Neuroscl Abstr , 17 (1991) 671 12 Moore, R Y., Halans, A.E and Jones, B A , Serotonm neurons of the imdbram raphe: ascending prolectmns, J Comp Neurol, 180 (1978) 417-438. 13 Morin, L P., Mlchels, K.M., Smale, L. and Moore, R.Y, Serotonm regulation of circadian rhythmicity, Ann N Y Acad
Sct , 458 (1990) 418-426 14 Nishlno, H., Kolzuml, K. and Brooks, C McC , The role of suprachlasmatlc nuclei of the hypothalamus in the production of circadian rhythm, Brain Res, 112 (1976) 45-59 15 Prosser, R A and Gillette, M U , The mammalian circadian clock In the suprachlasmat~c nuclei is reset in vitro by cAMP, J Neurosct., 9 (1989) 1073-1081. 16 Prosser, R A , Miller, J D. and Heller, H.C., A serotonm agorest phase-shifts the circadian clock m the suprachmsmauc nuclei in vitro, Bram Res , 534 (1990) 336-339 17 Prosser, R . A , Miller, J D and Heller, H.C., In vitro resetting of the suprachiasmatic circadian pacemaker by specific serotonergxc agomsts and antagomsts, Soc Neuroscz Abstr, 17 (1991) 672 18 Ramlrez, A D , Ramlrez, V D and Meyer, D C , The nature and magnitude of m vwo 5-hydroxymdoleaceUc acid output from 5-hydroxytryptamine terminals IS related to specific regions of the suprachlasmatlc nucleus, Neuroendocrmology, 46 (1987) 430-438. 19 Reebs, S.G and Mrosovsky, N., Effects of induced wheel runnmg on the c~rca&an activity rhythms of Syrian hamsters: entrainment and phase response curve, J Blol Rhythms, 4 (1989) 39-48 20 Rusak, B and Bma, K.G., Neurotransmltters m the mammallan circadian system, Annu Rev Neurosct , 13 (1990) 387-401 21 Schmldt, A W and Peroutka, S.J., 5-Hydroxytryptamme receptor 'famflles', FASEB J , 3 (1989) 2242-2249. 22 Shenker, A , Maayani, S., Wemstem, H. and Green, J P , Pharmacological characterization of two 5-hydroxytryptamme receptors coupled to adenylate cyclase in guinea pig h~ppocampal membranes, Mol. Pharmacol, 31 (1987) 337-341 23 Shlbata, S , Lmu, S.Y. and Uekl, S , Effects of amino acids and monoamines on the neuronal actwlty of suprachmsmatic nucleus m hypothalamlc slice preparations, Jpn J Pharmacol, 33 (1983) 1225-1231. 24 Tommaga, K , Shibata, S , Uekl, S. and Watanabe, S , Effects of inhibitory and excitatory drugs on the metabohc rhythm of hamster suprachiasmatlc nucleus in vitro, Eur J Pharmacol, in press 25 Tommaga, K , Shxbata, S , Uekl, S and Watanabe, S , Effects of 5-HT1A agomsts on the circadian rhythm of wheel-running activity in hamsters, Eur J Pharmacol, in press.
353
BRES 25214
Phase-resetting effect of 8-OH-DPAT, a serotoninlA receptor agonist, on the circadian rhythm of firing rate in the rat suprachiasmatic nuclei in vitro Shigenobu Shibata, Atsuko Tsuneyoshi, Toshiyuki Hamada, Keiko Tominaga and Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceuttcal Scwnces, Kyushu University, Fukuoka (Japan) (Accepted 3 March 1992) Key words 5-Hydroxytryptamine, 8-OH-DPAT, Serotonm 5-HT~A; Suprachmsmatlc nucleus; Circadian rhythm
The 5-HTergic neurons m the mesencephahc raphe nucle~ provide a robust projection to the hypothalam~c suprachlasmat~c nucleus (SCN), the s~te of a putatwe neuronal c~rcadian pacemaker. Although ~t has been suggested that 5-HT neurons may play a role m the circa&an timing system, this role has not yet been specified. Prosser et al. (Bram Res., 534 (1990) 336-339) reported that 1 h treatments with qmpazine reduce robust phase shifts in vitro, and that this effect depends upon the c~rcadmn t~me of treatment However, qmpazine is a nonspecific 5-HT agomst. Besides, it is reported that the 5-HT~A agomst, 8-hydroxy-2-(dl-n-propylamino)tetrahne hydrobromlde (8-OH-DPAT) affected a circadian rhythm of hamster wheel-running activity In the present study we investigated whether the 5-HT~A agomst 8-OH-DPAT can reset the phase of the SCN clock when it is isolated m vitro The present results show that 1 h treatments with 8-OH-DPAT induce robust phase advances m vitro when it was administered during the subjectwe day. This result suggests that 5HTtA receptor functioning may play a role m modulating the phase of SCN clock, especially during the subjective day The mammalian suprachiasmatic nuclei (SCN) have been identified as a circadian pacemaker for behavioral and physiological functions 13'2°. There is a dense serotonergic (5-HT) projection to the SCN from cells located primarily in the dorsal and medial raphe nuclei ~'12. However, the functional significance of the input from the raphe to the circadian system is controversial. To elucidate the role of 5-HT function, many investigators have used the chemical and electrical destruction of the 5-HT system 2'4-6. To study the function of this projection in the mammalian circadian system, Prosser et al. 16 reported that quipazine could reset the phase of the SCN circadian pacemaker. Treatments during subjective daytime induced phase advances, while nighttime treatments induced phase delays. Because quipazine is a non-specific 5-HT agonist, it is uncertain whether the phase change induced by quipazine is due to actions at 5-HT1, at 5-HT 2 or at 5-HT 3 receptor subtypes. Recently we reported that the administration of the 5-HT1A agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone and ipsapirone during subjective day induced a phase advance of wheel-running activity under constant light condition 2s. However, administration of these drugs during subjective night did not induce phase changes. Besides, we found that the high
uptake of 2-deoxyglucose in the SCN during subjective day was inhibited by the perfusion of 8-OH-DPAT, but the low uptake of 2-deoxyglucose during subjective night was unaffected 24. The purpose of the present experiment was therefore to examine whether 8-OH-DPAT, which produces an inhibitory action of the SCN neurons, could reset the phase of the SCN circadian pacemaker. Male Wistar rats were housed in a 12:12 light-dark cycle. Each animal was killed and the brain quickly removed from the skull. Coronal hypothalamic slices (450 /~m thickness) were prepared through the SCN and anterior hypothalamic area ( A H A ) , using a tissue chopper. The slices were placed on disk-type chambers and were continuously perfused with warmed Krebs Ringer solution at 36°C equilibrated with 95% 02/5% CO2. The composition of Krebs Ringer was (in mM): NaC1 129, MgSO 4 1.3, N a H C O 3 22.4, KH2PO 4 1.2, KC1 4.2, glucose 10.0, CaCI 2 1.5, H E P E S 10 and gentamycin (0.5%). The buffer was maintained with the p H at 7.3-7.4. At a specified circadian time (CT; time referenced to the donor colony light cycle since slices were kept in constant light, thus, CT0 refers to light-on and CT12 to light-off in the colony) during the first day in vitro, perfusion was stopped and the medium inside the small
Correspondence. S Shibata, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu Umverslty 62, Fukuoka 812, Japan
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Circadian Time( hr ) Fig. 1 Neuronal electrical actwlty rhythms recorded from SCN m vitro on day 2 Plotted are the 2 h means + S.E.M. of finng rates of single SCN neurons. Each plot represents a single experiment A' control expenment with vehicle treated slices. The electrical activity peaked at CT6 on day 2 B - D shces were treated with 8-OH-DPAT 10, 1 and 0 1 ~M at CT7-8 on day 1 m vitro. The subsequent time of peak occurs
at CT2, 4 and 5, re&eating 4, 2 and 1 h earher than m control shces. Horizontal bar, hghts off mammal colony, vertical bar, Ume of treatment, dotted line means ume of peak m vehicle treated slices (6.14 + 0 14 h, n = 7)
chamber was replaced with m e d i u m s u p p l e m e n t e d with 8 - O H - D P A T (Research Biochemicals Inc.) A f t e r a 1 h exposure, 8 - O H - D P A T containing m e d i u m was replaced by normal m e d i u m and perfusion was reinstated. The spontaneous electrical activity of single SCN cells as recorded extracellularly through glass electrodes filled with 2 M NaC1 ( D C resistance, 2-10 M f~ in Krebs solution) during the second day in wtro. Single unit activity that r e m a i n e d stable was recorded over 5 min intervals. The activities of all cells recorded during a single ex-
p e r i m e n t were averaged into 2 h intervals using 1 h lags. Previous studies have shown that this p r o c e d u r e yields a pattern of electrical activity for the population of SCN neurons that varies little between animals, and that the time of peak electrical activity in particular is a reliable m a r k e r of the phase of the SCN p a c e m a k e r ls'16. Previously, Prosser et al. r e p o r t e d that the mean time of p e a k for untreated slices was CT 6.0 + 0.32 h (n = 5) 16. This indicates that the process of changing the medium does not alter the phase of the clock. The p e a k in electrical activity in control slices occurs around CT6 on
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In control experiments slices were treated for 1 h on day 1 in vitro between CT6 and CT10 with perfusion medium not supplemented with 8-OH-DPAT. In these experiments, the mean time of peak on the subsequent day occurred at CT6:14 + 0.14 h (n = 7). When slices were treated at CT7-8 on day 1 in vitro with 10 /~M 8-OH-DPAT, the peak in electrical activity was around CT2 on day 2 (Figs. 1B and 2). The mean phaseadvance induced by 10 /~M 8-OH-DPAT for 1 h at CT6-10 was 3.3 ___ 0.4 h (n = 4). The changes induced by 10 f~M 8-OH-DPAT treatment at different times throughout the circadian cycle are summarized in Fig. 2. The overall pattern shows that 8-OH-DPAT advances the time-of-peak when applied during mid-subjective day. A slight delay or no change is seen after 8-OHDPAT treatment during subjective night. In the next experiment, we observed the dose-dependent effect of 8-OH-DPAT on the magnitude of phase shift at the time of maximal phase advance (CT7-8). Treatment with 8-OH-DPAT produced phase advance in a dose-depend a y 216.
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-log M
8-OH-DPAT Ftg. 3. Dose-response curve for 8-OH-DPAT Each bar is mean + S.E.M Plotted are the phase advances induced by different concentrat]ons of 8-OH-DPAT applied at CT7-8. Maximal advances of around 4 h were reduced by 10/tM 8-OH-DPAT. ***P < 0.001; *P < 0.05 (Student's t-test).
dent manner, as shown in Fig. 3. The concentrations of 1 and 10/~M caused significant effects. In the present study, we demonstrated that 8-OHDPAT, a 5-HT1A agonist, could reset the phase of the circadian pacemaker in the SCN. This suggested that the SCN circadian pacemaker can be modulated by excitation of 5-HTIA receptors in this nucleus. Actually, the importance of 5-HT1A receptors in the SCN in the circadian pacemaker mechanism was recently reported H'lT. Our present result is consistent with these observations. Recently we reported that intraperitoneal administration of the 5-HTIA agonists 8-OH-DPAT, buspirone and ipsapirone during subjective day, especially around CT8, induced a phase advance of wheel-running activity25, while administration of these drugs during subjective night did not induce phase changes. In addition, we reported that the high uptake of 2-deoxyglucose in the SCN during subjective day was inhibited by the perfusion of 8-OH-DPAT, but low uptake of 2-deoxyglucose during subjective night was unaffected24. Moreover, single unit activity in the SCN and SCN field potentials elicited by optic nerve stimulation were inhibited by the application of 5-HT and 8-OH-DPAT 7'9'14'23. Starting from our previous observations and present results, it is suggested that the phase of the SCN clock can be modulated by the excitation of 5-HTIA receptors in the SCN, and that a 5-HTIA receptor mechanism is involved in phase advances during subjective day. Prosser and Gillette ~5 demonstrated that application of cAMP induced only phase advances during subjective day. Our present 8-OH-DPAT result is consistent with the effects of cAMP. A stimulatory effect of 8-OH-DPAT on the activity of adenylate cyclase in the guinea pig hippocampus and rat cerebral cortex have been reported previously8'22. Therefore, the 8-OH-DPAT induced phase advance may be mediated through the activation of adenylate cyclase and a subsequent increase in endogenous cAMP in the SCN. Recently Prosser et a1.16 reported the phase-resetting effect of quipazine on SCN circadian pacemakers. In the case of quipazine, however, the treatment produces both phase advances and phase delays and so does not entirely mimic the effects of cAMP analogs and 8-OH-DPAT. Thus quipazine may affect the pacemaker mechanism in the SCN through 5-HT1, 5-HT 2 or 5-HT 3 receptor subtypes 21. Starting from the similarity of quipazine's effects to those of 8-OH-DPAT and cAMP in inducing phase advances during subjective day, quipazine may affect the clock through a 5TH1A receptor mechanism in the SCN. Recently it was reported that intense running-wheel activity induces phase-shifts ~9 and entrains the circadian rhythms of rodents. However, the treatment produces both big phase advance and small phase delays and so
356 d o e s n o t e n t i r e l y m i m i c t h e effects o f 8 - O H - D P A T
in
receptor
mechamsm
m
the
SCN.
In conclusion,
the
vitro. A n i n c r e a s e in 5 - H T e r g i c activity h a s b e e n c o r r e -
present result suggested
l a t e d w i t h i n c r e a s e d a r o u s a l in r a t s 1°, a n d 5 - H T m e t a b -
a g o n i s t , c o u l d r e s e t t h e p h a s e of t h e c i r c a d i a n p a c e -
o l i s m is h i g h d u r i n g t h e l a t e d a y a n d n i g h t 3"~8. T h e r e f o r e
m a k e r m t h e S C N e s p e c i a l l y d u r i n g t h e s u b j e c t i v e daytime.
it is p o s s i b l e t h a t t h e b e h a v i o r a l l y i n d u c e d p h a s e ad-
that 8-OH-DPAT,
a 5-HTtA
v a n c e s d u r i n g d a y t i m e a r e m e d i a t e d t h r o u g h a 5-HT~A
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