76
Brain Research, 96 (1975) 76-81 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Loss of circadian rhythm in sleep-wakefulness cycle in the rat by suprachiasmatic nucleus lesions
NOBUO IBUKA AND HIROSHI KAWAMURA Laboratory of Neurophysiology, Mitsubisbi-Kasei Institute of Life Sciences, Machida-shi, Tokyo 194 (Japan)
(Accepted June 3rd, 1975)
Rats exposed to environmental illumination with the normal day-night cycle exhibit the greater portion of each day's sleep during the light hours 1,6. Stephan and Zucker 7 demonstrated that bilateral lesions of the suprachiasmatic nucleus (SCN) in rats permanently eliminated circadian rhythm in drinking behavior and locomotor activity which were significantly more frequent during dark hours before the lesions. A possible anatomical basis of this phenomenon demonstrating a retinohypothalamic projection in the rat was described by Moore and Lenn 5. Loss of circadian adrenocorticosterone rhythm after SCN lesions was also reported by Moore and Eichler 4. However, the effects of SCN lesions on the sleep-wakefulness cycle, especially whether the lesions influence specifically the amount of slow-wave sleep or paradoxical sleep, have thus far not been shown. In this communication the effects of bilateral SCN lesions as well as bilateral enucleation on the sleep-wakefulness cycle in rats will be reported. Male albino rats weighing 300-380 g were used. Surgery was performed under pentobarbital anesthesia (40-50 mg/kg, i.p.). Complete bilateral electrolytic SCN lesions were confirmed by post mortem histological examination in 4 rats. Enucleation was performed in two rats. Control electrolytic lesions were made in the anterior hypothalamic area in 5 rats. Cortical EEG activity was recorded with stainless steel screw electrodes placed in the frontal and occipital areas of the skull. For recording E M G from neck muscles and EOG, silver needle electrodes were used. These electrodes were connected to a 9-pin Amphenol plug which was cemented to the skull. At least 7 days recovery time elapsed after implantation before placing the rat in a recording room. Forty-eight hours habituation time before starting recording was allowed after placing the rat in a sound attenuated box in which food pellets and water were available ad libitum. For brain stem lesions, a pair of insect pin electrodes insulated except for their tips (0.25 mm, distance between tips, 0.6-0.8 mm) were implanted stereotaxically using K6nig and Klippel's atlas 3. After control recording, electrolytic lesion was made
77 passing DC current of 2.5 mA for 30 sec through these electrodes under pentobarbital anesthesia. Illumination was controlled automatically with a 12/12 light--dark schedule (light was turned off from 9.00 p.m. to 9.00 a.m.). On the table where the rat was placed in the deep box, intensity of the illumination was 200 lux. Room temperature was kept between 22 and 24 °C. Within this range of the fluctuation of temperature, no significant effect on the circadian rhythm of the rat in the sleep-wakefulness cycle was shown. The slow paper speed (3 cm/min) polygraph records were divided into 3 stages: wakefulness with low-voltage fast EEG and high EMG activity, slow-wave sleep stage with high-amplitude slow-wave EEG, and paradoxical sleep stage with lowvoltage fast EEG and bursts of rapid eye movement in EOG with abolition of EMG activity except occasional twitches. The sum of the duration of each of the 3 stages were expressed as percentage in each hour. Most significant change observed after SCN lesions was the abolition of the difference in the amount of total sleep (slow-wave sleep plus paradoxical sleep) between light hours and dark hours. As shown in Fig. 1, consecutive 4 days' records before lesion indicated a high amount (69.2 ~) of total sleep per 12 h during illumination and a low amount (34.3 ~o) during dark hours. However, immediately after recovery from anesthesia, circadian rhythm of the Rat 15 Light
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Days before
Brain Research, 96 (1975) 76-81 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Loss of circadian rhythm in sleep-wakefulness cycle in the rat by suprachiasmatic nucleus lesions
NOBUO IBUKA AND HIROSHI KAWAMURA Laboratory of Neurophysiology, Mitsubisbi-Kasei Institute of Life Sciences, Machida-shi, Tokyo 194 (Japan)
(Accepted June 3rd, 1975)
Rats exposed to environmental illumination with the normal day-night cycle exhibit the greater portion of each day's sleep during the light hours 1,6. Stephan and Zucker 7 demonstrated that bilateral lesions of the suprachiasmatic nucleus (SCN) in rats permanently eliminated circadian rhythm in drinking behavior and locomotor activity which were significantly more frequent during dark hours before the lesions. A possible anatomical basis of this phenomenon demonstrating a retinohypothalamic projection in the rat was described by Moore and Lenn 5. Loss of circadian adrenocorticosterone rhythm after SCN lesions was also reported by Moore and Eichler 4. However, the effects of SCN lesions on the sleep-wakefulness cycle, especially whether the lesions influence specifically the amount of slow-wave sleep or paradoxical sleep, have thus far not been shown. In this communication the effects of bilateral SCN lesions as well as bilateral enucleation on the sleep-wakefulness cycle in rats will be reported. Male albino rats weighing 300-380 g were used. Surgery was performed under pentobarbital anesthesia (40-50 mg/kg, i.p.). Complete bilateral electrolytic SCN lesions were confirmed by post mortem histological examination in 4 rats. Enucleation was performed in two rats. Control electrolytic lesions were made in the anterior hypothalamic area in 5 rats. Cortical EEG activity was recorded with stainless steel screw electrodes placed in the frontal and occipital areas of the skull. For recording E M G from neck muscles and EOG, silver needle electrodes were used. These electrodes were connected to a 9-pin Amphenol plug which was cemented to the skull. At least 7 days recovery time elapsed after implantation before placing the rat in a recording room. Forty-eight hours habituation time before starting recording was allowed after placing the rat in a sound attenuated box in which food pellets and water were available ad libitum. For brain stem lesions, a pair of insect pin electrodes insulated except for their tips (0.25 mm, distance between tips, 0.6-0.8 mm) were implanted stereotaxically using K6nig and Klippel's atlas 3. After control recording, electrolytic lesion was made
77 passing DC current of 2.5 mA for 30 sec through these electrodes under pentobarbital anesthesia. Illumination was controlled automatically with a 12/12 light--dark schedule (light was turned off from 9.00 p.m. to 9.00 a.m.). On the table where the rat was placed in the deep box, intensity of the illumination was 200 lux. Room temperature was kept between 22 and 24 °C. Within this range of the fluctuation of temperature, no significant effect on the circadian rhythm of the rat in the sleep-wakefulness cycle was shown. The slow paper speed (3 cm/min) polygraph records were divided into 3 stages: wakefulness with low-voltage fast EEG and high EMG activity, slow-wave sleep stage with high-amplitude slow-wave EEG, and paradoxical sleep stage with lowvoltage fast EEG and bursts of rapid eye movement in EOG with abolition of EMG activity except occasional twitches. The sum of the duration of each of the 3 stages were expressed as percentage in each hour. Most significant change observed after SCN lesions was the abolition of the difference in the amount of total sleep (slow-wave sleep plus paradoxical sleep) between light hours and dark hours. As shown in Fig. 1, consecutive 4 days' records before lesion indicated a high amount (69.2 ~) of total sleep per 12 h during illumination and a low amount (34.3 ~o) during dark hours. However, immediately after recovery from anesthesia, circadian rhythm of the Rat 15 Light
/ ,'
II
II
II
'I
Dark
°h d S
r ~
-,r
0 Light
. . . . Dark
m~
iI
4
3
2
1
1
II
5
6
7
12 13 1/4 15"27 28 29"32 33 3/* 35 36"60 61 62 63
Days before
