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Circadian changes in long-term potentiation of rat suprachiasmatic field potentials elicited by optic nerve stimulation in vitro
BRAIN RESEARCH Brain Research 695 (1995) 158-162
ELSEVIER
Research report
Circadian changes in long-term potentiation of rat suprachiasmatic field potentials elicited by optic nerve stimulation in vitro Yukiko Nishikawa, Shigenobu Shibata *, Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812, Japan
Accepted 23 May 1995
Abstract Optic nerve stimulation caused a postsynaptic field potential in the rat suprachiasmatic nucleus (SCN) of hypothalamic slices. In the present experiment, we demonstrated whether tetanic stimulation of optic nerve can produce a long-term potentiation (LTP) in the SCN postsynaptic field potential. The amplitude of SCN field potential was higher in the subjective day animals than that in the subjective night animals. Tetanic stimulation of optic nerve (100 Hz, 1 s) at subjective daytime (projected zeitgeber time: ZT 0-8) produced a LTP in this field potential, although the onset of LTP was slow. When tetanic stimulation was applied at ZT4, the percent increase of amplitude was 116.6% immediately after, 159.8% 30 min after and 215.4% 120 min after tetanic stimulation, whereas tetanic stimulation of optic nerve at subjective night-time caused a weak LTP in the SCN. Although tetanic stimulation of Schaffer collaterals induced a LTP formation in the CA1 region of rat hippocampal slices, there were no obvious circadian changes in this LTP formation. The present results demonstrated that excitatory influence on the SCN caused a synaptic plasticity such as LTP. Although the physiological meaning of this LTP is uncertain at present, LTP may be related to adaptation mechanism to photic stimulation. Keywords: Suprachiasmatic nucleus; Long-term potentiation; Field potential; Circadian rhythm
1. Introduction The mammalian suprachiasmatic nucleus (SCN) is well known as a primary oscillator in the circadian systems (see [12] for review). Entrainment of circadian rhythms to the environmental light/dark cycle is mediated by the direct retinohypothalamic tract. Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors have been suggested to be involved in mediating photic information in the SCN. Optic nerve stimulation or application of N M D A and non-NMDA receptor agonists could produce changes in the phase of the firing rhythm of SCN neurons in vitro [18,19,22,24,26,29]. The shape of phase-response curves produced by these treatments is similar to that produced by light pulse in vivo. Lund et al. reported that the retinal projection terminates in the upper half of the superior colliculus [15]. The retinotectal pathway is also thought to be glutamatergic [4,6]. Electrophysiological studies have demonstrated the presence of long-term potentiation (LTP) in slices of the superior colliculus [11,16] and visual cortex [3,13]. A
* Corresponding author. Fax: (81) (92) 632-2752. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00717-2
N M D A receptor antagonist A P V is reported to block the induction of LTP in the superior colliculus [16]. LTP is considered as a model of synaptic plasticity in which short high-frequency stimulation of an afferent pathway produces a persistent increase in the efficacy of synaptic transmission. Tetanic optic nerve stimulation failed to induce LTP in the superficial layer of the intact rat, but elicited LTP when picrotoxin was administered prior to the tetanic stimulation or when the ispilateral visual cortex was removed [11,25]. This report has suggested that GABAergic inhibitory input may stop the formation of LTP in the superior colliculus. Therefore the aim of the present experiments was to investigate whether tetanic stimulation of optic nerve can induce LTP formation in the rat SCN in vitro. In addition we examined the circadian changes in the SCN LTP formation in comparison with the hippocampal LTP.
2. Materials and methods Wistar rats (200-300 g) were housed under a 12:12 h light/dark cycle. To eliminate effects of light on the physiological state of the slice preparations, all animals
Y. Nishikawa et al. / Brain Research 695 (1995) 158-162
were housed in constant darkness for 4 8 - 7 2 h prior to sacrifice. The animals were decapitated under ether anesthesia and brains quickly removed from the skull. Horizontal hypothalamic or coronal dorsal hippocampal slices (0.45 mm thickness) were prepared using a vibratome as reported previously. Slices were preincubated with warmed Krebs-Ringer solution equilibrated with 95% 0 2 / 5 % C O 2 at 32°C. The composition of the control Krebs-Ringer solution was (in raM): NaC1, 129; MgSO4, 1.3; NaHCO 3, 2 2 . 4 ; K H z P O 4 , 1.2; KC1, 4.2; D-glucose, 10.0 and CaCI 2, 2.5. This buffer was maintained at pH 7.3-7.4. The slices were obtained 2 - 3 h before stimulation of optic nerve or Schaffer collaterals at a specified projected zeitgeber time (ZT); ZT0 refers to lights-on and ZT12 to lights-off in the animal colony. After preincubation, the slices were transferred to a recording chamber and kept in a constant flow medium (4 ml/min). Optic nerve stimulation-induced postsynaptic field potentials [23] corresponding to EPSP [14,23] or Schaffer collaterals stimulation-induced CA1 compound population spikes were recorded from the SCN or hippocampal slices, respectively, using glass microelectrode. Insulated stainless wires (diameter 0.1 mm) were placed on the optic nerve approximately 1 mm rostral to the optic chiasm or on the CA3 area. A single pulse stimulation with duration of 0.08 ms at 0.5-1.5 mA for optic nerve stimulation and 0.1-0.4 mA for Schaffer collaterals stimulation at a rate of 0.l Hz was used, because this has previously been shown to produce field potential in the ventrolateral SCN and population spikes in the CA1 pyramidal layer, respectively. Before recording LTP, the strength of the optic nerve or Schaffer collateral stimulation was adjusted to obtain a response of about half of the maximal amplitude of population spikes. After observation of stable response from the SCN or CA1 regions over 30 min, tetanic stimulation of 100 Hz for 1 s was applied to induce LTP. Amplitude of field potential was recorded with 5 min interval for 120 min after tetanic stimulation. The amplitude of post-
A
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~220
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"~Ca" 2 0 0
160 ~
140
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Time(min) Fig. 2. Circadian changes in the time course of the appearance of LTP in suprachiasmatic nucleus after tetanic stimulation to the optic nerve. Tetanic stimulation was applied to optic nerve at time 0. % of amplitude after tetanic stimulation against that before tetanic stimulation. Each point indicates the average of the postsynaptic field potential amplitude from 3 (ZT1, ZTI2) to 5 (ZT8) animals. The vertical bars indicate the S.E.M. ZT, projected Zeitgeber time.
synaptic field potential before tetanus was set as 100%. Data were expressed as mean + S.E.M. The significant difference between groups was determined by ANOVA.
V
rime(min)
30
Time(min)
30
60
0ms
120 10ms
Fig. 1. Time course of the appearance of LTP in suprachiasmatic nucleus after tetanic stimulation to the optic nerve. The traces show the postsynaptic field potentials recorded before, 30 min, 60 rain and 120 min after tetanic stimulation. Each trace is an average of eight sweeps. The tetanic stimulation was applied at projected zeitgeber time 4 for upper traces and at projected zeitgeber time 16 for lower traces.
160
Y.
Nishikawa et al. /Brain Research 695 (1995) 158-162
3. R e s u l t s 180.
Optic nerve stimulation elicited a large negative field potential in the SCN. Similar to our previous reports, amplitude of field potential is increased by increasing stimulus intensity. Amplitude of this field potential induced by maximal stimulus intensity was significantly higher in subjective day animals ( 0 . 9 4 _ 0.075 mV, P < 0.05) than in subjective night ones ( 0 . 7 1 _ 0.073 mV). After decreasing the stimulus intensity from maximal responses to half responses, tetanic stimulation (100 Hz, 1 s) was applied to optic nerve. During subjective day, especially at ZT4, tetanic stimulation to optic nerve produced a LTP formation (Fig. 1, Fig. 2). Amplitude of field potential was rapidly increased until 60 min, and then slowly increased until 120 min after tetanic stimulation. On the other hand, during subjective night, tetanic stimulation to optic nerve failed to cause LTP formation (Fig. 1, Fig. 2). The circadian changes in the LTP formation were not seen at 30 min (ANOVA, F ( 5 , 1 7 ) = 2.27, P > 0.05), but were significant at 60 min (F(5,17) = 4.76, P < 0.05), 90 min (F(5,17) = 16.38, P < 0.01) and 120 min ( F ( 5 , 1 6 ) = 9.37, P < 0.01) after tetanic stimulation (Fig. 3). To determine regional specificity for circadian changes in LTP formation, we examined the LTP formation in the hippocampal CA1 obtained from day or night animals.
160. 140. 120. "*3
loo ~ .o
---o--- ZT08 •
ZT 20
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30
60
Time(rain) Fig. 4. Circadian changes in the time course of the appearance of LTP in hippocampal CA1 region after tetanic stimulation to the Schaffer collaterals at time 0. % of amplitude after tetanic stimulation against that before tetanic stimulation. Each point indicates the average of the amplitude from 3 animals and the vertical bars indicate the S.E.M. ZT, projected zeitgeber time.
Amplitude of CA1 population spike potentials induced by maximal intensity was 1.12 + 0.20 mV in both subjective day and night animals. Tetanic stimulation to Schaffer collaterals induced a LTP formation (160% of control) in CA1 region of hippocampal slices in both day and night animals. Thus there were no obvious circadian changes in hippocampal LTP formation (Fig. 4).
4. D i s c u s s i o n 260.
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F i g . 3. C i r c a d i a n c h a n g e s in L T P in s u p r a c h i a s m a t i c n u c l e u s a f t e r t e t a n i c
stimulation to the optic nerve. % of amplitude after tetanic stimulation against that before tetanic stimulation. Each point indicates the averageof the postsynaptic field potential amplitude 30 min, 60 min, 90 min and 120 min after tetanic stimulation. Each point indicates the averageof the postsynaptic field potential amplitude from 3 (ZT1, ZT12) to 5 (ZT8) animals, and the vertical bars indicate the S.E.M. Significant differences among groups indicated P > 0.05, P < 0.05, P < 0.01 and P < 0.01 (ANOVA). ZT, projected zeitgeber time.
In the present experiment, we demonstrated that tetanic stimulation to optic nerve induced LTP formation in the SCN slices obtained from subjective day, whereas tetanic stimulation to optic nerve failed to induce LTP in the SCN from subjective night. Although the tetanic stimulation to optic nerve caused a LTP in the SCN, the developmental pattern of LTP formation was different from that in the hippocampal CA1 region. In the hippocampal CA1 regions, tetanic stimulation potentiated the population spike potential just from 5 min after tetanus stimulation, and this potentiation was maintained for an observed 60 min. The amplitude of the SCN field potential increased slowly in the early phase (60 min after tetanus), and potentiation was maintained for 120 min in the late phase (60-120 min). At present we do not know the reasons for such different LTP formation between hippocampal CA1 and optic nerve-SCN synapses. Although both synapses are reported to use glutamate as a neurotransmitter [2,4,6,10], the amplitude of field population spikes is bigger in the hippocampal CA1 than in the SCN. Therefore the different time course of LTP formation between these two regions may be due to the difference in synaptic density. Interestingly, LTP formation, especially its induction phase in the superior colliculus, is reported to be of slow onset [11,16] and to be similar to LTP formation in the SCN.
Y. Nishikawa et al. / Brain Research 695 (1995) 158-162
Tetanic stimulation to Schaffer collaterals elicited LTP formation in hippocampal CA1 of rat from both subjective day and subjective night. Thus there are no circadian differences of LTP formation in the hippocampus. Previously it was reported that light/dark influences on hippocampal LTP are preserved in the in vitro environment [5,9]. Therefore, it is suggested that hippocampal LTP formation is not controlled by circadian oscillators but by light/dark changes. The present results showed that there were circadian influences on LTP formation in the SCN region. During subjective day, tetanic stimulation to optic nerve induced LTP formation in the SCN. The neuronal mechanisms underlying circadian influences on the SCN LTP are uncertain at present. Spontaneous neuronal activity [20] and also amplitude of field potentials in the SCN are high during subjective day. Thus, the synaptic efficacy of optic nerve-SCN synapses may be activated during subjective day rather than during subjective night. The SCN neuropil is an almost entirely GABAergic network [17,28]. Recently it was reported that GABA synthesis, release, uptake or content within the SCN showed a circadian pattern [1]. The timing of the peak GABA levels were found during the night under constant dim red light. These results have suggested that during night-time, GABAergic inhibition may stop the induction of LTP formation by tetanic stimulation to optic nerve, although this issue remains to be further studied. In fact, GABAergic inhibitory influences from cortex are reported to attenuate the induction of LTP in the superior colliculus by the stimulation of optic nerve [11,25]. Optic nerve stimulation or glutamate receptor agonist application are reported to produce phase-shifts of circadian rhythm in the SCN spontaneous neuronal activity, when stimulation was applied during subjective night-time [7,8,21,27]. Therefore the LTP formation during daytime may be required to maintain high firing activity of SCN neurons during daytime, but may not be related to produce photic stimulation-induced phase shifts. Although physiological implication of LTP formation in the SCN is uncertain at present, efficacy of synaptic transmission in the SCN may be a related adaptation mechanism to photic stimulation.
References [1] Aguilar-Roblero, R., Verduzco-Carbajal, L., Rodriguez, C., Mendez-Franco, J., Moran, J. and de la Mora, M.P., Circadian rhythmicity in the GABAergic system in the suprachiasmatic nuclei of the rat, Neurosci. Lett., 157 (1993) 199-202. [2] Andersen, P., Bliss, T.V., Lomo, T., Olsen, L.I. and Skrede, K.K., Lamellar organization of hippocampal excitatory pathways, Acta Physiol. Scand., 76 (1969) 4A-5A. [3] Br6cher, S., Artola, A. and Singer, W., Agonists of cholinergic receptors facilitate synergistically the induction of long-term potentiation in slices of rat visual cortex, Brain Res., 573 (1992) 27-36.
161
[4] Cahill, G.M. and Meneker, M., Effects of excitatory amino acid receptor antagnists and agonists on suprachiasmatic nucleus responses to retinohypothalamic tract volleys, Brain Res., 479 (1989) 76-82. [5] Dana, R.C. and Martinez, Jr., J.L., Effect of adrenalectomy on the circadian rhythm of LTP, Brain Res., 308 (1984) 392-395. [6] De Vries, M.J. and Meijer, J.H., Glutamate immunoreactivity in terminals of the retinohypothalamic tract of the brown Norwegian rat, Brain Res., 612 (1993) 231-237. [7] De Vries, M.J., Treep, J.A., de Pauw, ES.D. and Meijer, J.H., The effects of electrical stimulation of the optic nerves and anterior optic chiasm on the circadian activity of rhythm of the Syrian hamster, Brain Res., 642 (1994) 206-212. [8] Ding, J.M., Chen, D., Weber, E.T., Faiman, L.E., Rea, M.A. and Gillette, M.U., Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO, Science, 266 (1994) 17131717. [9] Harris, K.M. and Teyler, T.J., Age differences in a circadian influence on hippocampal LTP, Brain Res., 261 (1983) 69-73. [10] Hestrin, S., Nicoll, R.A., Perkel, D.J. and Sah, P., Analysis of excitatory synaptic action in pyramidal cells using whole-cell recording from rat hippocampal slices, J. Physiol., 422 (1990) 203-225. [11] Hirai, H. and Okada, Y., Ipsilateral corticotectal pathway inhibits the formation of long-term potentiation (LTP) in the rat superior colliculus through GABAergic mechanism, Brain Res., 629 (1994) 23-30. [12] Inouye, S.I.T. and Shibata, S., Neurochemical organization of circadian rhythm in the suprachiasmatic nucleus, Neurosci. Res.. 20 (1994) 109-130. [13] Kato, N. and Yoshimura, H., Facilitatory effect of substance P on the susceptibility to long-term potentiation in the visual cortex of adult rats, Brain Res., 617 (1993) 353-356. [14] Kim, Y.I. and Dudek, F.E., Intracellular electrophysiological study of suprachiasmatic nucleus neurons in rodents: excitatory synaptic mechanism, J. Physiol., 444 (1991) 269-287. [15] Lund, R.D., Anatomic studies on the superior colliculus, lm,est. Opthalmol., 11 (1992) 434-44l. [16] Miyamoto, T and Okada, Y., NMDA receptor, protein kinase C and calmodulin system participate in the long-term potentiation in guinea pig superior colliculus slices, Brain Res., 605 (1993) 287-292. [17] Moore, R.Y. and Speh, J.C., GABA is the principal neurotransmitter of the circadian system, Neurosci. Lett., 150 (1993) 112-116. [18] Rea, M.A., Buckley, B. and Lutton, L.M., Local administration of EEA antagonist blocks light-induced phase shifts and c-los expression in the hamster SCN, Am. J. Physiol., 265 (1993) RlI91-Rl198. [19] Shibata, S., Liou, S.Y. and Ueki, S., 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 (1986)403-409. [20] Shibata, S. and Moore, R.Y., Tetrodotoxin does not affect circadian rhythm in neuronal activity and metabolism in rodent suprachiasmatic nucleus in vitro, Brain Res., 606 (1993) 259-266. [21] Shibata, S. and Moore, R.Y., Neuropeptide Y and optic chiasm stimulation affect suprachiasmatic nucleus circadian function in vitro, Brain Res., 615 (1993) 95-100. [22] Shibata, S., Ono, M., Tominaga, K., Hamada, T., Watanabe, A. and Watanabe, S., Involvement of vasoactive intestinal polypeptide in NMDA-induced phase delay of firing activity rhythm in the suprachiasmatic nucleus in vitro. Neurosci. Biobehar. Rer., 18 (1994) 591-595. [23] Shibata, S., Oomura, Y., Kita, H., Liou, S.Y. and Ueki, S., Field potentials in the suprachiasmatic nucleus of rat hypothalamic slice produced by optic nerve stimulation, Brain Res. Bull., 12 (1984) 377-379. [24] Shibata, S., Hamada, T., Watauabe, A., Ono, M. and Watanabe, S., Neurotransmitter interaction in the SCN clock function. In T. Hiroshige and K. Honma (Eds.), E~,olution of Circadian Clock, Hokkaido University press, Sapporo, Japan, 1994, pp. 177-185.
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[25] Shibata, Y., Tomita, H. and Okada, Y., The effects of ablation of the visual cortical area on the formation of LTP in the superior colliculus of the rat, Brain Res., 537 (1990) 345-348. [26] Shibata, S., Watanabe, A., Hamada, T., Ono, M. and Watanabe, S., N-methyl-D-aspartate-induced phase shifts in circadian rhythm of neuronal activity of rat SCN in vitro, Am. J. Physiol., 267 (1994) R360-R364. [27] Shirakawa, T. and Moore, R.Y., Glutamate shifts the phase of the circadian firing rhythm in the suprachiasmatic nucleus in vitro, Neurosci. Lett., 178 (1994) 47-50.
[28] Van den Pol, A.N., Gamma-aminobutyrate, gastrin releasing peptide, serotonin, somatostatin and vasopressin: Ultrastructural immunocytochemical localization in presynaptic axons in the suprachiasmatic nucleus, Neuroscience, 17 (1986) 643-659. [29] Watanabe, A., Hamada, T., Shibata, S. and Watanabe, S., Effects of nitric oxide synthase inhibitors on N-methyl-D-aspartate-induced phase delay of circadian rhythm of neuronal activity in the rat. suprachiasmatic nucleus in vitro, Brain Res., 646 (1994) 161-164.
ELSEVIER
Research report
Circadian changes in long-term potentiation of rat suprachiasmatic field potentials elicited by optic nerve stimulation in vitro Yukiko Nishikawa, Shigenobu Shibata *, Shigenori Watanabe Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812, Japan
Accepted 23 May 1995
Abstract Optic nerve stimulation caused a postsynaptic field potential in the rat suprachiasmatic nucleus (SCN) of hypothalamic slices. In the present experiment, we demonstrated whether tetanic stimulation of optic nerve can produce a long-term potentiation (LTP) in the SCN postsynaptic field potential. The amplitude of SCN field potential was higher in the subjective day animals than that in the subjective night animals. Tetanic stimulation of optic nerve (100 Hz, 1 s) at subjective daytime (projected zeitgeber time: ZT 0-8) produced a LTP in this field potential, although the onset of LTP was slow. When tetanic stimulation was applied at ZT4, the percent increase of amplitude was 116.6% immediately after, 159.8% 30 min after and 215.4% 120 min after tetanic stimulation, whereas tetanic stimulation of optic nerve at subjective night-time caused a weak LTP in the SCN. Although tetanic stimulation of Schaffer collaterals induced a LTP formation in the CA1 region of rat hippocampal slices, there were no obvious circadian changes in this LTP formation. The present results demonstrated that excitatory influence on the SCN caused a synaptic plasticity such as LTP. Although the physiological meaning of this LTP is uncertain at present, LTP may be related to adaptation mechanism to photic stimulation. Keywords: Suprachiasmatic nucleus; Long-term potentiation; Field potential; Circadian rhythm
1. Introduction The mammalian suprachiasmatic nucleus (SCN) is well known as a primary oscillator in the circadian systems (see [12] for review). Entrainment of circadian rhythms to the environmental light/dark cycle is mediated by the direct retinohypothalamic tract. Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors have been suggested to be involved in mediating photic information in the SCN. Optic nerve stimulation or application of N M D A and non-NMDA receptor agonists could produce changes in the phase of the firing rhythm of SCN neurons in vitro [18,19,22,24,26,29]. The shape of phase-response curves produced by these treatments is similar to that produced by light pulse in vivo. Lund et al. reported that the retinal projection terminates in the upper half of the superior colliculus [15]. The retinotectal pathway is also thought to be glutamatergic [4,6]. Electrophysiological studies have demonstrated the presence of long-term potentiation (LTP) in slices of the superior colliculus [11,16] and visual cortex [3,13]. A
* Corresponding author. Fax: (81) (92) 632-2752. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00717-2
N M D A receptor antagonist A P V is reported to block the induction of LTP in the superior colliculus [16]. LTP is considered as a model of synaptic plasticity in which short high-frequency stimulation of an afferent pathway produces a persistent increase in the efficacy of synaptic transmission. Tetanic optic nerve stimulation failed to induce LTP in the superficial layer of the intact rat, but elicited LTP when picrotoxin was administered prior to the tetanic stimulation or when the ispilateral visual cortex was removed [11,25]. This report has suggested that GABAergic inhibitory input may stop the formation of LTP in the superior colliculus. Therefore the aim of the present experiments was to investigate whether tetanic stimulation of optic nerve can induce LTP formation in the rat SCN in vitro. In addition we examined the circadian changes in the SCN LTP formation in comparison with the hippocampal LTP.
2. Materials and methods Wistar rats (200-300 g) were housed under a 12:12 h light/dark cycle. To eliminate effects of light on the physiological state of the slice preparations, all animals
Y. Nishikawa et al. / Brain Research 695 (1995) 158-162
were housed in constant darkness for 4 8 - 7 2 h prior to sacrifice. The animals were decapitated under ether anesthesia and brains quickly removed from the skull. Horizontal hypothalamic or coronal dorsal hippocampal slices (0.45 mm thickness) were prepared using a vibratome as reported previously. Slices were preincubated with warmed Krebs-Ringer solution equilibrated with 95% 0 2 / 5 % C O 2 at 32°C. The composition of the control Krebs-Ringer solution was (in raM): NaC1, 129; MgSO4, 1.3; NaHCO 3, 2 2 . 4 ; K H z P O 4 , 1.2; KC1, 4.2; D-glucose, 10.0 and CaCI 2, 2.5. This buffer was maintained at pH 7.3-7.4. The slices were obtained 2 - 3 h before stimulation of optic nerve or Schaffer collaterals at a specified projected zeitgeber time (ZT); ZT0 refers to lights-on and ZT12 to lights-off in the animal colony. After preincubation, the slices were transferred to a recording chamber and kept in a constant flow medium (4 ml/min). Optic nerve stimulation-induced postsynaptic field potentials [23] corresponding to EPSP [14,23] or Schaffer collaterals stimulation-induced CA1 compound population spikes were recorded from the SCN or hippocampal slices, respectively, using glass microelectrode. Insulated stainless wires (diameter 0.1 mm) were placed on the optic nerve approximately 1 mm rostral to the optic chiasm or on the CA3 area. A single pulse stimulation with duration of 0.08 ms at 0.5-1.5 mA for optic nerve stimulation and 0.1-0.4 mA for Schaffer collaterals stimulation at a rate of 0.l Hz was used, because this has previously been shown to produce field potential in the ventrolateral SCN and population spikes in the CA1 pyramidal layer, respectively. Before recording LTP, the strength of the optic nerve or Schaffer collateral stimulation was adjusted to obtain a response of about half of the maximal amplitude of population spikes. After observation of stable response from the SCN or CA1 regions over 30 min, tetanic stimulation of 100 Hz for 1 s was applied to induce LTP. Amplitude of field potential was recorded with 5 min interval for 120 min after tetanic stimulation. The amplitude of post-
A
159
~240-
~220
z'r01 ----.o----- ZT04
"~Ca" 2 0 0
160 ~
140
•~
120
80 -30
B
0
before
0
60
90
120
. i • 90
• i 120
~-, 2 4 0 v "~ 2 2 0
[ 2oo
-----o----- ZT13 -----w---- ZT16 A ZT20
~ 14o '~
120
80
i • • i 0 30
-30
, i 60
Time(min) Fig. 2. Circadian changes in the time course of the appearance of LTP in suprachiasmatic nucleus after tetanic stimulation to the optic nerve. Tetanic stimulation was applied to optic nerve at time 0. % of amplitude after tetanic stimulation against that before tetanic stimulation. Each point indicates the average of the postsynaptic field potential amplitude from 3 (ZT1, ZTI2) to 5 (ZT8) animals. The vertical bars indicate the S.E.M. ZT, projected Zeitgeber time.
synaptic field potential before tetanus was set as 100%. Data were expressed as mean + S.E.M. The significant difference between groups was determined by ANOVA.
V
rime(min)
30
Time(min)
30
60
0ms
120 10ms
Fig. 1. Time course of the appearance of LTP in suprachiasmatic nucleus after tetanic stimulation to the optic nerve. The traces show the postsynaptic field potentials recorded before, 30 min, 60 rain and 120 min after tetanic stimulation. Each trace is an average of eight sweeps. The tetanic stimulation was applied at projected zeitgeber time 4 for upper traces and at projected zeitgeber time 16 for lower traces.
160
Y.
Nishikawa et al. /Brain Research 695 (1995) 158-162
3. R e s u l t s 180.
Optic nerve stimulation elicited a large negative field potential in the SCN. Similar to our previous reports, amplitude of field potential is increased by increasing stimulus intensity. Amplitude of this field potential induced by maximal stimulus intensity was significantly higher in subjective day animals ( 0 . 9 4 _ 0.075 mV, P < 0.05) than in subjective night ones ( 0 . 7 1 _ 0.073 mV). After decreasing the stimulus intensity from maximal responses to half responses, tetanic stimulation (100 Hz, 1 s) was applied to optic nerve. During subjective day, especially at ZT4, tetanic stimulation to optic nerve produced a LTP formation (Fig. 1, Fig. 2). Amplitude of field potential was rapidly increased until 60 min, and then slowly increased until 120 min after tetanic stimulation. On the other hand, during subjective night, tetanic stimulation to optic nerve failed to cause LTP formation (Fig. 1, Fig. 2). The circadian changes in the LTP formation were not seen at 30 min (ANOVA, F ( 5 , 1 7 ) = 2.27, P > 0.05), but were significant at 60 min (F(5,17) = 4.76, P < 0.05), 90 min (F(5,17) = 16.38, P < 0.01) and 120 min ( F ( 5 , 1 6 ) = 9.37, P < 0.01) after tetanic stimulation (Fig. 3). To determine regional specificity for circadian changes in LTP formation, we examined the LTP formation in the hippocampal CA1 obtained from day or night animals.
160. 140. 120. "*3
loo ~ .o
---o--- ZT08 •
ZT 20
8O -30
0
30
60
Time(rain) Fig. 4. Circadian changes in the time course of the appearance of LTP in hippocampal CA1 region after tetanic stimulation to the Schaffer collaterals at time 0. % of amplitude after tetanic stimulation against that before tetanic stimulation. Each point indicates the average of the amplitude from 3 animals and the vertical bars indicate the S.E.M. ZT, projected zeitgeber time.
Amplitude of CA1 population spike potentials induced by maximal intensity was 1.12 + 0.20 mV in both subjective day and night animals. Tetanic stimulation to Schaffer collaterals induced a LTP formation (160% of control) in CA1 region of hippocampal slices in both day and night animals. Thus there were no obvious circadian changes in hippocampal LTP formation (Fig. 4).
4. D i s c u s s i o n 260.
24o~ 3Omin(p>O.05)
90min(I>
220 -'
200 180 160 140 120 100
60min(p<0.05)
240 "o
220 21717 180 160 140-" 120' 100." 80
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I
8 12 16 20 24 ZT
F i g . 3. C i r c a d i a n c h a n g e s in L T P in s u p r a c h i a s m a t i c n u c l e u s a f t e r t e t a n i c
stimulation to the optic nerve. % of amplitude after tetanic stimulation against that before tetanic stimulation. Each point indicates the averageof the postsynaptic field potential amplitude 30 min, 60 min, 90 min and 120 min after tetanic stimulation. Each point indicates the averageof the postsynaptic field potential amplitude from 3 (ZT1, ZT12) to 5 (ZT8) animals, and the vertical bars indicate the S.E.M. Significant differences among groups indicated P > 0.05, P < 0.05, P < 0.01 and P < 0.01 (ANOVA). ZT, projected zeitgeber time.
In the present experiment, we demonstrated that tetanic stimulation to optic nerve induced LTP formation in the SCN slices obtained from subjective day, whereas tetanic stimulation to optic nerve failed to induce LTP in the SCN from subjective night. Although the tetanic stimulation to optic nerve caused a LTP in the SCN, the developmental pattern of LTP formation was different from that in the hippocampal CA1 region. In the hippocampal CA1 regions, tetanic stimulation potentiated the population spike potential just from 5 min after tetanus stimulation, and this potentiation was maintained for an observed 60 min. The amplitude of the SCN field potential increased slowly in the early phase (60 min after tetanus), and potentiation was maintained for 120 min in the late phase (60-120 min). At present we do not know the reasons for such different LTP formation between hippocampal CA1 and optic nerve-SCN synapses. Although both synapses are reported to use glutamate as a neurotransmitter [2,4,6,10], the amplitude of field population spikes is bigger in the hippocampal CA1 than in the SCN. Therefore the different time course of LTP formation between these two regions may be due to the difference in synaptic density. Interestingly, LTP formation, especially its induction phase in the superior colliculus, is reported to be of slow onset [11,16] and to be similar to LTP formation in the SCN.
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Tetanic stimulation to Schaffer collaterals elicited LTP formation in hippocampal CA1 of rat from both subjective day and subjective night. Thus there are no circadian differences of LTP formation in the hippocampus. Previously it was reported that light/dark influences on hippocampal LTP are preserved in the in vitro environment [5,9]. Therefore, it is suggested that hippocampal LTP formation is not controlled by circadian oscillators but by light/dark changes. The present results showed that there were circadian influences on LTP formation in the SCN region. During subjective day, tetanic stimulation to optic nerve induced LTP formation in the SCN. The neuronal mechanisms underlying circadian influences on the SCN LTP are uncertain at present. Spontaneous neuronal activity [20] and also amplitude of field potentials in the SCN are high during subjective day. Thus, the synaptic efficacy of optic nerve-SCN synapses may be activated during subjective day rather than during subjective night. The SCN neuropil is an almost entirely GABAergic network [17,28]. Recently it was reported that GABA synthesis, release, uptake or content within the SCN showed a circadian pattern [1]. The timing of the peak GABA levels were found during the night under constant dim red light. These results have suggested that during night-time, GABAergic inhibition may stop the induction of LTP formation by tetanic stimulation to optic nerve, although this issue remains to be further studied. In fact, GABAergic inhibitory influences from cortex are reported to attenuate the induction of LTP in the superior colliculus by the stimulation of optic nerve [11,25]. Optic nerve stimulation or glutamate receptor agonist application are reported to produce phase-shifts of circadian rhythm in the SCN spontaneous neuronal activity, when stimulation was applied during subjective night-time [7,8,21,27]. Therefore the LTP formation during daytime may be required to maintain high firing activity of SCN neurons during daytime, but may not be related to produce photic stimulation-induced phase shifts. Although physiological implication of LTP formation in the SCN is uncertain at present, efficacy of synaptic transmission in the SCN may be a related adaptation mechanism to photic stimulation.
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