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The role of calcium ions in circadian rhythm of suprachiasmatic nucleus neuron activity in rat hypothalamic slices
Neuroscience Letters, 52 (1984) 181-184 Elsevier Scientific Publishers Ireland Ltd.
181
NSL 03045
THE ROLE OF C A L C I U M IONS IN C I R C A D I A N R H Y T H M OF S U P R A C H I A S M A T I C N U C L E U S N E U R O N A C T I V I T Y I N RAT HYPOTHALAMIC SLICES
SHIGENOBU SHIBATA, AKIKO SHIRATSUCHI, SHYH YUH LIOU and SHOWA UEKI Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812 (Japan)
(Received August 13th, 1984; Revised version received and accepted September 17th, 1984)
Key words: suprachiasmatic nucleus - circadian rhythm - calcium ions - single unit activity - rat brain slice - synaptic transmission
The role of calcium ions in maintaining the circadian rhythm of suprachiasmatic nucleus (SCN) neuron activity was investigated using rat hypothalamic slice preparations. In normal Krebs solution, the firing rate of SCN neurons was higher in the light period than in the dark period. In Ca 2÷ -free Krebs solution, SCN neuron activity was low during all periods and did not show diurnal rhythm. These results suggest that the disappearance of circadian rhythmic change of SCN neuron activity in Ca 2÷-free Krebs solution may be due to the disappearance of synaptic transmission in the SCN.
M u l t i p l e unit activity r e c o r d s in the rat s u p r a c h i a s m a t i c nucleus (SCN) have s h o w n c i r c a d i a n r h y t h m i c changes, a n d these changes persisted a f t e r i s o l a t i o n o f the h y p o t h a l a m u s i n c l u d i n g the S C N [5]. In e x p e r i m e n t s in vitro, it was o b s e r v e d t h a t single-unit activity in S C N slices t a k e n f r o m a n i m a l s k e p t on a l i g h t - d a r k cycle (LD) was higher d u r i n g the light t h a n d u r i n g the d a r k p e r i o d [2, 6, 8]. These r e p o r t s suggest t h a t the activity o f S C N n e u r o n s show the c i r c a d i a n r h y t h m even after a f f e r e n t i n p u t s f r o m o t h e r c e n t r a l nuclei [9] were cut. In o r d e r to d e m o n s t r a t e the role o f the i n t e r n e u r o n s a n d a f f e r e n t n e u r o n s in the S C N in the c i r c a d i a n r h y t h m o f S C N n e u r o n activity in vitro, we r e c o r d e d the S C N n e u r o n activity in low C a 2 + K r e b s solution. A t o t a l o f 58 W i s t a r strain rats were used. A n i m a l s were h o u s e d with n o r m a l L D (light p e r i o d 0 7 . 0 0 - 1 9 . 0 0 h) for 2 m o n t h s . F o r p r e p a r i n g the slices, rats were killed b y a b l o w o n the b a c k o f the neck at v a r i o u s times o f day: 08.30, 12.30, 16.30, 20.30, 00.30 a n d 04.30 h; the cerebri were q u i c k l y r e m o v e d f r o m the skull [6, 8]. H y p o t h a l a m i c slices, a b o u t 300 # m thick, i n c l u d i n g the S C N a n d the a n t e r i o r h y p o t h a l a m u s , were t h e n c o r o n a l l y s e c t i o n e d with a v i b r a t o m e . T h e c o m p o s i t i o n o f the c o n t r o l Krebs s o l u t i o n , e q u i l i b r a t e d with 95°/0 O2 a n d 5°/o CO2, was (in m M final c o n c e n t r a t i o n ) : NaC1, 124; KC1, 5; KHEPO4, 1.24; CaC12, 2.4; MgSO4, 1.3; 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
182
NaHCO3, 26; and glucose, 10. Recording electrodes were filled with 2 M NaCI (DC resistance, 2-10 MS2) in Krebs solution. The calcium concentration of low Ca z + Krebs solution ranged between 0 and 1.3 mM, while the magnesium concentration was kept constant (10 mM). Single neuron activity which remained stable for at least 5 min was recorded; the recording was terminated within 8 h after preparing the slice. The mean firing rate was determined during each 4-h period depending on the number of spontaneous active neurons recorded. In our previous study [6], the firing rates o f neurons in the ventrolateral part of the SCN were compared with those in the dorsomedial part, and the firing rates in both areas of SCN taken from animals kept on LD were higher during the light than during the dark period. Therefore, in the present experiment, these two areas were combined to determine the mean firing rate. As mentioned in our previous report [6], the firing patterns of single neuron activity were divided into the following three types: type I, (Fig. 1A) regular firing characterized by a very constant interspike interval; type II, irregular firing (Fig. 1B); and type II1, burst firing (not shown in figure). The influence of low C a 2 + Krebs solution on the occurrence of the three firing patterns and firing rates is shown in Table I. The population of type I decreased and that of type 1I increased in decreasing Ca 2 + ions of the Krebs solution. Fourteen of 30 cells were facilitated by 1.3 mM Ca 2+ Krebs (Fig. ID). Many cells were facilitated then inhibited (Fig. 1C) or inhibited by 0.6 and 0.0 mM Ca 2+ Krebs. The firing rate of SCN neurons in normal Krebs solution was significantly higher in the light period than in the dark period (Fig. 2). On the other hand, in C a 2 + -free Krebs solution, SCN neuron activity was low during all periods and did not show
?•I B
~J
I.
Ca free
(O mM Ca, lO rnM Mr)
1 min
Fig. I. S p o n t a n e o u s n e u r o n a l d i s c h a r g e o f S C N n e u r o n s . A : r e g u l a r firing (type l). 13: i r r e g u l a r firing ( t y p e II). A a n d B: r e c o r d s in n o r m a l K r e b s s o l u t i o n (2.4 m M C a 24 ). C a n d D: effect o f low C a 2 + K r e b s s o l u t i o n o n S C N n e u r o n a c t i v i t y . Bar, a p p l i c a t i o n p e r i o d o f low C a z ' K r e b s s o l u t i o n . C: a shift in the d i s c h a r g e p a t t e r n f r o m t y p e I to t y p e II a n d a d e c r e a s e in the firing r a t e w e r e o b s e r v e d in C a z + -free K r e b s s o l u t i o n . D: a shift in the d i s c h a r g e p a t t e r n f r o m type I to t y p e 11 a n d a n i n c r e a s e in t h e firing r a t e were o b s e r v e d in 0.6 m M C a 2 * K r e b s s o l u t i o n .
183
Dark period
10
o
8
03
I ,,'""
~6
I.......( 467I )
co
T I
I
I
I
I
I
I
13:00
17:00
21:00
01:00
05:00
09:00
13:00
0
• ...... •
Normal
(2.6 mM Ca,1.3 mM Mg)
•
Ca free
(0 mM Ca, lO mM Mg)
•
Fig. 2. Effect of Ca 2 ÷-free Krebs solution on the circadian r h y t h m of neuronal activity in SCN. Each point is the average discharge rate (imp./s) of neurons during each 4-h period, and each point contains 68-103 neurons in normal Krebs solution and 24-53 in Ca 2 ÷ -free Krebs solution. Numbers in parentheses are the total n u m b e r of neurons recorded.
diurnal rhythm. The populations of the type I, II and III in normal Krebs solution were 84.9, 13.5 and 1.5°70 during the light period, and 68.3, 27.4 and 4.3o70 during dark period, respectively. On the other hand, the populations of the three types in Ca2+-free Krebs solution were 28.6, 65.2 and 6.3°70 during the light period, and 25.6, 67.4 and 7.0o-/o during the dark period, respectively. In the present experiment we demonstrated the shift of the discharge pattern from type I to type II and the loss of circadian rhythmic change of SCN neurons in low Ca 2+ Krebs solution. TABLE I E F F E C T OF C A L C I U M C O N C E N T R A T I O N ON S U P R A C H I A S M A T I C N U C L E U S N E U R O N ACTIVITY Type I--*II, a shift of discharge pattern from type I to type 11 by reducing the calcium concentration. No change, no change of discharge pattern. ~, augmentation; T$, augmentation then reduction; $, reduction; - , no change in firing frequency o f neuron. Numbers (n) represent the n u m b e r of neurons recorded. Calcium concentration (mM) 1.3 0.6 0.0
Neuron type (n)
Neuron activity (n)
Type 1 4 I I
No change
l
~
~
-
20 29 18
10 6 1
14 2 0
5 13 10
6 13 9
5 7 0
184
The low C a 2 + Krebs solution used in the present experiment was adequate to abolish the synaptic transmission because we [7] previously reported that only late negative wave in the SCN produced by optic nerve stimulation disappeared in 0.3 mM Ca 2 + plus 10 mM Mg 2 -~ Krebs solution. Therefore, neuronal interaction in the SCN, as revealed by histological studies [3, 4, 11], may be important for the occurrence of a regular firing pattern and the circadian rhythmic change of SCN neurons. Recently, we recorded SCN neurons intracellularly in rat hypothalamic slices [10]. Tetrodotoxin-sensitive fast action potentials had after-hyperpolarization comprised of - 70 to - 80 mV amplitude and the slow component of after-hyperpolarization was blocked by Co 2 +. Hyperpolarizing current pulses produced clear anomalous rectification with rebound depolarization and this anomalous rectification disappeared by replacing Ca 2 + with Mg 2+, Mn 2 ÷ or Cd z + ions. Therefore, the above set of conductance by after-hyperpolarization provided the necessary ionic mechanisms to generate a regular firing pattern like the type I neuron. The rhythmically firing neurons of the dorsal raphe nucleus have afterhyperpolarization, and the latter is thought to be mediated by a Ca 2 ÷ -dependent K + conductance [1]. The disappearance of a regular firing pattern and circadian rhythmic change of SCN neuron activity in low Ca 2 + Krebs solution may be due to the disappearance not only of synaptic transmission but also of after-hyperpolarization. The present investigation demonstrated that calcium ions in medium are important in both the circadian rhythmic change of SCN neuron activity and the regular discharge of SCN neurons. 1 Aghajanian, G.K., Regulation of serotonergic neuronal activity: autoreceptors and pacemaker potentials, Advanc. Biochem. Psychopharmacol., 34 (1982) 173-181. 2 Green, D.J. and Gillette, R., Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 3 GiJldner, F.H. and Wolff, J.R., Dendro-dendritic synapses in the suprachiasmatic nuclei of the rat hypothalamus, J. Neurocytol., 3 (1974) 245-250. 4 Gfildner, F.H. and Wolff, J.R., Self-innervation of dendrites in the rat suprachiasmatic nucleus, Exp. Brain Res., 32 (1978) 77-82. 5 lnouye, S.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic "island' containing the suprachiasmatic nucleus, Proc. Natl. Acad. Sci. USA, 76 (1979) 5962-5966. 6 Shibata, S., kiou, S.Y., Ueki, S. and Oomura, Y., Influence of environmental light-dark and enucleation on activity of suprachiasmatic neurons in slice preparations, Brain Res., 302 (1984) 75-81. 7 Shibata, S., Oomura, Y., Hattori, K. and Kita, H., Responses of suprachiasmatic nucleus neurons lo optic nerve stimulation in rat hypothalamic slice preparation, Brain Res., 302 (1984) 83-89. 8 Shibata, S., Oomura, Y., Kita, H. and Hattori, K., Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res., 247 (1982) 154-158. 9 Sofroniew, M.V. and Weindl, A., Neuroanatomical organization and connections of the suprachiasmatic nucleus. In J. Aschoff, S. Daan and G.A. Groos (Eds.), Vertebrate Circadian Systems, Springer-Verlag, Berlin, 1982, pp. 75-86. 10 Sugimori, M., Shibata, S. and Oomura, Y., Electrophysiological basis for biorhythmic activity in the suprachiasmatic nucleus of the rat: an in vitro study, Soc. Neurosci. Abstr., in press. 11 Van den Pol, A.N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. Comp. Neurol., (1980) 661-702.
181
NSL 03045
THE ROLE OF C A L C I U M IONS IN C I R C A D I A N R H Y T H M OF S U P R A C H I A S M A T I C N U C L E U S N E U R O N A C T I V I T Y I N RAT HYPOTHALAMIC SLICES
SHIGENOBU SHIBATA, AKIKO SHIRATSUCHI, SHYH YUH LIOU and SHOWA UEKI Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka 812 (Japan)
(Received August 13th, 1984; Revised version received and accepted September 17th, 1984)
Key words: suprachiasmatic nucleus - circadian rhythm - calcium ions - single unit activity - rat brain slice - synaptic transmission
The role of calcium ions in maintaining the circadian rhythm of suprachiasmatic nucleus (SCN) neuron activity was investigated using rat hypothalamic slice preparations. In normal Krebs solution, the firing rate of SCN neurons was higher in the light period than in the dark period. In Ca 2÷ -free Krebs solution, SCN neuron activity was low during all periods and did not show diurnal rhythm. These results suggest that the disappearance of circadian rhythmic change of SCN neuron activity in Ca 2÷-free Krebs solution may be due to the disappearance of synaptic transmission in the SCN.
M u l t i p l e unit activity r e c o r d s in the rat s u p r a c h i a s m a t i c nucleus (SCN) have s h o w n c i r c a d i a n r h y t h m i c changes, a n d these changes persisted a f t e r i s o l a t i o n o f the h y p o t h a l a m u s i n c l u d i n g the S C N [5]. In e x p e r i m e n t s in vitro, it was o b s e r v e d t h a t single-unit activity in S C N slices t a k e n f r o m a n i m a l s k e p t on a l i g h t - d a r k cycle (LD) was higher d u r i n g the light t h a n d u r i n g the d a r k p e r i o d [2, 6, 8]. These r e p o r t s suggest t h a t the activity o f S C N n e u r o n s show the c i r c a d i a n r h y t h m even after a f f e r e n t i n p u t s f r o m o t h e r c e n t r a l nuclei [9] were cut. In o r d e r to d e m o n s t r a t e the role o f the i n t e r n e u r o n s a n d a f f e r e n t n e u r o n s in the S C N in the c i r c a d i a n r h y t h m o f S C N n e u r o n activity in vitro, we r e c o r d e d the S C N n e u r o n activity in low C a 2 + K r e b s solution. A t o t a l o f 58 W i s t a r strain rats were used. A n i m a l s were h o u s e d with n o r m a l L D (light p e r i o d 0 7 . 0 0 - 1 9 . 0 0 h) for 2 m o n t h s . F o r p r e p a r i n g the slices, rats were killed b y a b l o w o n the b a c k o f the neck at v a r i o u s times o f day: 08.30, 12.30, 16.30, 20.30, 00.30 a n d 04.30 h; the cerebri were q u i c k l y r e m o v e d f r o m the skull [6, 8]. H y p o t h a l a m i c slices, a b o u t 300 # m thick, i n c l u d i n g the S C N a n d the a n t e r i o r h y p o t h a l a m u s , were t h e n c o r o n a l l y s e c t i o n e d with a v i b r a t o m e . T h e c o m p o s i t i o n o f the c o n t r o l Krebs s o l u t i o n , e q u i l i b r a t e d with 95°/0 O2 a n d 5°/o CO2, was (in m M final c o n c e n t r a t i o n ) : NaC1, 124; KC1, 5; KHEPO4, 1.24; CaC12, 2.4; MgSO4, 1.3; 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
182
NaHCO3, 26; and glucose, 10. Recording electrodes were filled with 2 M NaCI (DC resistance, 2-10 MS2) in Krebs solution. The calcium concentration of low Ca z + Krebs solution ranged between 0 and 1.3 mM, while the magnesium concentration was kept constant (10 mM). Single neuron activity which remained stable for at least 5 min was recorded; the recording was terminated within 8 h after preparing the slice. The mean firing rate was determined during each 4-h period depending on the number of spontaneous active neurons recorded. In our previous study [6], the firing rates o f neurons in the ventrolateral part of the SCN were compared with those in the dorsomedial part, and the firing rates in both areas of SCN taken from animals kept on LD were higher during the light than during the dark period. Therefore, in the present experiment, these two areas were combined to determine the mean firing rate. As mentioned in our previous report [6], the firing patterns of single neuron activity were divided into the following three types: type I, (Fig. 1A) regular firing characterized by a very constant interspike interval; type II, irregular firing (Fig. 1B); and type II1, burst firing (not shown in figure). The influence of low C a 2 + Krebs solution on the occurrence of the three firing patterns and firing rates is shown in Table I. The population of type I decreased and that of type 1I increased in decreasing Ca 2 + ions of the Krebs solution. Fourteen of 30 cells were facilitated by 1.3 mM Ca 2+ Krebs (Fig. ID). Many cells were facilitated then inhibited (Fig. 1C) or inhibited by 0.6 and 0.0 mM Ca 2+ Krebs. The firing rate of SCN neurons in normal Krebs solution was significantly higher in the light period than in the dark period (Fig. 2). On the other hand, in C a 2 + -free Krebs solution, SCN neuron activity was low during all periods and did not show
?•I B
~J
I.
Ca free
(O mM Ca, lO rnM Mr)
1 min
Fig. I. S p o n t a n e o u s n e u r o n a l d i s c h a r g e o f S C N n e u r o n s . A : r e g u l a r firing (type l). 13: i r r e g u l a r firing ( t y p e II). A a n d B: r e c o r d s in n o r m a l K r e b s s o l u t i o n (2.4 m M C a 24 ). C a n d D: effect o f low C a 2 + K r e b s s o l u t i o n o n S C N n e u r o n a c t i v i t y . Bar, a p p l i c a t i o n p e r i o d o f low C a z ' K r e b s s o l u t i o n . C: a shift in the d i s c h a r g e p a t t e r n f r o m t y p e I to t y p e II a n d a d e c r e a s e in the firing r a t e w e r e o b s e r v e d in C a z + -free K r e b s s o l u t i o n . D: a shift in the d i s c h a r g e p a t t e r n f r o m type I to t y p e 11 a n d a n i n c r e a s e in t h e firing r a t e were o b s e r v e d in 0.6 m M C a 2 * K r e b s s o l u t i o n .
183
Dark period
10
o
8
03
I ,,'""
~6
I.......( 467I )
co
T I
I
I
I
I
I
I
13:00
17:00
21:00
01:00
05:00
09:00
13:00
0
• ...... •
Normal
(2.6 mM Ca,1.3 mM Mg)
•
Ca free
(0 mM Ca, lO mM Mg)
•
Fig. 2. Effect of Ca 2 ÷-free Krebs solution on the circadian r h y t h m of neuronal activity in SCN. Each point is the average discharge rate (imp./s) of neurons during each 4-h period, and each point contains 68-103 neurons in normal Krebs solution and 24-53 in Ca 2 ÷ -free Krebs solution. Numbers in parentheses are the total n u m b e r of neurons recorded.
diurnal rhythm. The populations of the type I, II and III in normal Krebs solution were 84.9, 13.5 and 1.5°70 during the light period, and 68.3, 27.4 and 4.3o70 during dark period, respectively. On the other hand, the populations of the three types in Ca2+-free Krebs solution were 28.6, 65.2 and 6.3°70 during the light period, and 25.6, 67.4 and 7.0o-/o during the dark period, respectively. In the present experiment we demonstrated the shift of the discharge pattern from type I to type II and the loss of circadian rhythmic change of SCN neurons in low Ca 2+ Krebs solution. TABLE I E F F E C T OF C A L C I U M C O N C E N T R A T I O N ON S U P R A C H I A S M A T I C N U C L E U S N E U R O N ACTIVITY Type I--*II, a shift of discharge pattern from type I to type 11 by reducing the calcium concentration. No change, no change of discharge pattern. ~, augmentation; T$, augmentation then reduction; $, reduction; - , no change in firing frequency o f neuron. Numbers (n) represent the n u m b e r of neurons recorded. Calcium concentration (mM) 1.3 0.6 0.0
Neuron type (n)
Neuron activity (n)
Type 1 4 I I
No change
l
~
~
-
20 29 18
10 6 1
14 2 0
5 13 10
6 13 9
5 7 0
184
The low C a 2 + Krebs solution used in the present experiment was adequate to abolish the synaptic transmission because we [7] previously reported that only late negative wave in the SCN produced by optic nerve stimulation disappeared in 0.3 mM Ca 2 + plus 10 mM Mg 2 -~ Krebs solution. Therefore, neuronal interaction in the SCN, as revealed by histological studies [3, 4, 11], may be important for the occurrence of a regular firing pattern and the circadian rhythmic change of SCN neurons. Recently, we recorded SCN neurons intracellularly in rat hypothalamic slices [10]. Tetrodotoxin-sensitive fast action potentials had after-hyperpolarization comprised of - 70 to - 80 mV amplitude and the slow component of after-hyperpolarization was blocked by Co 2 +. Hyperpolarizing current pulses produced clear anomalous rectification with rebound depolarization and this anomalous rectification disappeared by replacing Ca 2 + with Mg 2+, Mn 2 ÷ or Cd z + ions. Therefore, the above set of conductance by after-hyperpolarization provided the necessary ionic mechanisms to generate a regular firing pattern like the type I neuron. The rhythmically firing neurons of the dorsal raphe nucleus have afterhyperpolarization, and the latter is thought to be mediated by a Ca 2 ÷ -dependent K + conductance [1]. The disappearance of a regular firing pattern and circadian rhythmic change of SCN neuron activity in low Ca 2 + Krebs solution may be due to the disappearance not only of synaptic transmission but also of after-hyperpolarization. The present investigation demonstrated that calcium ions in medium are important in both the circadian rhythmic change of SCN neuron activity and the regular discharge of SCN neurons. 1 Aghajanian, G.K., Regulation of serotonergic neuronal activity: autoreceptors and pacemaker potentials, Advanc. Biochem. Psychopharmacol., 34 (1982) 173-181. 2 Green, D.J. and Gillette, R., Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 3 GiJldner, F.H. and Wolff, J.R., Dendro-dendritic synapses in the suprachiasmatic nuclei of the rat hypothalamus, J. Neurocytol., 3 (1974) 245-250. 4 Gfildner, F.H. and Wolff, J.R., Self-innervation of dendrites in the rat suprachiasmatic nucleus, Exp. Brain Res., 32 (1978) 77-82. 5 lnouye, S.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic "island' containing the suprachiasmatic nucleus, Proc. Natl. Acad. Sci. USA, 76 (1979) 5962-5966. 6 Shibata, S., kiou, S.Y., Ueki, S. and Oomura, Y., Influence of environmental light-dark and enucleation on activity of suprachiasmatic neurons in slice preparations, Brain Res., 302 (1984) 75-81. 7 Shibata, S., Oomura, Y., Hattori, K. and Kita, H., Responses of suprachiasmatic nucleus neurons lo optic nerve stimulation in rat hypothalamic slice preparation, Brain Res., 302 (1984) 83-89. 8 Shibata, S., Oomura, Y., Kita, H. and Hattori, K., Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res., 247 (1982) 154-158. 9 Sofroniew, M.V. and Weindl, A., Neuroanatomical organization and connections of the suprachiasmatic nucleus. In J. Aschoff, S. Daan and G.A. Groos (Eds.), Vertebrate Circadian Systems, Springer-Verlag, Berlin, 1982, pp. 75-86. 10 Sugimori, M., Shibata, S. and Oomura, Y., Electrophysiological basis for biorhythmic activity in the suprachiasmatic nucleus of the rat: an in vitro study, Soc. Neurosci. Abstr., in press. 11 Van den Pol, A.N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. Comp. Neurol., (1980) 661-702.