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Sleep-promoting effect following intracerebroventricular injection of a phosphorylated analogue of delta sleep-inducing peptide (DSIP-P) in rats
Elsevier Scientilic Publishers Ireland I i,.{
NSL 05506
Sleep-promoting effect following intracerebroventricular injection of a phosphorylated analogue of delta sleep-inducing peptide (DSIP-P) in rats K e i k o N a k a g a k i , Shigemitsu Ebihara, S e t s u o U s u i , Y o s h i k o H o n d a and Yasuro Takahashi Department o[Psychology, Tokyo Metropolitan Institute[or Neuroseiences, Fuchu Cit)', Tokvo (Japan) (Received 4 January 1988; Revised version received 21 April 1988; Accepted 25 April 1988)
Key u'ord~v Delta sleep-inducing peptide; Slow wave sleep; Paradoxical sleep; lntracerebroventricular injection: Rat The effect of phosphorylated delta sleep-inducing peptide (DSIP-P) on sleep of rats was studied. DSIP-P (20 or 200 pmol/kg) was injected into the third cerebroventricle of male rats immediately before the onset of the dark period of a 12:12 h light-dark cycle. DSIP-P resulted in increases of slow-wave sleep (SWS) (17.3 %, P < 0.01) and paradoxical sleep (PS) (32.3 %, P < 0.05) during the subsequent dark period without shortening sleep latency in the dose of 200 pmol/kg. The SWS-promoting effect was carried over to the next light period. These changes returned to control levels on the second day. These results indicate that DSIP-P is a tong-lasting sleep-promoting substance in rats,
Delta sleep-inducing peptide (DSIP) was isolated from the dialysates of cerebral venous blood taken from rabbits receiving electrical stimulation of the thalamus [8]. DSIP has been reported to have sleep-inducing and -maintaining properties in several mammals and in man [3]. DSIP-P is an analogue of DSIP phosphorylated at the serine in position 7 and was proved to exist naturally in the rat brain [1]. Although it was reported that DSIP-P influenced the circadian locomotor activity rhythm of rats [2], there has so far been no report about the effect on rat sleep except for Kimura et al.'s abstract [6]. The present investigation was undertaken to examine the effect of DSIP-P on rat sleep. The DSIP-P used in this study was supplied by Dr. Schoenenberger, Switzerland. Six male Sprague-Dawley rats weighing 300-390g were chronically implanted with electrodes for cortical EEG and nuchal EMG and with a guide cannula (22
Correspondence: Y. Takahashi, Department of Psychology, Tokyo Metropolitan Institute for Neurosciences, Fuchu City, Tokyo 183, Japan, 0304-3940/881'$ 03.50 (D 1988 Elsevier Scientific Publishers Ireland Ltd.
161
gauge) into the 3rd cerebroventricle at the coordinates A: 1 mm posterior to the bregma, L: 0, V: dura - 7 mm. After surgery, the rats were housed individually in the recording cage in a sound-attenuated and electrically shielded room. These rats were handled daily and adapted to small boxes for injection. At least 2 weeks were allowed lk)r recovery before the experiments were started. The experimental environment was maintained at a temperature of 25-+- I~'C and a humidity of 40 +_ 5 % oll ~l 12:12 h light dark cycle (lights on at 06.00 h). Laboratory chow and water were supplied ad libitum. The rats were transferred to the small boxes for injection immediately before the onset of the dark period. Ten ltl of DSIP-P (20 or 200 pmol/kg), dissolved in physiological saline solution, or saline as a control was injected into the third ventricle of rats over 10 rain with the Terasaki dispensor (Hamilton). After injection, rats were returned to the recording cages and polygraphic recordings were started. All rats received 4 infusions in order of saline-l, saline-2, DSIP-P (20 pmol/ kg) and DSIP-P (200 pmol/kg) at an interval of a week. Saline was injected twice lk}r adaptation and saline-2 was used as a control. The EEG and E M G were recorded at a paper speed of 5 mm/s for 48 h following injection. Polygraphic recordings were scored in 30-s epochs and divided into 3 stages by visual inspection: wakefulness (W), slow wave sleep (SWS) and paradoxical sleep (PS). Following completion of the experiment, the placement of the intracerebroventricular cannula was checked by injection of methylene blue. A two-tailed paired t-test was applied for the statistical analysis of DSIP-P effects. In this experiment, DSIP-P was injected immediately before the onset of the dark period, because sleep-promoting substances are expected to be more effective when injected in the circadian active period [4, 5]. There was no significant difference between saline and DSIP-P in latencies of SWS and PS after injection (Fig. I). As shown in the left of Fig. 2, DSIP-P resulted in a dose-dependent increase in SWS
DSIP-P 50.
SWS
PS
2{i...a
u
Fig. 1. Sleep latencies of SWS and PS after intracerebroventricular injection of DSIP-P in rats.
162
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Fig, 2. Hourly distributions (upper) and cumulative curves (lower) of SWS (left) and PS (right) during 48 h after injection of DSIP-P in rats. Each value indicates mean _+ S.E.M. (n=6). An arrow indicates the injection time of DSIP-P or saline. during the first 12 h dark period following administration. A l t h o u g h there was no significant effect o f D S I P - P on hourly sleep distributions, the increment o f SWS was evident from the cumulative curve as SWS progressively increased in the rats administered with DSIP-P. The rats administered with 200 p m o l / k g o f D S I P - P showed 17.3 % (P < 0.01) o f increment o f SWS during the first 12 h following administration. This effect was carried over to the next 12 h, where the increment o f SWS was 6.0% ( P < 0 . 0 1 ) in the rats administered with 20 pmol/kg. On the second day, SWS
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returned to the control level. DSIP-P also resulted in a dose-dependent increase in PS during the first 12h following administration as shown in the cumulative curve (Fig. 2B). The increment of PS in the first 12h amounts was 32.3% ( P < 0 . 0 5 ) in the dosage group of 200 pmol/kg. In the next light period, there was no significant change. In the dark period of the second day, the final cumulative amount of PS was not statistically different from control although a statistically significant decrease was observed in some points of cumulative curve. The effects of intracerebroventricular administration of DSIP on rat sleep so far reported are contradictory. Ursin and Larsen [10] reported that a single injection of DSIP increased both SWS and PS. lnou6 et al. [4] also reported that continuous infu-
164
sion of D S I P increased both SWS a n d PS. O n the other hand, T o b l e r and Borbely [9] failed to find a n y s l e e p - p r o m o t i n g effect. Scherschlicht [7] e x a m i n e d the effects o f i n t r a v e n o u s injections of D S I P and D S I P - P on cat sleep, and f o u n d that the sleep-enhancing effect of D S I P - P was greater than that of DSIP. K i m u r a et al. [6] reported that c o n t i n u o u s infusion of D S I P - P into the 3rd cerebroventricle of rats e n h a n c e d SWS a n d PS. In both studies, the increment of PS was much greater than that of SWS. The results of our present study with D S I P - P are in a c c o r d a n c e with the above m e n t i o n e d findings. Moreover. the s l e e p - p r o m o t i n g effect o f D S I P - P lasted over 24 h after a single injection. The s l e e p - p r o m o t i n g effect o f D S I P was reported to be transient even in c o n t i n u o u s l y infused rats [4]. Thus, D S I P - P seems to have a long-lasting s l e e p - p r o m o t i n g effect in rats. This research was s u p p o r t e d by G r a n t s - i n - A i d for Scientific Research 571 14004 a n d 58106004 of the M i n i s t r y o f E d u c a t i o n of Japan. I Van Dijk, A.M.A. and Schoenenberger, G.A., Biochemical evidence for DSIP specific binding sites in the rat brain. In S. Inou6 and A.A. Borb61y(Eds.), Endogenous Sleep Substances and Sleep Regulation, Japan ScientificSocieties Press, Tokyo, 1985, pp. 167-178. 2 Graf, M.V., Christen, I-L, Tobler, H.J., Maier, P.F. and Schoenenberger, G.A., Effects of repeated DSIP and DSIP-P administration on the circadian locomoter activity of rats, Pharmacol. Biochem. Behav., 15 (1981) 717-721. 3 Graf, M.V. and Kastin, A.J., Delta-sleep-inducingpeptide (DSIP): a review, Neurosci. Biobehav. Rev.. 8 (1984) 83 93. 4 lnou6, S., Honda, K., Komoda, Y., Uchizono, K., Ueno, R. and Hayaishi, O., Differential sleep-promoting effects of five sleep substances nocturnally infused in unrestrained rats, Proc. Natl. Acad. Sci. U.S.A.. 81 (1984) 6240~6244. 5 lnou6, S., Honda, K., Komoda, Y., Uchizono, K., Ueno, R. and Hayaishi, O., Little sleep-promoting effect of three sleep substances diurnally infused in unrestrained rats, Neurosci. Lett., 49 (1984) 207211. 6 Kimura, M., Honda, K. and lnou& S., Effects of DSIP analogues on nocturnal sleep in unrestrained rats, J. Physiol. Soc. Jpn., 49 (1987) 444 (abstract). 7 Scherschlicht, R., Pharmacological profile of delta sleep-inducing peptide (DSIP) and a phosphorylated analogue, (Ser-PO) DSIP. In W.P. Koella (Ed.), Sleep 1982, Karger, Basel, 1983, pp. 109-111. 8 Schoenenberger, G.A. and Monnier, M., Characterization of a delta-electroencephalogram (sleep)inducing peptide, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 1282-1286. 9 Tobler, I. and Borb61y, A.A., Effect of delta sleep-inducing peptide (DSIP) and arginine vasotocin (AVT) on sleep and motor activity in the rat, Waking Sleeping,4 (1980) 139 153. 10 Ursin, R. and Larsen, M., Increased sleep followingintracerebroventricular injection of the delta sleepinducing peptide in rats. Neurosci. Lett., 40 (1983) t45- 149.
NSL 05506
Sleep-promoting effect following intracerebroventricular injection of a phosphorylated analogue of delta sleep-inducing peptide (DSIP-P) in rats K e i k o N a k a g a k i , Shigemitsu Ebihara, S e t s u o U s u i , Y o s h i k o H o n d a and Yasuro Takahashi Department o[Psychology, Tokyo Metropolitan Institute[or Neuroseiences, Fuchu Cit)', Tokvo (Japan) (Received 4 January 1988; Revised version received 21 April 1988; Accepted 25 April 1988)
Key u'ord~v Delta sleep-inducing peptide; Slow wave sleep; Paradoxical sleep; lntracerebroventricular injection: Rat The effect of phosphorylated delta sleep-inducing peptide (DSIP-P) on sleep of rats was studied. DSIP-P (20 or 200 pmol/kg) was injected into the third cerebroventricle of male rats immediately before the onset of the dark period of a 12:12 h light-dark cycle. DSIP-P resulted in increases of slow-wave sleep (SWS) (17.3 %, P < 0.01) and paradoxical sleep (PS) (32.3 %, P < 0.05) during the subsequent dark period without shortening sleep latency in the dose of 200 pmol/kg. The SWS-promoting effect was carried over to the next light period. These changes returned to control levels on the second day. These results indicate that DSIP-P is a tong-lasting sleep-promoting substance in rats,
Delta sleep-inducing peptide (DSIP) was isolated from the dialysates of cerebral venous blood taken from rabbits receiving electrical stimulation of the thalamus [8]. DSIP has been reported to have sleep-inducing and -maintaining properties in several mammals and in man [3]. DSIP-P is an analogue of DSIP phosphorylated at the serine in position 7 and was proved to exist naturally in the rat brain [1]. Although it was reported that DSIP-P influenced the circadian locomotor activity rhythm of rats [2], there has so far been no report about the effect on rat sleep except for Kimura et al.'s abstract [6]. The present investigation was undertaken to examine the effect of DSIP-P on rat sleep. The DSIP-P used in this study was supplied by Dr. Schoenenberger, Switzerland. Six male Sprague-Dawley rats weighing 300-390g were chronically implanted with electrodes for cortical EEG and nuchal EMG and with a guide cannula (22
Correspondence: Y. Takahashi, Department of Psychology, Tokyo Metropolitan Institute for Neurosciences, Fuchu City, Tokyo 183, Japan, 0304-3940/881'$ 03.50 (D 1988 Elsevier Scientific Publishers Ireland Ltd.
161
gauge) into the 3rd cerebroventricle at the coordinates A: 1 mm posterior to the bregma, L: 0, V: dura - 7 mm. After surgery, the rats were housed individually in the recording cage in a sound-attenuated and electrically shielded room. These rats were handled daily and adapted to small boxes for injection. At least 2 weeks were allowed lk)r recovery before the experiments were started. The experimental environment was maintained at a temperature of 25-+- I~'C and a humidity of 40 +_ 5 % oll ~l 12:12 h light dark cycle (lights on at 06.00 h). Laboratory chow and water were supplied ad libitum. The rats were transferred to the small boxes for injection immediately before the onset of the dark period. Ten ltl of DSIP-P (20 or 200 pmol/kg), dissolved in physiological saline solution, or saline as a control was injected into the third ventricle of rats over 10 rain with the Terasaki dispensor (Hamilton). After injection, rats were returned to the recording cages and polygraphic recordings were started. All rats received 4 infusions in order of saline-l, saline-2, DSIP-P (20 pmol/ kg) and DSIP-P (200 pmol/kg) at an interval of a week. Saline was injected twice lk}r adaptation and saline-2 was used as a control. The EEG and E M G were recorded at a paper speed of 5 mm/s for 48 h following injection. Polygraphic recordings were scored in 30-s epochs and divided into 3 stages by visual inspection: wakefulness (W), slow wave sleep (SWS) and paradoxical sleep (PS). Following completion of the experiment, the placement of the intracerebroventricular cannula was checked by injection of methylene blue. A two-tailed paired t-test was applied for the statistical analysis of DSIP-P effects. In this experiment, DSIP-P was injected immediately before the onset of the dark period, because sleep-promoting substances are expected to be more effective when injected in the circadian active period [4, 5]. There was no significant difference between saline and DSIP-P in latencies of SWS and PS after injection (Fig. I). As shown in the left of Fig. 2, DSIP-P resulted in a dose-dependent increase in SWS
DSIP-P 50.
SWS
PS
2{i...a
u
Fig. 1. Sleep latencies of SWS and PS after intracerebroventricular injection of DSIP-P in rats.
162
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Fig, 2. Hourly distributions (upper) and cumulative curves (lower) of SWS (left) and PS (right) during 48 h after injection of DSIP-P in rats. Each value indicates mean _+ S.E.M. (n=6). An arrow indicates the injection time of DSIP-P or saline. during the first 12 h dark period following administration. A l t h o u g h there was no significant effect o f D S I P - P on hourly sleep distributions, the increment o f SWS was evident from the cumulative curve as SWS progressively increased in the rats administered with DSIP-P. The rats administered with 200 p m o l / k g o f D S I P - P showed 17.3 % (P < 0.01) o f increment o f SWS during the first 12 h following administration. This effect was carried over to the next 12 h, where the increment o f SWS was 6.0% ( P < 0 . 0 1 ) in the rats administered with 20 pmol/kg. On the second day, SWS
163
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returned to the control level. DSIP-P also resulted in a dose-dependent increase in PS during the first 12h following administration as shown in the cumulative curve (Fig. 2B). The increment of PS in the first 12h amounts was 32.3% ( P < 0 . 0 5 ) in the dosage group of 200 pmol/kg. In the next light period, there was no significant change. In the dark period of the second day, the final cumulative amount of PS was not statistically different from control although a statistically significant decrease was observed in some points of cumulative curve. The effects of intracerebroventricular administration of DSIP on rat sleep so far reported are contradictory. Ursin and Larsen [10] reported that a single injection of DSIP increased both SWS and PS. lnou6 et al. [4] also reported that continuous infu-
164
sion of D S I P increased both SWS a n d PS. O n the other hand, T o b l e r and Borbely [9] failed to find a n y s l e e p - p r o m o t i n g effect. Scherschlicht [7] e x a m i n e d the effects o f i n t r a v e n o u s injections of D S I P and D S I P - P on cat sleep, and f o u n d that the sleep-enhancing effect of D S I P - P was greater than that of DSIP. K i m u r a et al. [6] reported that c o n t i n u o u s infusion of D S I P - P into the 3rd cerebroventricle of rats e n h a n c e d SWS a n d PS. In both studies, the increment of PS was much greater than that of SWS. The results of our present study with D S I P - P are in a c c o r d a n c e with the above m e n t i o n e d findings. Moreover. the s l e e p - p r o m o t i n g effect o f D S I P - P lasted over 24 h after a single injection. The s l e e p - p r o m o t i n g effect o f D S I P was reported to be transient even in c o n t i n u o u s l y infused rats [4]. Thus, D S I P - P seems to have a long-lasting s l e e p - p r o m o t i n g effect in rats. This research was s u p p o r t e d by G r a n t s - i n - A i d for Scientific Research 571 14004 a n d 58106004 of the M i n i s t r y o f E d u c a t i o n of Japan. I Van Dijk, A.M.A. and Schoenenberger, G.A., Biochemical evidence for DSIP specific binding sites in the rat brain. In S. Inou6 and A.A. Borb61y(Eds.), Endogenous Sleep Substances and Sleep Regulation, Japan ScientificSocieties Press, Tokyo, 1985, pp. 167-178. 2 Graf, M.V., Christen, I-L, Tobler, H.J., Maier, P.F. and Schoenenberger, G.A., Effects of repeated DSIP and DSIP-P administration on the circadian locomoter activity of rats, Pharmacol. Biochem. Behav., 15 (1981) 717-721. 3 Graf, M.V. and Kastin, A.J., Delta-sleep-inducingpeptide (DSIP): a review, Neurosci. Biobehav. Rev.. 8 (1984) 83 93. 4 lnou6, S., Honda, K., Komoda, Y., Uchizono, K., Ueno, R. and Hayaishi, O., Differential sleep-promoting effects of five sleep substances nocturnally infused in unrestrained rats, Proc. Natl. Acad. Sci. U.S.A.. 81 (1984) 6240~6244. 5 lnou6, S., Honda, K., Komoda, Y., Uchizono, K., Ueno, R. and Hayaishi, O., Little sleep-promoting effect of three sleep substances diurnally infused in unrestrained rats, Neurosci. Lett., 49 (1984) 207211. 6 Kimura, M., Honda, K. and lnou& S., Effects of DSIP analogues on nocturnal sleep in unrestrained rats, J. Physiol. Soc. Jpn., 49 (1987) 444 (abstract). 7 Scherschlicht, R., Pharmacological profile of delta sleep-inducing peptide (DSIP) and a phosphorylated analogue, (Ser-PO) DSIP. In W.P. Koella (Ed.), Sleep 1982, Karger, Basel, 1983, pp. 109-111. 8 Schoenenberger, G.A. and Monnier, M., Characterization of a delta-electroencephalogram (sleep)inducing peptide, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 1282-1286. 9 Tobler, I. and Borb61y, A.A., Effect of delta sleep-inducing peptide (DSIP) and arginine vasotocin (AVT) on sleep and motor activity in the rat, Waking Sleeping,4 (1980) 139 153. 10 Ursin, R. and Larsen, M., Increased sleep followingintracerebroventricular injection of the delta sleepinducing peptide in rats. Neurosci. Lett., 40 (1983) t45- 149.