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Sleep-promoting effect of the sleep-promoting substance (SPS) and delta sleep-inducing peptide (DSIP) in the mouse
276
Brain Research, 192 (1980) 276 2~0 ~:) Elsevier/North-Holland Biomedical Press
Sleep-promoting effect of the sleep-promoting substance (SPS) and delta sleep-inducing peptide (DSIP) in the mouse
HIROAKI NAGASAKI*, KUNIO KITAHAMA**, JEAN-LOUIS VALATX and MICHEL JOUVET Ddpartment de Mddecine Expdrhnenta[e, Facultd de Mddecine, Universitd Claude Bernard, 8 Avenue Rockefeller, 69373 Lyon (France)
(Accepted February 14th, 1980) Key words: sleep - - sleep-promoting substance - - delta sleep-inducing peptide
The sleep-enhancing effects of several 'sleep factors' differ according to the way of injection, the recipient animals and the sleep states. In previous studies, effected in the rat by Uchizono's group, the sleep factor extracted from the brain stem of sleepdeprived rats increased total sleep time per day by about 50-75 o~ during the first 24 h following i.p. injection ~. This factor also increased the total sleep time of mice following i.v.t, administration 10 and inhibited the spontaneous discharges of abdominal ganglia and the stretch receptor ~0 of the crayfish. In comparison, according to Monnier et al., the synthetic peptide delta sleep-inducing peptide (DSIP) induced slow wave sleep (SWS) when administered intraventricularly 7,9 and/or intravenously 3 in the rabbit, and increased SWS and paradoxical sleep (PS) in the rat 8. However the sleep factor S purified by Pappenheimer's group was only effective in promoting SWS when administered intraventricularly2, 6. In order to further examine the sleep enhancing properties of both a brain extract from sleep deprived rats and DSIP, we are now reporting a dose-response effect in mice following i.p. administration of the partially purified brain extract, which we call sleep-promoting factor (SPS) 11, and DSIP. C 5 7 B L / 6 0 r l mice (male, 6-month-old, n : 10) were chronically implanted with screw electrodes on frontal-occipital positions of the cortex for E E G and needle electrodes were inserted in dorsal neck muscles for EMG. E E G electrodes were fixed to the skull by dental cement and the mice were habituated to the cable for at least one week before the experiment began. In all the experiments, 10 animals were used repeatedly as described below. Recordings were carried out continuously for 24 h/24 h on a polygraph (Alvar-Minihuit), with a paper speed of 30 cm/min and analyzed visually. The SPS was dissolved in saline and injected i.p. at 0.05, 0.1, 0.2, 0.4 and 1.0 * Present address: Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo 173, Japan. ** To whom requests for reprints should be addressed.
277 U (1.00 U is equivalent to one brain stem of the rat deprived of total sleep for 24 h); extraction procedures for SPS were described elsewhere 11. The DSIP was injected i.p. at a dose of 30 nmol/kg at 19.00 h. The sequence of administration for various doses of the SPS on the 10 mice with an interval of 2 or 3 days between injections was as follows: the first group (5 mice) successively received saline (0.2 U), saline (1.0 U), saline (0.05 U); the second group (5 mice) received saline (0.1 U), saline-DSIP-saline (0.4 U). The number of experiments and animals is shown in Table I. The saline injection was carried out one day before each injection of the SPS or DS1P (Protein Research Foundation, Osaka), administered at 18.30-18.50 h under a lighting schedule of 19.00-07.00 h (dark) and 07.00-19.00 h (light). The SPS (0.05-0.4 units) produced a dose-dependent increase in SWS and PS without sedation or abnormal EEG. A significant increase in total sleep time was observed during the 24 h following administration of this extract, particularly at 0.2 and 0.4 U. Furthermore, a significant increase of PS appeared at 0.I and 0.2 units (Table I). SPS (1.0 U) decreased both SWS and PS for the first 3 h after administration but total sleep time during 24 h was similar to the control group. Figs. 1 and 2 show the amounts of SWS and PS for 24 h after administration. The maximal effect on SWS was observed after injection of 0.4 U of the SPS (Fig. 1). Significant differences in SWS duration between 0.4 U of the SPS and the control were observed during 1-7, 10-14 and 18-24 h after administration (P < 0.01). The latency of SWS increase was within the first 1.5 h. The maximal effect on PS was obtained with 0.2 U of the SPS (Fig. 2). Significant differences between 0.2 U of the SPS and the control were observed during 9-15 and 19-24 h after administration (P < 0.01). The beginning of PS increase occurred 9.5 h after administration, and the maximum of PS increase was observed during the lighted hours (07.00-19.00 h). TABLE I
Dose-response of the brain extract from sleep-deprived rat (SPS) and the delta sleep-inducing peptide (DSIP) on sleep length .following i.p. injection between 18.30 and 18.50 h The total sleep time was significantly increased following administration of the and the DSIP (30 nmol/kg) when compared to saline control group (P < 0.01). duration was also significant in 0.2 and 0.4 U of the SPS- and DSIP-treated Later PS increase was significant in 0.1 and 0.2 U of the SPS-treated group, DSIP on PS was not significant.
Examples/ animals
No treatment Saline 0.05 U 0.1 U 0.2 U 0.4 U DS1P
12/4 20/10 8/6 8/4 8/4 8/7 8/4
SPS (0.2 and 0.4 U) The increase of SWS groups (P < 0.01). but the effect of the
Total sleep time (min ± S.E.)
Slow w a v e sleep (min ± S.E.)
Paradoxical sleep (rain ± S.E.)
PS/SWS
756.3 814.1 791.9 835.4 910.6 966.9 901.1
687.5 741.7 710.6 746.8 817.7 890.1 827.3
68.8 77.2 81.9 88.7 109.9 78.9 76.1
10.0 10.4 11.5 11.8 13.4 8.9 9.2
± ± ± ± ± 4±
32.3 11.1 20.3 31.9 18.1" 30.9* 12.4"
± 29.5 zk 11.9 ± 20.0 :k 32.4 J_ 16.3" ± 31.0' ± 12.1"
* Significant difference (P < 0.01) from saline control group.
± 4± ± ± ± ±
5.3 2.0 4.5 5.3* 5.0* 2.4 2.4
(%)
278 rain 1
SWS
hour
19 7 18hour Fig. 1. Effectof sleep-promoting substance (SPS) of 0.4 U injected at 19.00h (~,), on slow wave sleep (SWS) duration per h in C57BL/6mice. A significantincrease in SWS was observed for 12 consecutive h during the dark period. Indicated areas reflect mean ± S.E.M. The 30 nmol/kg dose of DSIP significantly increased SWS duration, and corresponds to the effect of 0.2 U of the SPS. The effect of DSIP on PS was not significant (Table I). The present study reveals the dose-dependent alteration of SWS and PS provoked by the injection of SPS before the beginning of the dark period. It is noteworthy that a remarkable increase of SWS was seen in the first 1-7 h after administration whereas PS increased during the period 9-24 h after administration. And further, the optimal dose for effects on SWS (0.4 U) and PS (0.2 U) was different. On the other hand the higher dose of the SPS (0.4 U) produced prolonged SWS phases during which many short episodes of theta wave were observed (under 10 sec). Concerning the net effective amounts of the sleep factors, these were estimated to be 0.2 #g/0.2 U of the SPS, and 0.75/~g/mouse for the DSIP. In the case of the sleep factor S, the effective dose was less than 150 pmol/rabbit i.v.t. 2. From the fact that the effective doses of these substances are so small, it might be considered that their putative effect may be directly on the mechanism of sleep, especially when compared with tryptophan, which is effective only at a dose of 200 mg/rad. However the mechanism of the effects of these substances is unknown. The cerebrospinal fluid of a sleepdeprived rat inhibited the motor activity in a recipient rat a, and this suggests an indirect sleep promotion by an inhibition of motor activity.
279
P$ min 10 9 8 7 6 5 & 3 2 1 19
7
18 hour
Fig. 2. Effect of SPS of 0.2 U injected at 19.00 h (~) on PS duration per h in C57BL/6 mice. Note a later PS increase during the light period. Indicated areas reflect mean 4- S.E.M.
The long latency of PS was dependent on the injection time (19.00 h) since the latency of PS increase was within 3 h after administration at 09.00 h (unpublished data). This fact leads us to suggest that complex mechanisms, whether peripheral or central, could be involved in the PS triggering system. Further experiments are needed to check the transport of these substances across the blood-brain barrier and their localization in the brain. In summary, sleep-promoting substance (SPS), an extract from the brain stem of a sleep-deprived rat, more purified than an BE-SDR, induced an SWS increase for 12 h consecutively, with a retarded PS increase in C57BL/6 mice when injected i.p. at 19.00 h. It should be noted that the effect of this substance was dose-dependent. This work was supported by I N S E R M (Grant U.52), CNRS (Grant LA 162; ATP 2378) and D R M E (Grant 77093). The fellowship of H. Nagasaki was provided by INSERM. The SPS was generously donated by Dr. M. Ishikawa and Y. Komoda, Institute for Medical and Dental Engineering, Tokyo Medical and Dental University.
280 1 Hartmann, E., The Sleeping Pill, Yale University Press, 1978, pp. 162-181. 2 Krueger, J. M., Pappenheimer, J. R. and Karnovsky, M. L., Sleep-promoting factor S : purification and properties, Proc. nat. Aead. Sci. (Wash.), 75 (1978) 5235-5238. 3 Monnier, M., Dudler, L., G/i.chter, R. and Schoenenberger, G., Delta sleep-inducing peptide (DSIP), EEG and motor activity in rabbits following intravenous administration, Neurosci. lett., 6 (1977) 9-13. 4 Nagasaki, H., Iriki, M., lnou6, S. and Uchizono, K., The presence of sleep promoting material in the brain of sleep-deprived rats, Proc. Jap. Acad., 50 (1974) 241-246. 5 Nagasaki, H., Iriki, M. and Uchizono, K., Inhibitory effect of the brain extract from sleepdeprived rats (BE-SDR) on the spontaneous discharges of crayfish abdominal ganglion, Brain Research, 109 (1976) 202-205. 6 Pappenheimer, J. R., Koski, G., Fencl, V., Karnovsky, M. S. and Kruger, G., Extraction of sleeppromoting factor S from cerebrospinal fluid and from brains of sleep-derived animals, J. Neurophysiok, 38 (1978) 1299-1311. 7 Polc, P., Schneeberger, J. and Haefely, W., Effect of the delta sleep-inducing peptide (DSIP) on the sleep-wakefulness cycle of cats, Neurosci. Lett., 9 (1978) 33-36. 8 Sachs, J., Ungar, J., Waser, P. G. and Borbely, A. A., Factors in cerebrospinal fluid affecting motor activity in the rat, Neurosci. Lett., 2 (1976) 83-86. 9 Schoenenberger, G. A. and Monnier, M., Characterization of delta EEG sleep inducing peptide (DSIP), Plvc. nat. Acad. Sci. (Wash.), 74 (1977) 1282-1286. 10 Uchizono, K., Higashi, A., lriki, M., Nagasaki, H., Ishikawa, M., Komoda, Y., Inou6, S. and Honda, K., Sleep promoting fractions obtained from the brain stem of sleep deprived rats. In M. Ito (Ed.), Integrative Control Function of the Brain, Vol. 1, Kodansha, Tokyo, 1978, pp. 392-395. 11 Ucbizono, K., Inou6, S., Iriki, M., Ishikawa, M., Komoda, Y. and Nagasaki, H., Purification of the sleep promoting substances from sleep-deprived rat brain. In R. Walter and J. Meienhofer (Eds.) Peptide: Chemistry, Structure and Biology, Ann Arbor Science, 1975, pp. 667-671.
Brain Research, 192 (1980) 276 2~0 ~:) Elsevier/North-Holland Biomedical Press
Sleep-promoting effect of the sleep-promoting substance (SPS) and delta sleep-inducing peptide (DSIP) in the mouse
HIROAKI NAGASAKI*, KUNIO KITAHAMA**, JEAN-LOUIS VALATX and MICHEL JOUVET Ddpartment de Mddecine Expdrhnenta[e, Facultd de Mddecine, Universitd Claude Bernard, 8 Avenue Rockefeller, 69373 Lyon (France)
(Accepted February 14th, 1980) Key words: sleep - - sleep-promoting substance - - delta sleep-inducing peptide
The sleep-enhancing effects of several 'sleep factors' differ according to the way of injection, the recipient animals and the sleep states. In previous studies, effected in the rat by Uchizono's group, the sleep factor extracted from the brain stem of sleepdeprived rats increased total sleep time per day by about 50-75 o~ during the first 24 h following i.p. injection ~. This factor also increased the total sleep time of mice following i.v.t, administration 10 and inhibited the spontaneous discharges of abdominal ganglia and the stretch receptor ~0 of the crayfish. In comparison, according to Monnier et al., the synthetic peptide delta sleep-inducing peptide (DSIP) induced slow wave sleep (SWS) when administered intraventricularly 7,9 and/or intravenously 3 in the rabbit, and increased SWS and paradoxical sleep (PS) in the rat 8. However the sleep factor S purified by Pappenheimer's group was only effective in promoting SWS when administered intraventricularly2, 6. In order to further examine the sleep enhancing properties of both a brain extract from sleep deprived rats and DSIP, we are now reporting a dose-response effect in mice following i.p. administration of the partially purified brain extract, which we call sleep-promoting factor (SPS) 11, and DSIP. C 5 7 B L / 6 0 r l mice (male, 6-month-old, n : 10) were chronically implanted with screw electrodes on frontal-occipital positions of the cortex for E E G and needle electrodes were inserted in dorsal neck muscles for EMG. E E G electrodes were fixed to the skull by dental cement and the mice were habituated to the cable for at least one week before the experiment began. In all the experiments, 10 animals were used repeatedly as described below. Recordings were carried out continuously for 24 h/24 h on a polygraph (Alvar-Minihuit), with a paper speed of 30 cm/min and analyzed visually. The SPS was dissolved in saline and injected i.p. at 0.05, 0.1, 0.2, 0.4 and 1.0 * Present address: Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashi-ku, Tokyo 173, Japan. ** To whom requests for reprints should be addressed.
277 U (1.00 U is equivalent to one brain stem of the rat deprived of total sleep for 24 h); extraction procedures for SPS were described elsewhere 11. The DSIP was injected i.p. at a dose of 30 nmol/kg at 19.00 h. The sequence of administration for various doses of the SPS on the 10 mice with an interval of 2 or 3 days between injections was as follows: the first group (5 mice) successively received saline (0.2 U), saline (1.0 U), saline (0.05 U); the second group (5 mice) received saline (0.1 U), saline-DSIP-saline (0.4 U). The number of experiments and animals is shown in Table I. The saline injection was carried out one day before each injection of the SPS or DS1P (Protein Research Foundation, Osaka), administered at 18.30-18.50 h under a lighting schedule of 19.00-07.00 h (dark) and 07.00-19.00 h (light). The SPS (0.05-0.4 units) produced a dose-dependent increase in SWS and PS without sedation or abnormal EEG. A significant increase in total sleep time was observed during the 24 h following administration of this extract, particularly at 0.2 and 0.4 U. Furthermore, a significant increase of PS appeared at 0.I and 0.2 units (Table I). SPS (1.0 U) decreased both SWS and PS for the first 3 h after administration but total sleep time during 24 h was similar to the control group. Figs. 1 and 2 show the amounts of SWS and PS for 24 h after administration. The maximal effect on SWS was observed after injection of 0.4 U of the SPS (Fig. 1). Significant differences in SWS duration between 0.4 U of the SPS and the control were observed during 1-7, 10-14 and 18-24 h after administration (P < 0.01). The latency of SWS increase was within the first 1.5 h. The maximal effect on PS was obtained with 0.2 U of the SPS (Fig. 2). Significant differences between 0.2 U of the SPS and the control were observed during 9-15 and 19-24 h after administration (P < 0.01). The beginning of PS increase occurred 9.5 h after administration, and the maximum of PS increase was observed during the lighted hours (07.00-19.00 h). TABLE I
Dose-response of the brain extract from sleep-deprived rat (SPS) and the delta sleep-inducing peptide (DSIP) on sleep length .following i.p. injection between 18.30 and 18.50 h The total sleep time was significantly increased following administration of the and the DSIP (30 nmol/kg) when compared to saline control group (P < 0.01). duration was also significant in 0.2 and 0.4 U of the SPS- and DSIP-treated Later PS increase was significant in 0.1 and 0.2 U of the SPS-treated group, DSIP on PS was not significant.
Examples/ animals
No treatment Saline 0.05 U 0.1 U 0.2 U 0.4 U DS1P
12/4 20/10 8/6 8/4 8/4 8/7 8/4
SPS (0.2 and 0.4 U) The increase of SWS groups (P < 0.01). but the effect of the
Total sleep time (min ± S.E.)
Slow w a v e sleep (min ± S.E.)
Paradoxical sleep (rain ± S.E.)
PS/SWS
756.3 814.1 791.9 835.4 910.6 966.9 901.1
687.5 741.7 710.6 746.8 817.7 890.1 827.3
68.8 77.2 81.9 88.7 109.9 78.9 76.1
10.0 10.4 11.5 11.8 13.4 8.9 9.2
± ± ± ± ± 4±
32.3 11.1 20.3 31.9 18.1" 30.9* 12.4"
± 29.5 zk 11.9 ± 20.0 :k 32.4 J_ 16.3" ± 31.0' ± 12.1"
* Significant difference (P < 0.01) from saline control group.
± 4± ± ± ± ±
5.3 2.0 4.5 5.3* 5.0* 2.4 2.4
(%)
278 rain 1
SWS
hour
19 7 18hour Fig. 1. Effectof sleep-promoting substance (SPS) of 0.4 U injected at 19.00h (~,), on slow wave sleep (SWS) duration per h in C57BL/6mice. A significantincrease in SWS was observed for 12 consecutive h during the dark period. Indicated areas reflect mean ± S.E.M. The 30 nmol/kg dose of DSIP significantly increased SWS duration, and corresponds to the effect of 0.2 U of the SPS. The effect of DSIP on PS was not significant (Table I). The present study reveals the dose-dependent alteration of SWS and PS provoked by the injection of SPS before the beginning of the dark period. It is noteworthy that a remarkable increase of SWS was seen in the first 1-7 h after administration whereas PS increased during the period 9-24 h after administration. And further, the optimal dose for effects on SWS (0.4 U) and PS (0.2 U) was different. On the other hand the higher dose of the SPS (0.4 U) produced prolonged SWS phases during which many short episodes of theta wave were observed (under 10 sec). Concerning the net effective amounts of the sleep factors, these were estimated to be 0.2 #g/0.2 U of the SPS, and 0.75/~g/mouse for the DSIP. In the case of the sleep factor S, the effective dose was less than 150 pmol/rabbit i.v.t. 2. From the fact that the effective doses of these substances are so small, it might be considered that their putative effect may be directly on the mechanism of sleep, especially when compared with tryptophan, which is effective only at a dose of 200 mg/rad. However the mechanism of the effects of these substances is unknown. The cerebrospinal fluid of a sleepdeprived rat inhibited the motor activity in a recipient rat a, and this suggests an indirect sleep promotion by an inhibition of motor activity.
279
P$ min 10 9 8 7 6 5 & 3 2 1 19
7
18 hour
Fig. 2. Effect of SPS of 0.2 U injected at 19.00 h (~) on PS duration per h in C57BL/6 mice. Note a later PS increase during the light period. Indicated areas reflect mean 4- S.E.M.
The long latency of PS was dependent on the injection time (19.00 h) since the latency of PS increase was within 3 h after administration at 09.00 h (unpublished data). This fact leads us to suggest that complex mechanisms, whether peripheral or central, could be involved in the PS triggering system. Further experiments are needed to check the transport of these substances across the blood-brain barrier and their localization in the brain. In summary, sleep-promoting substance (SPS), an extract from the brain stem of a sleep-deprived rat, more purified than an BE-SDR, induced an SWS increase for 12 h consecutively, with a retarded PS increase in C57BL/6 mice when injected i.p. at 19.00 h. It should be noted that the effect of this substance was dose-dependent. This work was supported by I N S E R M (Grant U.52), CNRS (Grant LA 162; ATP 2378) and D R M E (Grant 77093). The fellowship of H. Nagasaki was provided by INSERM. The SPS was generously donated by Dr. M. Ishikawa and Y. Komoda, Institute for Medical and Dental Engineering, Tokyo Medical and Dental University.
280 1 Hartmann, E., The Sleeping Pill, Yale University Press, 1978, pp. 162-181. 2 Krueger, J. M., Pappenheimer, J. R. and Karnovsky, M. L., Sleep-promoting factor S : purification and properties, Proc. nat. Aead. Sci. (Wash.), 75 (1978) 5235-5238. 3 Monnier, M., Dudler, L., G/i.chter, R. and Schoenenberger, G., Delta sleep-inducing peptide (DSIP), EEG and motor activity in rabbits following intravenous administration, Neurosci. lett., 6 (1977) 9-13. 4 Nagasaki, H., Iriki, M., lnou6, S. and Uchizono, K., The presence of sleep promoting material in the brain of sleep-deprived rats, Proc. Jap. Acad., 50 (1974) 241-246. 5 Nagasaki, H., Iriki, M. and Uchizono, K., Inhibitory effect of the brain extract from sleepdeprived rats (BE-SDR) on the spontaneous discharges of crayfish abdominal ganglion, Brain Research, 109 (1976) 202-205. 6 Pappenheimer, J. R., Koski, G., Fencl, V., Karnovsky, M. S. and Kruger, G., Extraction of sleeppromoting factor S from cerebrospinal fluid and from brains of sleep-derived animals, J. Neurophysiok, 38 (1978) 1299-1311. 7 Polc, P., Schneeberger, J. and Haefely, W., Effect of the delta sleep-inducing peptide (DSIP) on the sleep-wakefulness cycle of cats, Neurosci. Lett., 9 (1978) 33-36. 8 Sachs, J., Ungar, J., Waser, P. G. and Borbely, A. A., Factors in cerebrospinal fluid affecting motor activity in the rat, Neurosci. Lett., 2 (1976) 83-86. 9 Schoenenberger, G. A. and Monnier, M., Characterization of delta EEG sleep inducing peptide (DSIP), Plvc. nat. Acad. Sci. (Wash.), 74 (1977) 1282-1286. 10 Uchizono, K., Higashi, A., lriki, M., Nagasaki, H., Ishikawa, M., Komoda, Y., Inou6, S. and Honda, K., Sleep promoting fractions obtained from the brain stem of sleep deprived rats. In M. Ito (Ed.), Integrative Control Function of the Brain, Vol. 1, Kodansha, Tokyo, 1978, pp. 392-395. 11 Ucbizono, K., Inou6, S., Iriki, M., Ishikawa, M., Komoda, Y. and Nagasaki, H., Purification of the sleep promoting substances from sleep-deprived rat brain. In R. Walter and J. Meienhofer (Eds.) Peptide: Chemistry, Structure and Biology, Ann Arbor Science, 1975, pp. 667-671.