Brain Research, 122 (1977) 33--47
33
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
ANALYSIS OF SLEEP-WAKEFULNESS RHYTHMS IN MALE RATS AFTER SUPRACHIASMATIC NUCLEUS LESIONS AND OCULAR ENUCLEATION
NOBUO IBUKA, SHIN-ICHI T. INOUYE and HIROSHI KAWAMURA Laboratory of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, Machida-shi, Tokyo 194 (Japan)
(Accepted June 8th, 1976)
SUMMARY To determine quantitatively characteristics of sleep-wakefulness rhythms in male albino rats, computer analysis of long term polygraphic records (24 h/day) of cortical EEG activity, neck EMG and EOG taken from 23 rats under 12 : 12 lightdark schedule was performed. After bilateral suprachiasmatic nucleus (SCN) lesions, the circadian rhythm in sleep-wakefulness was completely eliminated, although no attenuation or even slight enhancement of the ultradian rhythms with 2-4 h periodicity was observed. After enucleation of both eyes, the circadian rhythm was freerunning with a phase shift in the range from -- 12 to +22 min/day in 6 rats. A gradual decrease of the spectral value of the circadian rhythm and inverse enhancement of the ultradian rhythms with 4--7 h periodicity (predominantly 6 h in 4 out of 6 rats) were also shown. In the spectral diagram, the appearance of paradoxical sleep (PS) paralleled slow-wave sleep (SWS), in the cases of the circadian rhythm and ultradian rhythms with 4-7 h periodicity. Behaviorally blind rats with bilateral primary optic tract (POT) lesions maintained the circadian rhythm in sleep-wakefulness entrained to the environmental light--dark cycle. Power spectral analysis showed no characteristic difference from normal rats. Based on these data, the role of the SCN as a pacemaker of endogenous circadian rhythm in sleep-wakefulness is discussed.
INTRODUCTION The neural control mechanism that regulates diurnal rhythms in hormonal activity of internal millieu as well as animal behavior such as eating, drinking, locomotor activity and sleep-wakefulness was not known until quite recently in mammals 15,26. Moore and Lenntg, Moore 14 clarified retino-hypothalamic fibers terminating in the suprachiasmatic nuclei (SCN) located just above the optic chiasma in the rat and in the other mammals. Hendrickson et al. a also showed similar results.
34 Lesions involving these nuclei abolished the circadian rhythm in drinking and locomotor activity 29, adrenal corticosterone 16 and pineal serotonin N-acetyltransferase activity is in the rat. We have reported in a previous communication 1° the loss of circadian rhythm in sleep-wakefulness after SCN lesions, and indicated the abolition of percentage differences of SWS and PS between the light and dark hours under LD 12 : 12 schedule. In this study, an attempt was made to describe further results from a computer analysis of the changes of the circadian and ultradian rhythms after SCN lesions, bilateral ocular enucleation and primary optic tract (POT) lesions, to clarify their influences on sleep-wakefulness rhythms. MATERIALS AND METHODS Wistar strain male albino rats with body weight ranging from 300 to 390 g were used. Rats were mated and reared in the animal quarter of our Institute. Several of the rats used in the initial stage of the experiments were purchased from Shizuoka Laboratory Animal Coop. Analyzed data were obtained from 4 experimental groups, which were composed of 6 rats with bilateral eye enucleation, 7 with bilateral SCN lesions, 6 with hypothalamic area lesions not involving SCN and 4 with bilateral POT lesions. The operations were performed under pentobarbital anesthesia (40-50 mg/kg, i.p.). Stainless steel screw electrodes for EEG and silver-wire electrodes for neck muscle E M G and EOG recordings were implanted and connected to a 9-pin Amphenol plug which was cemented to the skull. SCN lesions were made by passing 2.5 mA DC current for 30 sec through a pair of insect-pin electrodes, with 0.4 mm in diameter and insulated except for 0.25 mm at the tip, which were implanted stereotaxically using KSnig and Klippel's atlas 11. POT lesions were made through a pair of insect-pin electrodes with 0.5 mm in diameter and tip exposure length 0.3 mm, according to a method described by Moore et al. 17. Because of the relatively large size of the lesions in the case of POT destruction, recording electrodes were not implanted beforehand. Therefore, no control record was taken before the POT lesions. A sound attenuated chamber with an open-top recording box (30 cm × 30 cm × 50 cm) with bedding for the rat was placed on a table which was illuminated by white fluorescent lights under 12 : 12 light--dark schedule. The lights were turned off from 9.00 p.m. to 9.00 next morning. The intensity of illumination was 200 lux on top of the table. Room temperature was kept between 22 and 24 °C. Food and water were available ad libitum. Before starting the recording, each rat was allowed at least 7 days to recover from surgery. The animal was then habituated to the recording environment at least for 2 days. A Nihon-Kohden polygraph with paper speed of 30 mm/min was outside the sound attenuated chamber. Polygraphic records were scored by visual inspection of the data into 3 stages - wakefulness, slow-wave sleep (SWS) and paradoxical sleep (PS). The longest stage in every 20 sec was taken as the representative stage for
35
Rat 25 before
Lesion
5 th
I
I
E',"l,',', ~
E
l
~
[ _ _ ~
r~
E~ ~ l
SCN Lesion I
Ld','~'d~-~-~
Eba,,",u~
after
~ r&
a~LJ;,'L ,,,~
81 st d a y
I
I
E~,~,'," ,,h~',,',' ;-h, E.i'',,,._~
E [Ln
s
,=__n
~ ~
r
II
E
ru
[:':,"~,__~n~
md~Ftt_ E~
m~L.~,
Fig. 1. Sleep stage diagram. Distribution of slow-wave sleep (SWS), paradoxical sleep (PS) and wakefulness (W) before and after SCN lesion. Open bars, light hour; filled bars, dark hours. Each row represents 3 h. From top to bottom, records from 9.00 a.m. to 9.00 a.m. next day. Note sleep occurred predominantly during light hours before SCN lesion. After SCN lesion, distribution of SWS and PS is equal during light and dark hours in each day. that particular period. The score was serially fed into a PDP 12 computer, which could display graphically sleep stage diagrams as shown in Fig. 1, together with displays of the results of statistical calculation of the distribution and percentages of sleep stages in each hour (such as used in Figs. 2, 5, 6). For further analysis of the rhythms, a M E L C O M 7000 general-purpose computer was used. To clarify temporal characteristics of the sleep-wakefulness rhythms, statistical analysis of the power spectrum was employed. For each stage of sleep, a time series was obtained from a proportion of the sleep duration in each 30 min (in some cases 1 h) during consecutive 4 or 5 days. The spectrum was computed by means of Fourier transform of the autocovariance function of the series. For each frequency, the spectrum was proportional to the sum of squared coefficients of sinusoidal components of the frequency, which was interpreted as the intensity of the rhythm. In the case of free-running circadian rhythm after enucleation of the eyes, it was necessary to compute a phase shift of the rhythm. This was defined as a value of the phase of the cross spectrum between the free-running activity and single sinusoidal wave of an exact 1/24 h frequency as a reference. We calculated the averaged phase difference between consecutive 4 or 5 days records and environmental 12 : 12 lightdark cycle. It should be noted that the bandwidth in our spectral diagram is a little greater than 1/24 cycle per hour. Therefore, two peaks lying within a range less than the bandwidth cannot be discriminated. At the completion of each lesion experiment, the rat brain was perfused with 10 ~o formalin and examined histologically, using thionin stained serial 15/~m sections.
36 RAT 25 100Jr
~
Days before Lesion
w
1
--SWS
"l
i
v
Days after SCN Lesion
1,0~I..
4
5
6
7
-ioI
t lr~ 371
A 17
81
'
18
82
'
19
'
20
"
83
Fig. 2. Hourly distribution of SWS and PS before and after SCN lesion. Each point represents percentage of SWS (solid line) and PS (broken line) in each hour. Thick lines on the abscissae indicate dark hours.
RESULTS
(1) SCN lesions A most dramatic change, that revealed itself following small hypothalamic lesions involving bilateral SCN, was the immediate and persistent abolition of the circadian rhythm in sleep-wakefulness. This is apparent even from the simple sleep stage diagram shown in Fig. 1. Sample data taken from rat No. 25 show that before the lesion, a greater portion of sleep was recorded during the light hours, whereas after SCN lesion, the distribution of sleep was equal between the light and dark hours as shown on the 5th and 81st day's records after the lesion. In Fig. 2, distribution of SWS and PS is demonstrated as percentages in each hour. Disappearance of the circadian rhythm immediately after SCN lesion is apparent. This continued up to the end of 83 days of recordings, although the ultradian rhythms of SWS with 2-4 h periodicity were not abolished after the lesion. Power spectral analysis of the sleep rhythms in the rat evidently demonstrated the complete loss of 24 h peak circadian rhythm in both SWS and PS stages after SCN lesion. Fig. 3 illustrates a summary of the data of power spectral analysis taken from 4 rats with SCN lesions which were recorded for a sufficiently long period for computer analysis. Only results of total sleep analysis are shown. In the diagram, a tendency towards enhancement of ultradian rhythms with 2-4 h periodicity and the disappearance of the 24 h periodicity circadian rhythm are obvious after SCN lesions. Appearance of PS was parallel with SWS in the case of the circadian rhythm and ultradian rhythms with 4-7 h periodicity
37
before
lesion
TS
°o2°5
/i '
o :
:
/
0.10
d !
-
Ti i il.~i
~
~--. Rot 20
~
,---4 Rat 37
?~i
ta
OO5
2412 8 6 4 ;3 after SCN lesion . 0.10
?,
2
hours
AA,'A °
. _
x
_
'
2412 8 6
4
3
2
hours
Fig. 3. Power spectrum of total sleep rhythms before and after SCN lesions. Summarized data from 4 rats. Tendency of enhancement of the ultradian rhythms with 2--4 h periodicity is apparent. Circadian rhythms before SCN lesions are not shown, because of their very high intensities. Ordinates: arbitrary unit used in all spectral intensity in this paper. Abscissae: period.
in the spectral diagram. In the ultradian rhythms with 2-4 h periodicity, they were not always parallel. Although, the power spectral diagram is not shown, this can easily be recognized in the percentage diagram of PS and SWS shown in Fig. 2. Fig. 4 illustrates the extent of the lesions involving SCN taken from 3 rats which had relatively smaller hypothalamic lesions. In all 7 cases when SCN lesions resulted in the abolition of the circadian rhythm, SCN was always completely destroyed bilaterally. Equal distribution of SWS and PS during the light and dark hours in each day was very characteristic after SCN lesions. Rats with SCN lesions lost their circadian rhythm immediately after surgery and it was not restored until the end of the recordings, which lasted up to 3 months. However, the amount of sleep (total, SWS and PS) in a day did not change before and after SCN lesions. As shown in Table I, in rat No. 25 as an example, the average amount of total sleep per day calculated from 4 consecutive days' records was 603.5 min before SCN lesion and an average of all 3 postoperative periods was 592.4 min/day. Summing up the results obtained from the 7 rats with SCN lesions, the total amount of daily sleep in both SWS and PS stages did not change significantly before and after lesions
38
f~
Fig. 4. Size of hypothalamic lesions involving bilateral SCN taken from 3 rats. Two blank areas inside hatched lesion areas illustrate SCN. Vertical hatching demonstrates hypothalamic lesion in the rat No. 15; oblique hatching, the rat No. 25; and horizontal hatching, the rat No. 39, respectively. A, B and C: rostral, middle and caudal portion of the SCN. according to a statistical test (in SWS, t ~ 1.286, df -----6, P > 0.05; in PS, t ~ 1.678, d f ---- 6, P > 0.05). O n the contrary, anterior h y p o t h a l a m i c area ( A H A ) lesions close to SCN or involving unilateral S C N affected to some extent the d i s t r i b u t i o n of SWS a n d PS i n a day, d i m i n i s h i n g the difference of a m o u n t of SWS a n d PS between the light
39 TABLE I Changes of sleep amounts after brain lesions and blinding
After surgery, averaged amounts of sleep in each period of postoperative days are shown (not necessarily 4 but 2-5 days). PS/TS: proportion of paradoxical sleep to total sleep (SWS + PS) in 24-h records. Control(average of 4 days)
After surgery
SWS (min)
post-op. SWS (min) days 12L 12D
12L
12D
PS (rain) PS/TS (%) - 24 h 12L 12D
PS (rain) PS[TS (%) 24 h 12L 12D
SCNlesion (rat No. 25)
420.8 85.5 76.6 20.5 16.1
4-7 17-20 81-83
224.7 248.4 50.6 49.9 17.5 219.8 258.3 38.7 46.8 15.2 292.6 246.2 52.1 49.7 15.9
AHA lesion (rat No. 17)
443.5 152.6 91.4 23.0 16.1
4-6 13-14 25-27
375.1 253.4 87.1 44.6 17.3 339.8 223.2 85.7 33.1 17.4 426.9 188.6 97.9 22.3 16.3
POT lesion (rat No. 34)
no control recording
15-16 28-30 48-52
363.2 117.7 86.0 20.2 18.1 378.7 151.2 75.6 29.0 16.5 386.8 140.4 71.7 26.4 15.7
Blinding (rat No. 44)
437.3 142.3 90.0 24.9 16.5
9-13 357.7 183.6 36--40 242.9 350.9 57-61 309.7 289.7 71-75 362.6 240.9
94.3 35.3 70.4 71.3
31.4 79.1 43.5 36.0
18.8 16.2 16.0 15.1
and dark hours. A case of the rat No. 17 is also shown in Table I. In such cases, the amplitude of 24 h periodicity in the power spectrum was attenuated but gradually recovered its amplitude to the prelesion level. (2) P O T lesions
When bilateral P O T were destroyed at the level where the optic tract sent its fibers to. the lateral geniculate body, pretectum and superior colliculus, the rat had dilated pupils and could not respond to visual stimulus which moved in front of the eyes. As shown in Fig. 5 (rat No. 34), no change of the circadian rhythm in SWS a n d PS could be seen. There was no free-running of the circadian rhythm in sleep--wakefulness, unlike in the rats with enucleation of bilateral eyes, even though they seemed behaviorally blind. To examine blindness more thoroughly, light-dark discrimination test in the modified Y-shaped two choice apparatus was carried out in rats No. 34 and No. 35. The testing apparatus was composed of a starting box and adjacent two goal boxes with a swing-door in each box. One of the door-panels was painted black and another was white. A protruded partition panel separated these two doors. A rat had to push a correct door (either black or white) with his nose to get a reward food pellet. When light-dark discrimination was impaired, the rat was unable to push the correct door successfully. Unfortunately, however, both rats failed to pass through the swing-door in pretraining stage even after many trials, running frequently against and hitting the protruded partition panel of the choice
40 Rat 34 Days after POT Lesion
~q
--Sw5 ---
p$
u')
15
16
~;8
/+9
++
~so o 1o0 a °I.
_~so o lOO %
'
5O
51
52
Fig. 5. Hourly distribution of SWS and PS after POT lesion. Circadian rhythm is entrained to the environmental light-dark cycle without deterioration by the 52nd day.
point. For this reason, the test was discontinued. Normal control rats learned the light-dark discrimination to criterion (90 ~o in consecutive two days) in 70-100 trials. No significant difference from normal rats was revealed in the power spectral diagrams. In the other two rats with POT lesions, similar results were obtained. The rats with POT lesions clearly entrain their circadian rhythm in sleep-wakefulness to the environmental light-dark cycle in both SWS and PS. (3) Enucleation of bilateral eyes Fig. 6 illustrates a diagram of the hourly distribution of sleep for each day taken from long term records of the rat No. 44. Before enucleation, a larger amount of sleep was obvious during the light hours compared to the dark hours. On the 27th day after ocular enucleation, almost equal distribution of SWS is seen both in the dark and light hours. On the 40th day, apparently the distribution of sleep during the light and dark hours reversed and a larger amount of sleep was shown during the dark hours. Thus, phase shift of the circadian rhythm is obvious, clearly indicating free-running of the circadian rhythm. Averaged daily delay of the circadian rhythm compared to exact 24 h cycle was 19.7 min during the recording lasted 96 days, although daily delay was not always quite uniform. Spectral value for 6 h periodicity increased as though it has an inverse relationship to the attenuation of the circadian rhythm. This is shown in Fig. 7 taken from rat No. 44. In Fig. 8, a summary of the data demonstrating a phase shift of free-running circadian rhythm taken from 6 rats after blinding is shown. Only rat No. 4 shows advancement of the phase of the circadian rhythm (average free-running periodicity was 23 h 48 rain in 16 days). Attenuation of the circadian rhythm indicated as a decrease of the power spectral intensity is also shown in Fig. 9. In the rat No. 4, the intensity of the circadian
• RAT 44
0 8O
Do
b e f o r e Blinding
2
0
:
'
~ SWS .... PS
3
'
4
'
g
'
1(I
'
11
"
12
'
UU" 0.% ~50' ,V)
0
64
65
66
67
68
73
74
'
96
"
G,50' U~
o
8.,O"
71
'
72
93
'
94
75
r,5o.
o ~50' (/1
'
95
'
Fig. 6. Distribution of SWS and PS before and after enucleation of both eyes recorded more than 3 months. Free-running activity of the circadian rhythm is clearly shown. At the end of 96 days recording after enucleation, the circadian rhythm became less marked.
42
Rot 44
/
0J0~" A
|
PS
before blinding,
/ \
after blinding
I//~ U
,,
~--'~ /,
2'/.
0.5-
/~
SWS
'"~
33
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
ANALYSIS OF SLEEP-WAKEFULNESS RHYTHMS IN MALE RATS AFTER SUPRACHIASMATIC NUCLEUS LESIONS AND OCULAR ENUCLEATION
NOBUO IBUKA, SHIN-ICHI T. INOUYE and HIROSHI KAWAMURA Laboratory of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, Machida-shi, Tokyo 194 (Japan)
(Accepted June 8th, 1976)
SUMMARY To determine quantitatively characteristics of sleep-wakefulness rhythms in male albino rats, computer analysis of long term polygraphic records (24 h/day) of cortical EEG activity, neck EMG and EOG taken from 23 rats under 12 : 12 lightdark schedule was performed. After bilateral suprachiasmatic nucleus (SCN) lesions, the circadian rhythm in sleep-wakefulness was completely eliminated, although no attenuation or even slight enhancement of the ultradian rhythms with 2-4 h periodicity was observed. After enucleation of both eyes, the circadian rhythm was freerunning with a phase shift in the range from -- 12 to +22 min/day in 6 rats. A gradual decrease of the spectral value of the circadian rhythm and inverse enhancement of the ultradian rhythms with 4--7 h periodicity (predominantly 6 h in 4 out of 6 rats) were also shown. In the spectral diagram, the appearance of paradoxical sleep (PS) paralleled slow-wave sleep (SWS), in the cases of the circadian rhythm and ultradian rhythms with 4-7 h periodicity. Behaviorally blind rats with bilateral primary optic tract (POT) lesions maintained the circadian rhythm in sleep-wakefulness entrained to the environmental light--dark cycle. Power spectral analysis showed no characteristic difference from normal rats. Based on these data, the role of the SCN as a pacemaker of endogenous circadian rhythm in sleep-wakefulness is discussed.
INTRODUCTION The neural control mechanism that regulates diurnal rhythms in hormonal activity of internal millieu as well as animal behavior such as eating, drinking, locomotor activity and sleep-wakefulness was not known until quite recently in mammals 15,26. Moore and Lenntg, Moore 14 clarified retino-hypothalamic fibers terminating in the suprachiasmatic nuclei (SCN) located just above the optic chiasma in the rat and in the other mammals. Hendrickson et al. a also showed similar results.
34 Lesions involving these nuclei abolished the circadian rhythm in drinking and locomotor activity 29, adrenal corticosterone 16 and pineal serotonin N-acetyltransferase activity is in the rat. We have reported in a previous communication 1° the loss of circadian rhythm in sleep-wakefulness after SCN lesions, and indicated the abolition of percentage differences of SWS and PS between the light and dark hours under LD 12 : 12 schedule. In this study, an attempt was made to describe further results from a computer analysis of the changes of the circadian and ultradian rhythms after SCN lesions, bilateral ocular enucleation and primary optic tract (POT) lesions, to clarify their influences on sleep-wakefulness rhythms. MATERIALS AND METHODS Wistar strain male albino rats with body weight ranging from 300 to 390 g were used. Rats were mated and reared in the animal quarter of our Institute. Several of the rats used in the initial stage of the experiments were purchased from Shizuoka Laboratory Animal Coop. Analyzed data were obtained from 4 experimental groups, which were composed of 6 rats with bilateral eye enucleation, 7 with bilateral SCN lesions, 6 with hypothalamic area lesions not involving SCN and 4 with bilateral POT lesions. The operations were performed under pentobarbital anesthesia (40-50 mg/kg, i.p.). Stainless steel screw electrodes for EEG and silver-wire electrodes for neck muscle E M G and EOG recordings were implanted and connected to a 9-pin Amphenol plug which was cemented to the skull. SCN lesions were made by passing 2.5 mA DC current for 30 sec through a pair of insect-pin electrodes, with 0.4 mm in diameter and insulated except for 0.25 mm at the tip, which were implanted stereotaxically using KSnig and Klippel's atlas 11. POT lesions were made through a pair of insect-pin electrodes with 0.5 mm in diameter and tip exposure length 0.3 mm, according to a method described by Moore et al. 17. Because of the relatively large size of the lesions in the case of POT destruction, recording electrodes were not implanted beforehand. Therefore, no control record was taken before the POT lesions. A sound attenuated chamber with an open-top recording box (30 cm × 30 cm × 50 cm) with bedding for the rat was placed on a table which was illuminated by white fluorescent lights under 12 : 12 light--dark schedule. The lights were turned off from 9.00 p.m. to 9.00 next morning. The intensity of illumination was 200 lux on top of the table. Room temperature was kept between 22 and 24 °C. Food and water were available ad libitum. Before starting the recording, each rat was allowed at least 7 days to recover from surgery. The animal was then habituated to the recording environment at least for 2 days. A Nihon-Kohden polygraph with paper speed of 30 mm/min was outside the sound attenuated chamber. Polygraphic records were scored by visual inspection of the data into 3 stages - wakefulness, slow-wave sleep (SWS) and paradoxical sleep (PS). The longest stage in every 20 sec was taken as the representative stage for
35
Rat 25 before
Lesion
5 th
I
I
E',"l,',', ~
E
l
~
[ _ _ ~
r~
E~ ~ l
SCN Lesion I
Ld','~'d~-~-~
Eba,,",u~
after
~ r&
a~LJ;,'L ,,,~
81 st d a y
I
I
E~,~,'," ,,h~',,',' ;-h, E.i'',,,._~
E [Ln
s
,=__n
~ ~
r
II
E
ru
[:':,"~,__~n~
md~Ftt_ E~
m~L.~,
Fig. 1. Sleep stage diagram. Distribution of slow-wave sleep (SWS), paradoxical sleep (PS) and wakefulness (W) before and after SCN lesion. Open bars, light hour; filled bars, dark hours. Each row represents 3 h. From top to bottom, records from 9.00 a.m. to 9.00 a.m. next day. Note sleep occurred predominantly during light hours before SCN lesion. After SCN lesion, distribution of SWS and PS is equal during light and dark hours in each day. that particular period. The score was serially fed into a PDP 12 computer, which could display graphically sleep stage diagrams as shown in Fig. 1, together with displays of the results of statistical calculation of the distribution and percentages of sleep stages in each hour (such as used in Figs. 2, 5, 6). For further analysis of the rhythms, a M E L C O M 7000 general-purpose computer was used. To clarify temporal characteristics of the sleep-wakefulness rhythms, statistical analysis of the power spectrum was employed. For each stage of sleep, a time series was obtained from a proportion of the sleep duration in each 30 min (in some cases 1 h) during consecutive 4 or 5 days. The spectrum was computed by means of Fourier transform of the autocovariance function of the series. For each frequency, the spectrum was proportional to the sum of squared coefficients of sinusoidal components of the frequency, which was interpreted as the intensity of the rhythm. In the case of free-running circadian rhythm after enucleation of the eyes, it was necessary to compute a phase shift of the rhythm. This was defined as a value of the phase of the cross spectrum between the free-running activity and single sinusoidal wave of an exact 1/24 h frequency as a reference. We calculated the averaged phase difference between consecutive 4 or 5 days records and environmental 12 : 12 lightdark cycle. It should be noted that the bandwidth in our spectral diagram is a little greater than 1/24 cycle per hour. Therefore, two peaks lying within a range less than the bandwidth cannot be discriminated. At the completion of each lesion experiment, the rat brain was perfused with 10 ~o formalin and examined histologically, using thionin stained serial 15/~m sections.
36 RAT 25 100Jr
~
Days before Lesion
w
1
--SWS
"l
i
v
Days after SCN Lesion
1,0~I..
4
5
6
7
-ioI
t lr~ 371
A 17
81
'
18
82
'
19
'
20
"
83
Fig. 2. Hourly distribution of SWS and PS before and after SCN lesion. Each point represents percentage of SWS (solid line) and PS (broken line) in each hour. Thick lines on the abscissae indicate dark hours.
RESULTS
(1) SCN lesions A most dramatic change, that revealed itself following small hypothalamic lesions involving bilateral SCN, was the immediate and persistent abolition of the circadian rhythm in sleep-wakefulness. This is apparent even from the simple sleep stage diagram shown in Fig. 1. Sample data taken from rat No. 25 show that before the lesion, a greater portion of sleep was recorded during the light hours, whereas after SCN lesion, the distribution of sleep was equal between the light and dark hours as shown on the 5th and 81st day's records after the lesion. In Fig. 2, distribution of SWS and PS is demonstrated as percentages in each hour. Disappearance of the circadian rhythm immediately after SCN lesion is apparent. This continued up to the end of 83 days of recordings, although the ultradian rhythms of SWS with 2-4 h periodicity were not abolished after the lesion. Power spectral analysis of the sleep rhythms in the rat evidently demonstrated the complete loss of 24 h peak circadian rhythm in both SWS and PS stages after SCN lesion. Fig. 3 illustrates a summary of the data of power spectral analysis taken from 4 rats with SCN lesions which were recorded for a sufficiently long period for computer analysis. Only results of total sleep analysis are shown. In the diagram, a tendency towards enhancement of ultradian rhythms with 2-4 h periodicity and the disappearance of the 24 h periodicity circadian rhythm are obvious after SCN lesions. Appearance of PS was parallel with SWS in the case of the circadian rhythm and ultradian rhythms with 4-7 h periodicity
37
before
lesion
TS
°o2°5
/i '
o :
:
/
0.10
d !
-
Ti i il.~i
~
~--. Rot 20
~
,---4 Rat 37
?~i
ta
OO5
2412 8 6 4 ;3 after SCN lesion . 0.10
?,
2
hours
AA,'A °
. _
x
_
'
2412 8 6
4
3
2
hours
Fig. 3. Power spectrum of total sleep rhythms before and after SCN lesions. Summarized data from 4 rats. Tendency of enhancement of the ultradian rhythms with 2--4 h periodicity is apparent. Circadian rhythms before SCN lesions are not shown, because of their very high intensities. Ordinates: arbitrary unit used in all spectral intensity in this paper. Abscissae: period.
in the spectral diagram. In the ultradian rhythms with 2-4 h periodicity, they were not always parallel. Although, the power spectral diagram is not shown, this can easily be recognized in the percentage diagram of PS and SWS shown in Fig. 2. Fig. 4 illustrates the extent of the lesions involving SCN taken from 3 rats which had relatively smaller hypothalamic lesions. In all 7 cases when SCN lesions resulted in the abolition of the circadian rhythm, SCN was always completely destroyed bilaterally. Equal distribution of SWS and PS during the light and dark hours in each day was very characteristic after SCN lesions. Rats with SCN lesions lost their circadian rhythm immediately after surgery and it was not restored until the end of the recordings, which lasted up to 3 months. However, the amount of sleep (total, SWS and PS) in a day did not change before and after SCN lesions. As shown in Table I, in rat No. 25 as an example, the average amount of total sleep per day calculated from 4 consecutive days' records was 603.5 min before SCN lesion and an average of all 3 postoperative periods was 592.4 min/day. Summing up the results obtained from the 7 rats with SCN lesions, the total amount of daily sleep in both SWS and PS stages did not change significantly before and after lesions
38
f~
Fig. 4. Size of hypothalamic lesions involving bilateral SCN taken from 3 rats. Two blank areas inside hatched lesion areas illustrate SCN. Vertical hatching demonstrates hypothalamic lesion in the rat No. 15; oblique hatching, the rat No. 25; and horizontal hatching, the rat No. 39, respectively. A, B and C: rostral, middle and caudal portion of the SCN. according to a statistical test (in SWS, t ~ 1.286, df -----6, P > 0.05; in PS, t ~ 1.678, d f ---- 6, P > 0.05). O n the contrary, anterior h y p o t h a l a m i c area ( A H A ) lesions close to SCN or involving unilateral S C N affected to some extent the d i s t r i b u t i o n of SWS a n d PS i n a day, d i m i n i s h i n g the difference of a m o u n t of SWS a n d PS between the light
39 TABLE I Changes of sleep amounts after brain lesions and blinding
After surgery, averaged amounts of sleep in each period of postoperative days are shown (not necessarily 4 but 2-5 days). PS/TS: proportion of paradoxical sleep to total sleep (SWS + PS) in 24-h records. Control(average of 4 days)
After surgery
SWS (min)
post-op. SWS (min) days 12L 12D
12L
12D
PS (rain) PS/TS (%) - 24 h 12L 12D
PS (rain) PS[TS (%) 24 h 12L 12D
SCNlesion (rat No. 25)
420.8 85.5 76.6 20.5 16.1
4-7 17-20 81-83
224.7 248.4 50.6 49.9 17.5 219.8 258.3 38.7 46.8 15.2 292.6 246.2 52.1 49.7 15.9
AHA lesion (rat No. 17)
443.5 152.6 91.4 23.0 16.1
4-6 13-14 25-27
375.1 253.4 87.1 44.6 17.3 339.8 223.2 85.7 33.1 17.4 426.9 188.6 97.9 22.3 16.3
POT lesion (rat No. 34)
no control recording
15-16 28-30 48-52
363.2 117.7 86.0 20.2 18.1 378.7 151.2 75.6 29.0 16.5 386.8 140.4 71.7 26.4 15.7
Blinding (rat No. 44)
437.3 142.3 90.0 24.9 16.5
9-13 357.7 183.6 36--40 242.9 350.9 57-61 309.7 289.7 71-75 362.6 240.9
94.3 35.3 70.4 71.3
31.4 79.1 43.5 36.0
18.8 16.2 16.0 15.1
and dark hours. A case of the rat No. 17 is also shown in Table I. In such cases, the amplitude of 24 h periodicity in the power spectrum was attenuated but gradually recovered its amplitude to the prelesion level. (2) P O T lesions
When bilateral P O T were destroyed at the level where the optic tract sent its fibers to. the lateral geniculate body, pretectum and superior colliculus, the rat had dilated pupils and could not respond to visual stimulus which moved in front of the eyes. As shown in Fig. 5 (rat No. 34), no change of the circadian rhythm in SWS a n d PS could be seen. There was no free-running of the circadian rhythm in sleep--wakefulness, unlike in the rats with enucleation of bilateral eyes, even though they seemed behaviorally blind. To examine blindness more thoroughly, light-dark discrimination test in the modified Y-shaped two choice apparatus was carried out in rats No. 34 and No. 35. The testing apparatus was composed of a starting box and adjacent two goal boxes with a swing-door in each box. One of the door-panels was painted black and another was white. A protruded partition panel separated these two doors. A rat had to push a correct door (either black or white) with his nose to get a reward food pellet. When light-dark discrimination was impaired, the rat was unable to push the correct door successfully. Unfortunately, however, both rats failed to pass through the swing-door in pretraining stage even after many trials, running frequently against and hitting the protruded partition panel of the choice
40 Rat 34 Days after POT Lesion
~q
--Sw5 ---
p$
u')
15
16
~;8
/+9
++
~so o 1o0 a °I.
_~so o lOO %
'
5O
51
52
Fig. 5. Hourly distribution of SWS and PS after POT lesion. Circadian rhythm is entrained to the environmental light-dark cycle without deterioration by the 52nd day.
point. For this reason, the test was discontinued. Normal control rats learned the light-dark discrimination to criterion (90 ~o in consecutive two days) in 70-100 trials. No significant difference from normal rats was revealed in the power spectral diagrams. In the other two rats with POT lesions, similar results were obtained. The rats with POT lesions clearly entrain their circadian rhythm in sleep-wakefulness to the environmental light-dark cycle in both SWS and PS. (3) Enucleation of bilateral eyes Fig. 6 illustrates a diagram of the hourly distribution of sleep for each day taken from long term records of the rat No. 44. Before enucleation, a larger amount of sleep was obvious during the light hours compared to the dark hours. On the 27th day after ocular enucleation, almost equal distribution of SWS is seen both in the dark and light hours. On the 40th day, apparently the distribution of sleep during the light and dark hours reversed and a larger amount of sleep was shown during the dark hours. Thus, phase shift of the circadian rhythm is obvious, clearly indicating free-running of the circadian rhythm. Averaged daily delay of the circadian rhythm compared to exact 24 h cycle was 19.7 min during the recording lasted 96 days, although daily delay was not always quite uniform. Spectral value for 6 h periodicity increased as though it has an inverse relationship to the attenuation of the circadian rhythm. This is shown in Fig. 7 taken from rat No. 44. In Fig. 8, a summary of the data demonstrating a phase shift of free-running circadian rhythm taken from 6 rats after blinding is shown. Only rat No. 4 shows advancement of the phase of the circadian rhythm (average free-running periodicity was 23 h 48 rain in 16 days). Attenuation of the circadian rhythm indicated as a decrease of the power spectral intensity is also shown in Fig. 9. In the rat No. 4, the intensity of the circadian
• RAT 44
0 8O
Do
b e f o r e Blinding
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:
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~ SWS .... PS
3
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4
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g
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11
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12
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0
64
65
66
67
68
73
74
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96
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71
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72
93
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94
75
r,5o.
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95
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Fig. 6. Distribution of SWS and PS before and after enucleation of both eyes recorded more than 3 months. Free-running activity of the circadian rhythm is clearly shown. At the end of 96 days recording after enucleation, the circadian rhythm became less marked.
42
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