European Journal of Pharmacology, 217 (1992) 79-84 0 1992 Elsevier Science Publishers B.V. All rights reserved
79 0014-2999/92/$05.00
EJP 52507
Effects of inhibitory and excitatory drugs on the metabolic rhythm of the hamster suprachiasmatic nucleus in vitro Keiko Tominaga, Shigenobu Shibata, Showa Ueki and Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceuticul Sciences, Kyushu University 62, Fukuoka 812, Japan
Received 13 January 1992, revised MS received 24 March 1992, accepted 25 March 1992
In order to elucidate the role of excitatory and inhibitory transmitters within the suprachiasmatic nucleus (SCN) in the circadian change of 2-deoxyglucose (2-DG) uptake in this nucleus, the effects of %hydroxy-2-(di-n-propylamino) tetralin hydrobromide (&OH-DPAT), muscimol, flurazepam, pentobarbital and glutamate on uptake of 2-DG by hamster SCN were examined in hypothalamic slice preparations. 2-DG uptake in the SCN was high during the subjective day and low during the subjective night. The high uptake of 2-DG in the SCN during the daytime was inhibited by the superfusion of &OH-DPAT, muscimol, flurazepam and pentobarbital in a dose-dependent manner, but the low uptake of 2-DG during the night was unaffected. The low uptake during the night was significantly increased by treatment with glutamate, whereas 2-DG uptake during the day was unaffected. In contrast to the above results, 20 mM KC1 and 1 /IM tetrodotoxin increased and decreased 2-DG uptake during both the day and night, respectively. The present results strongly suggest that agonists of S-HT,, receptors and GABA,-benzodiazepine-barbiturate complex receptors regulate the function of the SCN through their inhibitory action on 2-DG uptake during the day, and that glutamate also regulates SCN function through it stimulatoty action on 2-DG uptake during the night. Circadian
rhythm;
Suprachiasmatic
nucleus; 2-Deoxyglucose uptake; 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin); GABA, receptors; Benzodiazepine receptors
1. Introduction
The mammalian suprachiasmatic nucleus (SCN) has been identified as a circadian pacemaker for behavioral and physiological functions (Moore, 1983). An endogeneous circadian rhythm in SCN neuronal firing rate and in 2-deoxyglucose (2-DG) uptake in vitro has been reported (Shibata et al., 1982; Newman and Hospod, 1986). There is a dense serotonergic projection to the SCN from the dorsal and medial raphe (Moore et al., 1978) and axones containing r-amino butyric acid (GABA) projection throughout the SCN (FraqoisBellan et al., 1990). We have previously demonstrated that GABA, benzodiazepines and serotonin (5HT) strongly inhibit firing discharge of SCN neurons in vitro (Shibata et al., 1983; Liou et al., 1990). Thus the SCN may be a brain site for an action of 5-HT,, receptor agonists and GABA,-benzodiazepinebarbiturate receptor agonists on the circadian system.
Correspondence to: K. Tominaga, Faculty of Pharmaceutical Sciences, 812, Japan.
Department of Pharmacology, Kyushu University 62, Fukuoka
The first aim of the present experiment was to examine whether superfusion of drugs which produce an inhibitory action on SCN neurons affects the rhythm of 2-DG uptake in the SCN in a hypothalamic slice preparation. The retinohypothalamic tract is one of the neural projections involved in entraining circadian rhythms to the light-dark cycle (Moore and Card, 1985). With regard to chemical neurotransmission in the retinohypothalamic tract, several pieces of evidence have been reported which indicate that excitatory amino acids (Cahill and Menaker, 1989; Liou et al., 1986; Shibata et al., 1986) and/or N-acetylaspartylglutamate (Moffett et al., 1990) may be involved in the transduction of photic information to the SCN. At present these are the most likely candidates to function as neurotranmitters in the the retinohypothalamic tract. Light pulses produce large phase changes in the rythm of circadian locomotor activity rhythm light pulses are given during the subjective night. If glutamate mediates photic information to the SCN, one would expect that glutamate could affect 2-DG uptake by the SCN, especially during the subjective night. In addition, acetylcholine receptor agonists and glutamate increase the sponta-
80
neous discharge rate of most SCN neurons (Nishino and Koizumi, 1977; Shibata et al., 1983). A second experiment was undertaken to examine whether perfusion of glutamate affects the rhythm of 2-DG uptake in the SCN. The results from above two experiments were compared with those obtained with high KC1 and tetrodotoxin.
2. Materials
and methods
2.1. Procedure Male golden hamsters, weighing 90-120 g, were housed in a normal (light-on 09:OO)or reversed (light-on 21:00) 12: 12 h light-dark cycle for at least 2 weeks before they were used. For the 2-DG uptake study, each animal was killed and the brain was quickly removed from the skull. Coronal hypothalamic slices (450 pm thick) were prepared through the SCN and anterior hypothalamic area (AI-IA) using a tissue chopper. In this experiment, CT refers to clock time, with the time of lights-on arbitrarily designated as CTO. The light phase of the light-dark cycle (CTO-12) is referred to as ‘subjective day’, whereas the dark phase (CT1224) is referred to as ‘subjective night.’ Slices to be studied during the subjective day or the subjective night were prepared at CT3 or CT15, respectively. The slices were preincubated in oxygenated Krebs-Ringer solution for 3 h. After the preincubation in KrebsRinger solution, the slices, which were held between two meshes, were removed into an incubation chamber. The composition of the control Krebs-Ringer solution, equilibrated with 95% O,-5% CO,, was (in mM): NaCl 129, MgSO, 1.3, NaHCO, 22.4, KH*PO, 1.2, KC1 4.2, glucose 10.0 and CaCI, 1.5. The buffer had a pH of 7.3-7.4. The incubation medium contained 1 pCi/ml of 2-DG (2-deoxy-D-[‘4C]-glucose, specific activity, 50 mCi/m mol; Amersham) and each of the drugs. Incubations were carried out for 45 min at 37°C at CT6 or CT18, respectively. The slices were then removed from the chamber and rinsed with 20 ml of warm, gassed preincubation buffer and returned to the original preincubation chamber for 30 min. At the end of washout, slices were placed on dry ice to stop metabolism. The SCN was punched out as described by Murakami et al. (1984). The SCN was homogenized in 1 ml phophate buffer containing 0.5% perchloric acid, and 450 ~1 of the homogenate was used to determine the total amount of protein. The radioactivity in another 450 ~1 of the homogenate was measured in a liquid scintillation counter after the homogenate was solubilized.
2.2. Drugs The drugs used in this study were: 8-hydroxy-2-(din-propylamino) tetralin hydrobromide (8-OH-DPAT, Funakoshi Japan), muscimol (Sigma), pentobarbital (Tanabe, Japan), flurazepam (Takeda, Japan), tetrodotoxin (TTX, Sankyo, Japan), glutamate (Sigma), carbachol (Tokyokasei, Japan) and ( - )-pindolol (Funakosi Inc., Japan). All drugs were dissolved in distilled water. Control preparations were treated with vehicle (distilled water). The application of vehicle at CT6 had no effect on 2-DG uptake (97 f 1.7%, n = 3 of untreated group). (-)-Pindolol was applied to the slices for 10 min before the addition of [14C]2-DG and 8-OH-DPAT, and was present throughout the incubation (45 min). 2.3. Statistical analysis Data are expressed as the means + S.E. Significant differences between groups were determined with a two way analysis of variance followed by the Wilcoxon or Mann-Whitney test for individual comparisons.
3. Results 3.1. Inhibitory drugs
We examined the effects of 8-OH-DPAT, muscimol, flurazepam and pentobarbital on 2-DG uptake by the SCN during the subjective day and the subjective night. During the subjective day (CT6), 2-DG uptake by the SCN was 28 & 1.6 (n = 15) dpm/mg total protein and was significantly (P < 0.01) higher than that during the subjective night (CT18) (19 + 1.1, n = 16). If the 2-DG uptake of control animals at CT6 was set at lOO%, the 2-DG uptake at CT18 became 69 + 3.6% (n = 16). The values of 2-DG uptake in control hamsters at CT6 or CT18 were set at lOO%, respectively. Superfusion of 8-OH-DPAT produced a significant and dose-related inhibition of 2-DG uptake by the SCN at CT6 (F(3,61) = 8.6, P < 0.01) (fig. l), while only a high dose of 8-OH-DPAT (100 PM) decreased 2-DG uptake at CT18 (fig. 1). The inhibitory effect of 8-OH-DPAT on 2-DG uptake was more pronounced at CT6 than at CT18 (compared to dose-response curve at CT18, F(3,61) = 3.2, P < 0.05). Because 10 PM 8-OH-DPAT significantly decreased 2-DG uptake at CT6 but not at CT18, we examined the effect of a 5-HT,, receptor antagonist, (- )-pindolol, on the decrease in 2-DG uptake caused by 10 PM 8-OH-DPAT. Pretreatment with 10 PM (-)-pindolol significantly antagonized the B-OH-DPAT-induced decrease in 2-DG uptake (P < O.OS), while 10 PM (--)-pindolol had no effect on
81 (7) (6)
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Pf
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(16)
f*
(6)
P** P** # 1
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i! : m g
(7)
Al 1 4** f** (4)
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i
(6)
(5)
50-
:: N
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I
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I
6
5
4
-log
t O-c’
td
0-OH-DPAT
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5
4
-log
M
Musclmol
Fig. 1. Effect of E-OH-DPAT on the 2-deoxyglucose uptake rhythm of hamster suprachiasmatic nucleus in vitro. The 2-DG uptake of control animals at CT6 or CT18 was set at 100%. Each point shows the mean+S.E. Numbers in brackets indicate the number of animals. Open circle, subjective day fCT6); closed circle, subjective night (CT18). # P < 0.05 compared to dose-response curve at CT18 (two way ANOVA); * P < 0.05, ** P < 0.01 vs. control animals (Cl at CT6 or CT18 (Williams-Wicoxon test).
Fig. 3. Effect of muscimol on 2-deoxyglucose uptake in the hamster suprachiasmatic nucleus in vitro. The 2-DG uptake of control animals at CT6 or CT18 was set at 100%. Each point shows the mean &SE. Numbers in brackets indicate the number of animals. Open circle, subjective day (CT6); closed circle, subjective night (CTIE). # P < 0.05 compared to dose-response curve at CT18 (two way ANOVA); ** P < 0.01 vs. control animals at CT6 (WilliamsWilcoxon test).
2-DG uptake at CT6 (P > 0.05) (fig. 2). At CT6, 2-DG uptake by the SCN was significantly inhibited by treatment with muscimol at 10 and 100 PM (compared to dose-response curve at CT18, F(2,47) = 4.6, P < 0.051, flurazepam at 100 FM (time x drug interaction, F(1,24) = 7.8, P < 0.01) and pentobarbital at 100 PM (time X drug interaction, F(1,27) = 9.1, P < 0.011,
whereas at CT18 2-DG uptake was unaffected by these treatments (fig. 3 and table 1). In contrast to the above drugs, TTX at 1 PM significantly reduced 2-DG uptake at both CT6 and CT18 (table 11, and there was no significant difference between CT6 and CT18 (time x drug interaction, F(1,36) = 0.03, P > 0.05). 3.2. Excitatory drugs 2-DG uptake by the SCN at CT18 was significantly increased by the perfusion of 10 PM glutamate (time x drug interaction, F&30) = 13.8, P < 0.01) (table 11, TABLE 1 Effects of various drugs on 2-deoxyglucose uptake by the suprachiasmatic nucleus in vitro [‘4C]-2-Deoxyglucose uptake was calculated as normalized radioactivity (dpm per mg of protein). The 2-DG uptake of control animals at CT6 or CT18 was set at 100%. Numbers in brackets indicate the number of animals. Drug
10
10
NM
Dose (PM)
2-DG uptake (%, mean k SE., n) CT6 CT18
10 20 mM 100 100 1
loo+ 10.0 (10) lOl+ll.O (8) 134 + 12.0 (8) ’ 61+ 4.0 (6)’ 56+ 13.9 (4) ’ 39+ 3.5 (IO) h
6-OH-DPAT 10
10
JIM
Plndolol
Fig. 2. Effect of (- )-pindolol on the E-OH-DPAT-induced decrease in 2-deoxyglucose uptake in the hamster suprachiasmatic nucleus in vitro. The 2-DG uptake of control animals at CT6 was set at 100%. Each column shows the mean f S.E. Numbers in brackets indicate the number of animals. Open columns, subjective day (CT6); stippled columns, subjective night (CT18). # P < 0.01 vs. control animals at CT6; * P < 0.05 vs. E-OH-DPAT injected group at CT6 (WilliamsWilcoxon test).
Control Glutamate KCI Pentobarbital Flurazepam Tetrodotoxin
lOOk3.4 (10) a 139k 10.1 (6) h 154 f 18.0 (7) h 91 f 9.0 (5) 104+21.0 (4) 42 + 3.6 (10) ’
a When the 2-DG uptake of control animals at CT6 was set at lOO%, the 2-DG uptake at CT18 (70 f 7.0%, n = 10) was significantly lower (P < 0.01, Mann-Whitney test) than the 2-DG uptake at CT6. b P < 0.01 vs. control group (Mann-Whitney test).
82
but at CT6, 2-DG uptake was unaffected by this treatment. On the other hand, 20 mM KC1 significantly increased 2-DG uptake at both CT6 and CT18, and there was no significant difference between CT6 and CT18 (time X drug interaction, F(1,31) = 0.8, P > 0.05) (table 1).
4. Discussion An inhibitory action of 8OH-DPAT, muscimol, flurazepam and pentobarbital was observed on SCN 2-DG uptake at CT6 but not at CT18. The present results suggest that the SCN is directly affected by muscimol, flurazepam, pentobarbital and 5-HT,, agonists. In previous experiments, GABA, muscimol, anxiolytics and 5-HT were found to inhibit SCN single unit activity (Lieu et al., 1986, 1990; Mason et al., 1991) and the field potentials in the SCN evoked by optic nerve stimulation (Shibata et al., 1983, 1986). From the localization study of Smith et al. (1989) and the above-mentioned facts, the SCN is the most likely brain site of action for most of these drugs. We recently reported that the phase-response curve produced by the 5-HT,, agonists 8-HT-DPAT, buspirone and ipsapirone was very similar to that produced by muscimol, triazolam and NPY, and that the large phase change caused by these drugs was observed only during the subjective day (Tominaga et al., 1992). At present we do not know the mechanism of action of 8-OH-DPAT on SCN neurons. It has been reported that 8-OH-DPAT has potent 5-HT,, agonistic activity on both post- and pre-synaptic 5-HT,, receptors in the brain (Hamon et al., 1984). In our experiment, 8-OHDPAT decreased 2-DG uptake by the SCN in a dosedependent manner. A biphasic stimulatory effect of 8-OH-DPAT on the activity of adenylate cyclase in the guinea pig hippocampus and rat cerebral cortex has been reported (Shenker et al., 1987; Dumuis et al., 1988; Fayolle et al., 1988; Mark and Geisler, 1990). In the presence of Ca ions, low concentrations of 8-OHDPAT potentiate adenylate cyclase activity, while higher concentrations inhibit the activity of the enzyme (Mork and Geisler, 1990). In addition, the results of electrophysiological studies suggest that high concentrations of 8-OH-DPAT (50-100 PM) are required to alter the amplitude of population spike in the CA1 evoked by the stimulation of Schaffer collaterals (Peroutka et al., 19871, and lo-30 PM 8-OH-DPAT induces hyperpolarization in pyramidal cells of the prefrontal cortex (Araneda and Andrade, 1991). These results suggest that the 5-HT,, receptor subtype in the hamster SCN seems to belong to the low affinity subtype of 5-HT,, receptor. Pretreatment with (--Ipindolol, an antagonist for 5-HT,, receptor (Oksenberg and Peroutka, 19881, markedly blocked the de-
crease in 2-DG uptake produced by 8-OH-DPAT. In our behavioral experiment, the phase advance of wheel-running activity induced by 8-OH-DPAT in hamsters was blocked by pretreatment with t-jpindolol (Tominaga et al., 1992). Melatonin, the indole amine hormone produced in the pineal gland, inhibits firing discharge in the rat SCN in vitro (Shibata et al., 1989). In behavioral studies, melatonin has effects on circadian rhythms during the late subjective day but not at other time of day (for review: Cassone, 1990). These results are similar to the present result obtained with 8-OH-DPAT. In addition, an inhibitory effect of melatonin on cyclic nucleotides has been observed (Carlson et al., 1989; Vanecek and Vollrath, 1989). It is therefore, possible that 8-OHDPAT affects SCN metabolic activity via same mechanism as melatonin. Pentobarbital also produces phase changes and the phase response curve for this drug is similar to that for triazolam and muscimol (Ebihara et al., 1988). However, GABA, benzodiazepines and pentobarbital may not act via 5-HT neurons, because the 5-HT projection is not involved in the triazolam-induced acceleration of re-entrainment to a phase-advanced light dark cycle @male et al., 1990). With regard to chemical transmission in the retinohypothalamic tract, it has been reported that excitatory amino acids (Cahill and Menaker, 1989; Liou et al., 1986; Shibata et al., 1986) may be involved in the function of the retinohypothalamic tract at the SCN. In addition, MK-801, a N-methyl-D-aspartate receptor antagonist, prevents both the phase shifts in the rhythm of hamster wheel-running activity (Colwell et al., 1990) and the induction of Fos protein by light pulses (Abe et al., 1991). Thus glutamte may mediate photic information to the SCN. In the present experiment, glutamate increased 2-DG uptake by the SCN during the subjective night, in contrast. to 8-OH-DPAT, which had a significant effect during the subjective day. However, in behavioral experiments, glutamate injected into the SCN produces a phase change (Meijer et al., 1988) resembling to the phase response curve for dark pulses (Boulos and Rusak, 1982). At present, we do not know the reasons for the contradictory results. In vivo, bolus injection of glutamate may swamp the uptake mechanisms that normally clear glutamate from the vicinity of receptors, causing a long-lasting depolarization. Thus in vivo glutamate may cause a depolarization block that prevents further firing. SCN field potentials are blocked by glutamate (1 mM) added to the bathing medium (Shibata et al., 1986). The question arises whether the inhibitory effects of 8-OH-DPAT, muscimol, flurazepam and pentobarbital, and the excitatory effect of glutamate reflect a specific involvement of these drugs in the time dependence of treatment. The excitatory effect of glutamate during
83
the subjective night, and the inhibitory effects of 8 OH-DPAT, muscimol, flurazepam and pentobarbital during the subjective day seem only to reflect that activity is already high during the day and low during the nights, thereby preventing further excitation during the day and further inhibition during the night. However, TTX significantly inhibited 2-DG uptake at both CT6 and CT18, whereas 20 mM KC1 significantly increased it at both CT6 and CT18. Thus general inhibitory and stimulatory agents affected 2-DG uptake without affecting the time dependence. The effect of TTX and 20 mM KC1 may therefore be non-specific, and the effect of the other drugs may be more specific. In summary, the present results strongly support a receptors, GABA,-benzodiazepinerole for SHT,, barbiturate receptors and glutamate receptor function in the control of the circadian rhythm of SCN activity.
References Abe, H., B. Rusak and H.A. Robertson, 1991, Photic induction of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK-801, Neurosci. Lett. 127, 9. Araneda, R. and R. Andrade, 1991,5-Hydroxytryptaminez and 5-hyreceptors mediate opposing responses on droxytryptamine,, membrane excitability in rat association cortex, Neuroscience, 40, 399. Boulos, Z. and B. Rusak, 1982, Circadian phase response curves for dark pulses in the hamster, J. Comp. Physiol. 146, 411. Cahill, G.M. and M. Menaker, 1989, Effects of excitatory amino acid receptor antagonists and agonists on suprachiasmatic nucleus responses to retino hypothalamic tract volleys, Brain Res. 479, 76. Carlson, L.L., D.R. Weaver and SM. Reppert, 1989, Melatonin signal transduction in hamster brain: inhibition of adenyl cyclase by a pertussis toxin-sensitive G protein, Endocrinology 125, 2670. Cassone, V.M., 1990, Effects of melatonin on vertebrate circadian systems, Trends Neurosci. 13, 457. Colwell, C.S., M.R. Ralph and M. Menaker, 1990, Do NMDA receptors mediate the effects of light on circadian behavior?, Brain Res. 523, 117. Dumuis, A., R. Bouhelal, M. Sebben, R. Gory and J. Bockaert, 1988, A nonclassical 5-hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system, Mol. Pharmacol. 34, 880. Ebihara, S., M. Goto and I. Oshima, 1988, Different responses of the circadian system to GABA-active drugs in two strains of mice, J. Biol. Rhythms 3, 357. Fayolle, C., M.-P. Fillion, P. Barone, P. Oudar, J.-C. Rousselle and G. Fillion, 1988, 5-Hydroxytryptamine stimulates two distinct adenylate cyclase activities in rat brain: High-affinity activation is related to a 5-HT, subtype different from 5-HT,,, 5-HT,, and 5-HT,,, Fundam. Clin. Pharmacol. 2, 195. FranSois-Bellan, A.M., P. Kachidian, G. Dusticier, M.C. Tonon, H. Vaudry and 0. Bosler, 1990, GABA neurons in the rat suprachiasmatic nucleus: involvement in chemospecific synaptic circuitry and evidence for GAD-peptide colocalization, J. Neurocytol. 19, 937. Hamon, M., S. Bourgoin, H. Gozlan, M.D. Hall, C. Goetz, F. Artaud and A.S. Horn, 1984, Biochemical evidence for the 5-HT agonist properties of rat (8-hydroxy-2-(di-n-propylamino)tetraIin) in the rat brain, Eur. J. Pharmacol. 100, 263.
Liou, S.Y., S. Shibata, K. Iwasaki and S. Ueki, 1986, Optic nerve stimulation-induced increase of release of ‘H-glutamate and 3Haspartate but not 3H-GABA from the suprachiasmatic nucleus in slices of rat hypothalamus, Brain Res. Bull. 16, 527. Liou, S.Y., S. Shibata and S. Ueki, 1986, Effect of monoamines on field potentials in the suprachiasmatic nucleus of slices of hypothalamus of the rat evoked by stimulation of the optic nerve, Neuropharmacology 25, 1009. Liou, S.Y., S. Shibata, H.E. Albers and S. Ueki, 1990, Effects of GABA and anxolytics on the single unit discharge of suprachiasmatic neurons in rat hypothalamic slices, Brain Res. Bull. 25, 103. Mason, R.S., SM. Bills and M.E. Harriongton, 1991, The effects of GABA and benzodiazepines on neurons in the suprachiasmatic nucleus (SCN) of Syrian hamsters, Brain Res., 552, 53. Meijer, J.H., E.A. Van der Zee and M. Dietz, 1988, Glutamate phase shifts circadian activity rhythms in hamsters, Neurosci. Lett. 86, 177. Moffett, J.R., L. Williamson, M. Palkovits and M.A.A. Namboodiri, 1990, N-Acetylaspartylglutamate: A transmitter candidate for the retinohypothalamic tract, Proc. Natl. Acad. Sci. U.S.A. 87, 8065. Moore, R.Y., 1983, Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc. 42, 2783. Moore, R.Y. and J.P. Card, 1985, Visual pathways and the entrainment of circadian rhythms, Ann. N. Y. Acad. Sci. 453, 123. Moore, R.Y., A.E. Halaris and B.A. Jones, 1978, Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol. 180, 417. Mark, A. and A. Geisler, 1990, 5-Hydroxytryptamine receptor agonists influence calcium-stimulated adenylate cyclase activity in the cerebral cortex and hippocampus of the rat, Eur. J. Pharmacol. 175, 237. Murakami, N., K. Takahashi and K. Kawashima, 1984, Effect of light on the acetylcholine concentrations of the suprachiasmatic nucleus in the rat, Brain Res. 311, 358. Newman, G.G. and F.E. Hospod, 1986, Rhythm of suprachiasmatic nucleus 2-deoxyglucose uptake in vitro, Brain Res. 381, 345. Nishino, H. and K. Koizumi, 1977, Responses of neurons in the suprachiasmatic nuclei of the hypothalamus to putative transmitters, Brain Res. 120, 167. Oksenberg, D. and S.J. Peroutka, 1988, Antagonism of 5-hydroxytryptamme,+, (5-HT,,) receptor-mediated modulation of adenylate cyclase activity by pindolol and propranolol isomers, Biochem. Pharmacol. 37, 3429. Peroutka, S.J., M.D. Mauk and J.D. Kocsis, 1987, Modulation of neuronal activity in the hippocampus by 5-hydroxytryptamine and 5-hydroxytryptamme,. selective drugs, Neuropharmacology 26, 139. Shenker, A., S. Maayani, H. Weinstein and J.P. Green, 1987, Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes, Mol. Pharmacol. 31, 357. Shibata, S., Y. Oomura, H. Kita and K. Hattori, 1982, Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res. 247, 154. Shibata, S., S.Y. Liou and S. Ueki, 1983, Effect of amino acids and monoamines on the neuronal activity of suprachiasmatic nucleus in hypothalamic slice preparations, Jap. J. Pharmacol. 33, 1225. Shibata, S., S.Y. Liou and S. Ueki, 1986, Influence of excitatory amino acid receptor antagonists and of baclofen on synaptic transmission in the optic nerve to the suprachiasmatic nucleus in slices of rat hypothalamus, Neuropharmacology 25, 403. Shibata, S., V.M. Cassone and R.Y. Moore, 1989, Effects of melatonin on neuronal activity in the rat suprachiasmatic nucleus in vitro, Neurosci. Lett. 97, 140. Smale, L., K.M. Michels, R.Y. Moore and L.P. Morin, 1990, Destruction of the hamster serotonergic system by 5,7-DHT: effects
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5-HT,, agonists on the circadian rhythm of wheel-running activity in hamsters. Eur. J. Pharmacol. 214, 79. Vanecek, J. and L. Vollrath, 1989, Melatonin inhibits cyclic AMP and cyclic GMP accumulation in the rat pituitary, Brain Res. 505, 157.
79 0014-2999/92/$05.00
EJP 52507
Effects of inhibitory and excitatory drugs on the metabolic rhythm of the hamster suprachiasmatic nucleus in vitro Keiko Tominaga, Shigenobu Shibata, Showa Ueki and Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceuticul Sciences, Kyushu University 62, Fukuoka 812, Japan
Received 13 January 1992, revised MS received 24 March 1992, accepted 25 March 1992
In order to elucidate the role of excitatory and inhibitory transmitters within the suprachiasmatic nucleus (SCN) in the circadian change of 2-deoxyglucose (2-DG) uptake in this nucleus, the effects of %hydroxy-2-(di-n-propylamino) tetralin hydrobromide (&OH-DPAT), muscimol, flurazepam, pentobarbital and glutamate on uptake of 2-DG by hamster SCN were examined in hypothalamic slice preparations. 2-DG uptake in the SCN was high during the subjective day and low during the subjective night. The high uptake of 2-DG in the SCN during the daytime was inhibited by the superfusion of &OH-DPAT, muscimol, flurazepam and pentobarbital in a dose-dependent manner, but the low uptake of 2-DG during the night was unaffected. The low uptake during the night was significantly increased by treatment with glutamate, whereas 2-DG uptake during the day was unaffected. In contrast to the above results, 20 mM KC1 and 1 /IM tetrodotoxin increased and decreased 2-DG uptake during both the day and night, respectively. The present results strongly suggest that agonists of S-HT,, receptors and GABA,-benzodiazepine-barbiturate complex receptors regulate the function of the SCN through their inhibitory action on 2-DG uptake during the day, and that glutamate also regulates SCN function through it stimulatoty action on 2-DG uptake during the night. Circadian
rhythm;
Suprachiasmatic
nucleus; 2-Deoxyglucose uptake; 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin); GABA, receptors; Benzodiazepine receptors
1. Introduction
The mammalian suprachiasmatic nucleus (SCN) has been identified as a circadian pacemaker for behavioral and physiological functions (Moore, 1983). An endogeneous circadian rhythm in SCN neuronal firing rate and in 2-deoxyglucose (2-DG) uptake in vitro has been reported (Shibata et al., 1982; Newman and Hospod, 1986). There is a dense serotonergic projection to the SCN from the dorsal and medial raphe (Moore et al., 1978) and axones containing r-amino butyric acid (GABA) projection throughout the SCN (FraqoisBellan et al., 1990). We have previously demonstrated that GABA, benzodiazepines and serotonin (5HT) strongly inhibit firing discharge of SCN neurons in vitro (Shibata et al., 1983; Liou et al., 1990). Thus the SCN may be a brain site for an action of 5-HT,, receptor agonists and GABA,-benzodiazepinebarbiturate receptor agonists on the circadian system.
Correspondence to: K. Tominaga, Faculty of Pharmaceutical Sciences, 812, Japan.
Department of Pharmacology, Kyushu University 62, Fukuoka
The first aim of the present experiment was to examine whether superfusion of drugs which produce an inhibitory action on SCN neurons affects the rhythm of 2-DG uptake in the SCN in a hypothalamic slice preparation. The retinohypothalamic tract is one of the neural projections involved in entraining circadian rhythms to the light-dark cycle (Moore and Card, 1985). With regard to chemical neurotransmission in the retinohypothalamic tract, several pieces of evidence have been reported which indicate that excitatory amino acids (Cahill and Menaker, 1989; Liou et al., 1986; Shibata et al., 1986) and/or N-acetylaspartylglutamate (Moffett et al., 1990) may be involved in the transduction of photic information to the SCN. At present these are the most likely candidates to function as neurotranmitters in the the retinohypothalamic tract. Light pulses produce large phase changes in the rythm of circadian locomotor activity rhythm light pulses are given during the subjective night. If glutamate mediates photic information to the SCN, one would expect that glutamate could affect 2-DG uptake by the SCN, especially during the subjective night. In addition, acetylcholine receptor agonists and glutamate increase the sponta-
80
neous discharge rate of most SCN neurons (Nishino and Koizumi, 1977; Shibata et al., 1983). A second experiment was undertaken to examine whether perfusion of glutamate affects the rhythm of 2-DG uptake in the SCN. The results from above two experiments were compared with those obtained with high KC1 and tetrodotoxin.
2. Materials
and methods
2.1. Procedure Male golden hamsters, weighing 90-120 g, were housed in a normal (light-on 09:OO)or reversed (light-on 21:00) 12: 12 h light-dark cycle for at least 2 weeks before they were used. For the 2-DG uptake study, each animal was killed and the brain was quickly removed from the skull. Coronal hypothalamic slices (450 pm thick) were prepared through the SCN and anterior hypothalamic area (AI-IA) using a tissue chopper. In this experiment, CT refers to clock time, with the time of lights-on arbitrarily designated as CTO. The light phase of the light-dark cycle (CTO-12) is referred to as ‘subjective day’, whereas the dark phase (CT1224) is referred to as ‘subjective night.’ Slices to be studied during the subjective day or the subjective night were prepared at CT3 or CT15, respectively. The slices were preincubated in oxygenated Krebs-Ringer solution for 3 h. After the preincubation in KrebsRinger solution, the slices, which were held between two meshes, were removed into an incubation chamber. The composition of the control Krebs-Ringer solution, equilibrated with 95% O,-5% CO,, was (in mM): NaCl 129, MgSO, 1.3, NaHCO, 22.4, KH*PO, 1.2, KC1 4.2, glucose 10.0 and CaCI, 1.5. The buffer had a pH of 7.3-7.4. The incubation medium contained 1 pCi/ml of 2-DG (2-deoxy-D-[‘4C]-glucose, specific activity, 50 mCi/m mol; Amersham) and each of the drugs. Incubations were carried out for 45 min at 37°C at CT6 or CT18, respectively. The slices were then removed from the chamber and rinsed with 20 ml of warm, gassed preincubation buffer and returned to the original preincubation chamber for 30 min. At the end of washout, slices were placed on dry ice to stop metabolism. The SCN was punched out as described by Murakami et al. (1984). The SCN was homogenized in 1 ml phophate buffer containing 0.5% perchloric acid, and 450 ~1 of the homogenate was used to determine the total amount of protein. The radioactivity in another 450 ~1 of the homogenate was measured in a liquid scintillation counter after the homogenate was solubilized.
2.2. Drugs The drugs used in this study were: 8-hydroxy-2-(din-propylamino) tetralin hydrobromide (8-OH-DPAT, Funakoshi Japan), muscimol (Sigma), pentobarbital (Tanabe, Japan), flurazepam (Takeda, Japan), tetrodotoxin (TTX, Sankyo, Japan), glutamate (Sigma), carbachol (Tokyokasei, Japan) and ( - )-pindolol (Funakosi Inc., Japan). All drugs were dissolved in distilled water. Control preparations were treated with vehicle (distilled water). The application of vehicle at CT6 had no effect on 2-DG uptake (97 f 1.7%, n = 3 of untreated group). (-)-Pindolol was applied to the slices for 10 min before the addition of [14C]2-DG and 8-OH-DPAT, and was present throughout the incubation (45 min). 2.3. Statistical analysis Data are expressed as the means + S.E. Significant differences between groups were determined with a two way analysis of variance followed by the Wilcoxon or Mann-Whitney test for individual comparisons.
3. Results 3.1. Inhibitory drugs
We examined the effects of 8-OH-DPAT, muscimol, flurazepam and pentobarbital on 2-DG uptake by the SCN during the subjective day and the subjective night. During the subjective day (CT6), 2-DG uptake by the SCN was 28 & 1.6 (n = 15) dpm/mg total protein and was significantly (P < 0.01) higher than that during the subjective night (CT18) (19 + 1.1, n = 16). If the 2-DG uptake of control animals at CT6 was set at lOO%, the 2-DG uptake at CT18 became 69 + 3.6% (n = 16). The values of 2-DG uptake in control hamsters at CT6 or CT18 were set at lOO%, respectively. Superfusion of 8-OH-DPAT produced a significant and dose-related inhibition of 2-DG uptake by the SCN at CT6 (F(3,61) = 8.6, P < 0.01) (fig. l), while only a high dose of 8-OH-DPAT (100 PM) decreased 2-DG uptake at CT18 (fig. 1). The inhibitory effect of 8-OH-DPAT on 2-DG uptake was more pronounced at CT6 than at CT18 (compared to dose-response curve at CT18, F(3,61) = 3.2, P < 0.05). Because 10 PM 8-OH-DPAT significantly decreased 2-DG uptake at CT6 but not at CT18, we examined the effect of a 5-HT,, receptor antagonist, (- )-pindolol, on the decrease in 2-DG uptake caused by 10 PM 8-OH-DPAT. Pretreatment with 10 PM (-)-pindolol significantly antagonized the B-OH-DPAT-induced decrease in 2-DG uptake (P < O.OS), while 10 PM (--)-pindolol had no effect on
81 (7) (6)
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Fig. 1. Effect of E-OH-DPAT on the 2-deoxyglucose uptake rhythm of hamster suprachiasmatic nucleus in vitro. The 2-DG uptake of control animals at CT6 or CT18 was set at 100%. Each point shows the mean+S.E. Numbers in brackets indicate the number of animals. Open circle, subjective day fCT6); closed circle, subjective night (CT18). # P < 0.05 compared to dose-response curve at CT18 (two way ANOVA); * P < 0.05, ** P < 0.01 vs. control animals (Cl at CT6 or CT18 (Williams-Wicoxon test).
Fig. 3. Effect of muscimol on 2-deoxyglucose uptake in the hamster suprachiasmatic nucleus in vitro. The 2-DG uptake of control animals at CT6 or CT18 was set at 100%. Each point shows the mean &SE. Numbers in brackets indicate the number of animals. Open circle, subjective day (CT6); closed circle, subjective night (CTIE). # P < 0.05 compared to dose-response curve at CT18 (two way ANOVA); ** P < 0.01 vs. control animals at CT6 (WilliamsWilcoxon test).
2-DG uptake at CT6 (P > 0.05) (fig. 2). At CT6, 2-DG uptake by the SCN was significantly inhibited by treatment with muscimol at 10 and 100 PM (compared to dose-response curve at CT18, F(2,47) = 4.6, P < 0.051, flurazepam at 100 FM (time x drug interaction, F(1,24) = 7.8, P < 0.01) and pentobarbital at 100 PM (time X drug interaction, F(1,27) = 9.1, P < 0.011,
whereas at CT18 2-DG uptake was unaffected by these treatments (fig. 3 and table 1). In contrast to the above drugs, TTX at 1 PM significantly reduced 2-DG uptake at both CT6 and CT18 (table 11, and there was no significant difference between CT6 and CT18 (time x drug interaction, F(1,36) = 0.03, P > 0.05). 3.2. Excitatory drugs 2-DG uptake by the SCN at CT18 was significantly increased by the perfusion of 10 PM glutamate (time x drug interaction, F&30) = 13.8, P < 0.01) (table 11, TABLE 1 Effects of various drugs on 2-deoxyglucose uptake by the suprachiasmatic nucleus in vitro [‘4C]-2-Deoxyglucose uptake was calculated as normalized radioactivity (dpm per mg of protein). The 2-DG uptake of control animals at CT6 or CT18 was set at 100%. Numbers in brackets indicate the number of animals. Drug
10
10
NM
Dose (PM)
2-DG uptake (%, mean k SE., n) CT6 CT18
10 20 mM 100 100 1
loo+ 10.0 (10) lOl+ll.O (8) 134 + 12.0 (8) ’ 61+ 4.0 (6)’ 56+ 13.9 (4) ’ 39+ 3.5 (IO) h
6-OH-DPAT 10
10
JIM
Plndolol
Fig. 2. Effect of (- )-pindolol on the E-OH-DPAT-induced decrease in 2-deoxyglucose uptake in the hamster suprachiasmatic nucleus in vitro. The 2-DG uptake of control animals at CT6 was set at 100%. Each column shows the mean f S.E. Numbers in brackets indicate the number of animals. Open columns, subjective day (CT6); stippled columns, subjective night (CT18). # P < 0.01 vs. control animals at CT6; * P < 0.05 vs. E-OH-DPAT injected group at CT6 (WilliamsWilcoxon test).
Control Glutamate KCI Pentobarbital Flurazepam Tetrodotoxin
lOOk3.4 (10) a 139k 10.1 (6) h 154 f 18.0 (7) h 91 f 9.0 (5) 104+21.0 (4) 42 + 3.6 (10) ’
a When the 2-DG uptake of control animals at CT6 was set at lOO%, the 2-DG uptake at CT18 (70 f 7.0%, n = 10) was significantly lower (P < 0.01, Mann-Whitney test) than the 2-DG uptake at CT6. b P < 0.01 vs. control group (Mann-Whitney test).
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but at CT6, 2-DG uptake was unaffected by this treatment. On the other hand, 20 mM KC1 significantly increased 2-DG uptake at both CT6 and CT18, and there was no significant difference between CT6 and CT18 (time X drug interaction, F(1,31) = 0.8, P > 0.05) (table 1).
4. Discussion An inhibitory action of 8OH-DPAT, muscimol, flurazepam and pentobarbital was observed on SCN 2-DG uptake at CT6 but not at CT18. The present results suggest that the SCN is directly affected by muscimol, flurazepam, pentobarbital and 5-HT,, agonists. In previous experiments, GABA, muscimol, anxiolytics and 5-HT were found to inhibit SCN single unit activity (Lieu et al., 1986, 1990; Mason et al., 1991) and the field potentials in the SCN evoked by optic nerve stimulation (Shibata et al., 1983, 1986). From the localization study of Smith et al. (1989) and the above-mentioned facts, the SCN is the most likely brain site of action for most of these drugs. We recently reported that the phase-response curve produced by the 5-HT,, agonists 8-HT-DPAT, buspirone and ipsapirone was very similar to that produced by muscimol, triazolam and NPY, and that the large phase change caused by these drugs was observed only during the subjective day (Tominaga et al., 1992). At present we do not know the mechanism of action of 8-OH-DPAT on SCN neurons. It has been reported that 8-OH-DPAT has potent 5-HT,, agonistic activity on both post- and pre-synaptic 5-HT,, receptors in the brain (Hamon et al., 1984). In our experiment, 8-OHDPAT decreased 2-DG uptake by the SCN in a dosedependent manner. A biphasic stimulatory effect of 8-OH-DPAT on the activity of adenylate cyclase in the guinea pig hippocampus and rat cerebral cortex has been reported (Shenker et al., 1987; Dumuis et al., 1988; Fayolle et al., 1988; Mark and Geisler, 1990). In the presence of Ca ions, low concentrations of 8-OHDPAT potentiate adenylate cyclase activity, while higher concentrations inhibit the activity of the enzyme (Mork and Geisler, 1990). In addition, the results of electrophysiological studies suggest that high concentrations of 8-OH-DPAT (50-100 PM) are required to alter the amplitude of population spike in the CA1 evoked by the stimulation of Schaffer collaterals (Peroutka et al., 19871, and lo-30 PM 8-OH-DPAT induces hyperpolarization in pyramidal cells of the prefrontal cortex (Araneda and Andrade, 1991). These results suggest that the 5-HT,, receptor subtype in the hamster SCN seems to belong to the low affinity subtype of 5-HT,, receptor. Pretreatment with (--Ipindolol, an antagonist for 5-HT,, receptor (Oksenberg and Peroutka, 19881, markedly blocked the de-
crease in 2-DG uptake produced by 8-OH-DPAT. In our behavioral experiment, the phase advance of wheel-running activity induced by 8-OH-DPAT in hamsters was blocked by pretreatment with t-jpindolol (Tominaga et al., 1992). Melatonin, the indole amine hormone produced in the pineal gland, inhibits firing discharge in the rat SCN in vitro (Shibata et al., 1989). In behavioral studies, melatonin has effects on circadian rhythms during the late subjective day but not at other time of day (for review: Cassone, 1990). These results are similar to the present result obtained with 8-OH-DPAT. In addition, an inhibitory effect of melatonin on cyclic nucleotides has been observed (Carlson et al., 1989; Vanecek and Vollrath, 1989). It is therefore, possible that 8-OHDPAT affects SCN metabolic activity via same mechanism as melatonin. Pentobarbital also produces phase changes and the phase response curve for this drug is similar to that for triazolam and muscimol (Ebihara et al., 1988). However, GABA, benzodiazepines and pentobarbital may not act via 5-HT neurons, because the 5-HT projection is not involved in the triazolam-induced acceleration of re-entrainment to a phase-advanced light dark cycle @male et al., 1990). With regard to chemical transmission in the retinohypothalamic tract, it has been reported that excitatory amino acids (Cahill and Menaker, 1989; Liou et al., 1986; Shibata et al., 1986) may be involved in the function of the retinohypothalamic tract at the SCN. In addition, MK-801, a N-methyl-D-aspartate receptor antagonist, prevents both the phase shifts in the rhythm of hamster wheel-running activity (Colwell et al., 1990) and the induction of Fos protein by light pulses (Abe et al., 1991). Thus glutamte may mediate photic information to the SCN. In the present experiment, glutamate increased 2-DG uptake by the SCN during the subjective night, in contrast. to 8-OH-DPAT, which had a significant effect during the subjective day. However, in behavioral experiments, glutamate injected into the SCN produces a phase change (Meijer et al., 1988) resembling to the phase response curve for dark pulses (Boulos and Rusak, 1982). At present, we do not know the reasons for the contradictory results. In vivo, bolus injection of glutamate may swamp the uptake mechanisms that normally clear glutamate from the vicinity of receptors, causing a long-lasting depolarization. Thus in vivo glutamate may cause a depolarization block that prevents further firing. SCN field potentials are blocked by glutamate (1 mM) added to the bathing medium (Shibata et al., 1986). The question arises whether the inhibitory effects of 8-OH-DPAT, muscimol, flurazepam and pentobarbital, and the excitatory effect of glutamate reflect a specific involvement of these drugs in the time dependence of treatment. The excitatory effect of glutamate during
83
the subjective night, and the inhibitory effects of 8 OH-DPAT, muscimol, flurazepam and pentobarbital during the subjective day seem only to reflect that activity is already high during the day and low during the nights, thereby preventing further excitation during the day and further inhibition during the night. However, TTX significantly inhibited 2-DG uptake at both CT6 and CT18, whereas 20 mM KC1 significantly increased it at both CT6 and CT18. Thus general inhibitory and stimulatory agents affected 2-DG uptake without affecting the time dependence. The effect of TTX and 20 mM KC1 may therefore be non-specific, and the effect of the other drugs may be more specific. In summary, the present results strongly support a receptors, GABA,-benzodiazepinerole for SHT,, barbiturate receptors and glutamate receptor function in the control of the circadian rhythm of SCN activity.
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