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Proc. NatI. Acad. Sci. USA Vol. 85, pp. 3653-3656, May 1988 Physiological Sciences Evidence for a role of delta -inducing in slow-wave sleep and sleep-related growth release in the rat (delta sleep-inducing peptide antiserum/passive immunization/plasma /third ventricle injections/sleep deprivation) KANAK S. IYER*, G. A. MARKSt, A. J. KASTINt, AND S. M. MCCANN*§ *Department of , Division, and tDepartment of , University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235; and Section, Veteran's Administration Medical Center and Tulane University School of Medicine, 1601 Perdido Street, New Orleans, LA 70146 Contributed by S. M. McCann, February 8, 1988

ABSTRACT To examine the role of delta sleep-inducing appeared to be mediated by dopamine and were at least in peptide (DSIP) in sleep-related growth hormone (GH) release, part the result of diminished release of (22). male rats were deprived of sleep for 4 hr by placing them on a In the human there is a prominent release of GH during the slowly rotating wheel. Sleep deprivation by this method caused early hours ofsleep, which is associated with SWS (23). Since a significant increase in GH release, as indicated by the increase DSIP induces SWS, it occurred to us that it might also be in plasma GH concentrations (P < 0.01), and also in the involved in sleep-related GH release. Because the duration of amount of slow-wave sleep (SWS) (P < 0.001) above initial sleep is short in the rat, it has been difficult to demonstrate values after removal of the animals from the rotating wheel. a sleep-related GH release in this species (24, 25); however, These increases were blocked by microinjection into the third Kawakami et al. (25) have shown that periods of sleep cerebral ventricle of highly specific antiserum to DSIP. In deprivation in rats suppress GH release, which is followed by control rats receiving an equal volume of normal rabbit serum, a high level GH secretory burst upon cessation of sleep the significant increase in plasma GH as well as SWS remained deprivation. Consequently, it was necessary first to deter- after removal of the rats from the wheel. The increased release mine whether sleep-related GH release could be evoked in of endogenous DSIP in the sleep-deprived animals may have rats by depriving the animals of sleep. This was achieved by caused an increase in SWS as well as plasma GH. Since DSIP placing them in a vertical wheel that was slowly rotating. This increases plasma GH after its injection into the third cerebral required the animals to walk continuously to avoid falling ventricle and since passive immunization against DSIP blocks back as the wheel rotated. After removal of the animals from the increase in SWS and GH release that follows the 4 hr of the wheel, they indeed slept and this was associated with a sleep deprivation, the results suggest that DSIP can be a large increase in SWS and plasma GH. This made it possible physiological stimulus for sleep-related GH release as well as to (i) evaluate the role of DSIP in sleep-related GH release by for the induction of SWS. injection of a highly specific antiserum against DSIP into the third ventricle of sleep-deprived rats and (ii) determine the Delta sleep-inducing peptide (DSIP) is a neuropeptide that effect ofDSIP on sleep and plasma GH. Control animals were was isolated and characterized several years ago (ref. 1; see injected intraventricularly with normal rabbit serum (NRS). ref. 2 for a recent review of its present status). Its ability to induce slow-wave (delta) sleep (SWS) has been shown in MATERIALS AND METHODS several species, including humans (3), rabbits (4), rats (5, 6), and mice (7). Adult male Sprague-Dawley-derived (Sasco, Omaha, NB) DSIP-like immunoreactivity has been found in adult and rats (250-300 g) were maintained initially in group cages in a even fetal rat brain by RIA (8, 9) followed by HPLC (9). room with controlled lighting (lights on 0500-1700 hr) and Further evidence for the occurrence of DSIP in the rat is its temperature (23 + 1PC) and given the free access to food and localization in the brain by immunocytochemistry in two water. After 5-10 days, they were implanted with a third ma- ventricular cannula while anesthetized with tribromoethanol independent studies (10-12). DSIP-like immunoreactive (26). At the same time, electrodes were implanted for terial confirmed by HPLC has been found in plasma of the recording the electroencephalogram (EEG) and electromyo- human, rabbit, rat, and dog (13, 14) and in human breast milk gram (EMG). Thereafter, the animals were housed singly. (15) and (14). Seven to 10 days later, a silastic cannula was implanted in the In addition to increasing SWS (16), it has been shown to right external jugular vein (27) while the rat was anesthetized reduce the firing rate of in mammals and to produce with ether. Experiments were performed 3 days later. The a hyperpolarization of the resting membrane potential in cannula was used for withdrawal of heparinized (100 neurons of the snail (17). A direct action of DSIP on single units/ml) blood samples (0.5 ml). After withdrawal of sam- neurons of the brainstem in rats and rabbits has also been ples, the blood volume was restored by an injection of an observed (18). Behavioral effects of the peptide include equal volume of heparinized (20 units/ml) physiological decreased locomoter activity (16) and an altered circadian saline. After every four samples, the blood was centrifuged activity rhythm of rats (19). It also can protect against stress at low speed at 40C. The plasma was separated and frozen for (20). subsequent RIA and the erythrocytes were resuspended in There have been few studies of the effects of DSIP on the saline and injected back into the animal. release of . Third ventricular The cortical EEG was obtained from two stainless steel injection of DSIP caused a dose-dependent increase in screws tapped into the skull; the hippocampal EEG was growth hormone (GH) release in the rat (21). These effects recorded from a single stainless steel wire insulated except at

The publication costs of this article were defrayed in part by page charge Abbreviations: DSIP, delta sleep-inducing peptide; GH, growth payment. This article must therefore be hereby marked "advertisement" hormone; SWS, slow-wave sleep; NRS, normal rabbit serum. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed.

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the tip (diameter; 0.25 mm); the nuchal EMG was recorded from a bipolar spring electrode (Plastic Products, Roanoke, VA). Uninsulated portions of the electrode were surgically placed between the levator scapulae dorsalis and trapezius muscles. These three sources of electrical activity were used c for determination of the state of sleep. The occurrence of SWS and the waking state were determined based on stan- dard criteria determined from the EMG and cortical and C,) C r- hippocampal EEG potentials (28). To increase detection of ..I (IrE the occurrence of SWS, 1-min epochs were divided into 12 periods. Each epoch was "hand" scored for the number of 3- periods in which SWS occupied at least half of the period. cn The scorer was unaware of the experimental conditions. Animals were adapted to the recording situation for 48 hr. Rats were sleep-deprived by placing them into a slowly rotating drum (1 rpm; diameter, 40 cm) to which they had -15 0 15 30 45 60 75 90 405420 been previously adapted and where they had free access to food and water. Time (min) Sleep-Deprivation Experiments. In the first experiment, FIG. 1. Effect ofsleep deprivation from 1030 to 1445 hr on plasma were made for 6 hr from 1000 to 1700 polygraphic recordings GH (ng/ml) and SWS (min/15 min) after placement of the rats in a were every hr. Simultaneously, blood samples withdrawn 15 revolving wheel (n = 6). In this and Figs. 2-4, time zero is the time min for GH RIA. when the rats were removed from the wheel. *, Plasma GH In the second experiment, rats were sleep-deprived from concentrations; bars, time spent in SWS per 15 min. Vertical lines 1030 to 1445 hr. Then they were transferred to their home represent 1 SEM. cages and polygraph measurements and blood sampling for GH were carried out as described above. maximum time spent in SWS by 15 min. After the peak in GH In the third experiment, the rats were placed on stationary release, values declined to a minimum by 75 min, which was wheels from 1030 to 1445 hr and the rest of the experiment not different from the initial values at -15 and 0 min. was performed as in experiment 2. Similarly, the time spent in SWS diminished significantly by In the fourth experiment, rats were injected in the third 60 min and remained statistically unchanged thereafter to the ventricle with 3 gl of antiserum against DSIP (anti-DSIP) 24 end of the experiment. hr before sleep deprivation. They were then sleep-deprived The animals that were placed on the stationary wheel as in experiment 2. These animals received another dose of (experiment 3) did not show any significant increase in SWS 3 ptl of antiserum to DSIP 30 min before removal of blood at any time; however, they showed a significant elevation in samples. This is a highly specific anti-DSIP, which has been plasma GH at 90 min in comparison with earlier values (Fig. thoroughly characterized (8, 29). Control animals received an 2). equal volume ofNRS instead of anti-DSIP at the same times. Blockage of Effect of Sleep Deprivation on SWS and GH by RIA. Plasma samples were assayed in duplicate for GH Third Ventricular Injection of Antiserum to DSIP. Third concentration with RIA kits supplied by the National Insti- ventricular injection of 3 Al of anti-DSIP antiserum in two tute of Diabetes and Digestive and Kidney Diseases doses, one 24 hr before sleep deprivation and the other 30 min (NIDDK). Hormonal values are expressed in terms of the before blood sampling, completely blocked the increase in NIDDK rat RP-1 standard. SWS and plasma GH that were observed after sleep depri- Statistics. Periodicity of GH and SWS was determined by vation in experiment 2 (Fig. 3). Sleep-deprived control analysis of the power spectrum using the least-squares animals receiving NRS instead of antiserum showed a sig- were method (28). Cross-correlation analyses performed nificant increase in SWS as well as GH in comparison with between the time series ofthe amount ofSWS and plasma GH initial values (Fig. 4). Plasma GH values increased within 15 concentrations (28). Analysis of variance with repeated min after removal of the rats from the wheel and peaked at 60 measure the Neuman-Keul test was used to followed by min. The values then declined gradually to a minimum at 120 determine of during multiple sampling of significance changes min. The pattern was similar to that of rats that did not the rats. Student's t test was used to determine statistical receive third ventricular injections (Fig. 1). SWS also in- significance of differences between means of two groups. creased significantly between 15 and 30 min after removal RESULTS 80 10 Relationship Between GH and SWS in Control Rats. The method 3- - c power spectral analysis and the least-squares applied E to the time series of plasma GH concentrations and amount 60 IL') of SWS in 15-min periods in the control rats of experiment 1, E I .r_ C: which were not sleep-deprived, determined that the mean C3 40 5 .E periodicity for pulses ofplasma GH was 3 hr and that of SWS (91 was 2 hr in the five rats examined over the period from 1000 to 1700 hr. 20 ° m1C0 En Effect of Sleep Deprivation on Plasma GH and SWS. Fig. 1 shows the effect of sleep deprivation on plasma GH concen- trations and SWS in experiment 2. There was a significant 445 30 45 60 75 90 x05 420 increase in the amount of GH released, as evidenced by Time (min) increases in plasma GH from the initial values, as well as in the amount of SWS within 30-45 min after removing the FIG. 2. Plasma GH (ng/ml) and SWS (min/15 min) measured in animals from the rotating wheel and returning them to their rats that were previously placed on a stationary wheel from 1030 to home cages. The peak plasma GH at 45 min preceded the 1445 hr (n = 6). Downloaded by guest on September 24, 2021 Physiological Sciences: Iyer et A Proc. Natl. Acad. Sci. USA 85 (1988) 3655 100

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Time (min) FIG. 3. Effect of 3 of DSIP antiserum injected into the third Al Stationary DSIP- NRS Stationary DSIP- NRS ventricle 24 hr before and 30 min before sampling on plasma GH and AS AS SWS in rats previously subjected to sleep deprivation by being placed in a rotating wheel from 1030 to 1445 hr (n = 9). Revolving Revolving FIG. 5. Comparison of total amount of SWS and total sampled from the wheel and reached a plateau at 45 min. This plateau plasma GH in different groups. The mean amount of GH and SWS was maintained until 90 min, after which the values declined was significantly greater in sleep-deprived NRS-treated rats com- to a minimum at 120 min. Again, the pattern was similar to pared to anti-DSIP-treated rats; **, P < 0.001. The three groups that seen in animals that did not receive third ventricular were (i) on stationary wheel, (ii) on rotating wheel and anti-DSIP- injections, but the increase in SWS was more sustained. treated (sleep-deprived), (iii) on rotating wheel and NRS-treated The mean amount of SWS in NRS-treated animals after (sleep-deprived). sleep deprivation was significantly greater (P < 0.001) than < them in the 14 that of animals treated with anti-DSIP (Fig. 5). The anti- significant (P 0.0001) relationship between DSIP-treated rats exhibited no significant difference in SWS sleep-deprived NRS-treated rats. from that observed in animals that were not sleep deprived. The concentration of plasma GH measured in previously DISCUSSION sleep-deprived NRS-treated rats was significantly higher (P Our finding of a 3-hr periodicity of GH release is consistent < 0.001) than that of previously sleep-deprived anti-DSIP- with previously reported results (24, 25). However, unlike a treated rats and was also significantly greater than that ofthe study in immature male rats (25), we were unable to find a rats on the stationary wheel (P < 0.01) (Fig. 5). The latency 3-hr periodicity for SWS sleep. Our finding of a different to peak plasma GH was significantly less (P < 0.001) in periodicity for SWS and GH release is similar to that reported previously sleep-deprived, NRS-treated rats compared to earlier by others (24). this same latency for rats placed on the stationary wheel. There have been some questions raised about the relation- The temporal distribution of SWS was measured as the ship between SWS and GH release in the rat (24). The rat has time to reach 50% of total sleep during the 2 hr after removal shorter sleep/wakefulness cycles than the human. This may from the wheel. The time was significantly less (P < 0.05) in be the reason why we could not relate plasma GH and SWS previously sleep-deprived, NRS-treated rats compared to in animals that were not sleep depdved in our study; previously sleep-deprived anti-DSIP-treated rats as well as however, when the animals were sleep deprived by placing compared to rats previously on the stationary wheel (P < them in the rotating wheel, there was a clear linkage between 0.01). Cross-correlation analysis performed between plasma the increase in plasma GH and SWS that followed removal of GH and the amount of SWS (min/15 min) revealed a the rats from the wheel. This provided a model to study the role of DSIP in sleep-related GH release. The increase in plasma GH and SWS was not altered by the intraventricular injections of NRS in control sleep-deprived rats, whereas 80 I 10C both the GH release and increase in SWS were abolished by intraventricular injection of the highly specific antiserum E 60 - I directed against DSIP. These findings allow the suggestion that after sleep depri- C 6 h vation, there is increased release ofDSIP, which then acts on ~40 H H the hypothalamic systems controlling GH release on the one hand, and on the mechanisms inducing SWS on the other, to 20 C result in an increase in plasma GH and SWS. Passive neutralization of this released DSIP prevents both occur- rences. Presumably, the antiserum directed against DSIP is -45 0 15 30 45 60 75 90 405 420 taken up from the ventricle either passively or by active transport by ependymal cells and then diffuses to its sites of Time (min) action in the brain. FIG. 4. Effect of injection of 3 A.l of NRS into the third ventricle It is possible that the increased locomoter activity during 24 hr before and 30 min before blood sampling on plasma GH and sleep deprivation could be involved in the subsequent induc- SWS in rats previously subjected to sleep deprivation from 1030 to tion of growth hormone release in SWS. It is also possible 1445 hr (n = 14). that the failure of SWS to occur in the DSIP antiserum- Downloaded by guest on September 24, 2021 3656 Physiological Sciences: Iyer et al. Proc. Natl. Acad. Sci. USA 85 (1988)

injected animals might be related to the failure of growth 10. Kastin, A. J., Dickson, J. & Fischman, A. J. (1984) Physiol. hormone to increase. Behav. 33, 427-431. In the case of GH, we have shown that there is an increase 11. Feldman, S. C. & Kastin, A. J. (1984) 11, 303- in plasma GH after intraventricular injection ofDSIP that can 317. 12. Constantinidis, J., Bouras, C., Guntern, R., Taban, C. H. & be blocked by the dopamine receptor blocker pimozide, Tissot, R. (1983) Neuropsychobiology 10, 94-100. indicating that there is a dopaminergic step involved (21). In 13. Kastin, A. J., Castellanos, P. F., Banks, W. A. & Coy, D. H. vitro experiments showed that addition of DSIP to the media (1981) Pharmacol. Biochem. Behav. 15, 969-977. ofhypothalamic fragments in vitro led to decreased release of 14. Graf, M. V., Kastin, A. J. & Fischman, A. J. (1984) Pharma- somatostatin (22). Consequently, it is possible that the DSIP col. Biochem. Behav. 21, 761-766. neurons have axons that synapse with dopaminergic neurons 15. Graf, M. V., Hunter, C. A. & Kastin, A. J. (1984) J. Clin. to release dopamine, which in turn brings about a decrease in Endocrinol. Metab. 59, 127-132. the release of somatostatin. This diminished release of 16. Graf, M. V. & Kastin, A. J. (1984) Neurosci. Biobehav. Rev. 8, somatostatin into the hypophyseal portal vessels would 83-93. remove its inhibitory action on the somatotropes with result- 17. Sargsyan, A. S., Sumskaya, L. V., Alexandrova, I. Y., Bes- rukov, M. V., Mikhaleva, I. I., Iranov, V. T. & Balaban, P. M. ant release of GH and a subsequent increase in plasma GH. (1981) Bioorg. Khim. 7, 1125-1149. A parallel stimulatory effect of-DSIP on (gH-releasing factor) 18. Normanton, J. R. & Gent, J. P. (1983) Neuroscience 8, 107- GRF release has not been ruled out. DSIP terminals could 114. also interact with the neuronal systems that induce SWS 19. Monnier, M., Dudler, L., Gaechter, R. & Schoenenberger, sleep. G. A. (1972) Neurosci. Lett. 6, 9-13. 20. Graf, M., Christen, H., Tobler, H. J., Maier, P. F. & Schoe- We thank Judy Scott for preparing the manuscript. This work was nenberger, G. A. (1981) Pharmacol. Biochem. Behav. 15, supported by National Institutes of Health Grant DK10073. 717-721. 1. Schoenenberger, G. A. & Mennier, M. (1977) Proc. Natl. 21. Iyer, S. K. & McCann, S. M. (1987) 8, 45-48. Acad. Sci. USA 74, 1282-1286. 22. Iyer, S. K. & McCann, S. M. (1987) Neuroendocrinology 46, 2. Graf, M. V. & Kastin, A. J. (1986) Peptides 7, 1165-1187. 93-96. 3. Schneider-Heilmert, D. & Schoenenberger, G. A. (1981) Ex- 23. Takahashi, Y., Kipnis, D. & Daughaday, W. (1967) J. Clin. perientia-37, 913-917. Endocrinol. Metab. 27, 501-505. 4. Ye, Y. H., Lin, Y., Lu, Y. J., Li, C. X., Ji, A. X., Xing, Q. Y., 24. Willoughby, J. O., Martin, J. B., Renaud, L. P. & Brazeau, P. Liu, S. Y., Zhang, W. Y., Wang, Z. S. & Dai, X. J. (1984) (1970) Endocrinology 98, 991-996. Acta. Sci. Natl. Univ. Pek. 4, 63-68. 25. Kawakami, M., Kimura, F. & Tsai, C. W. (1983) J. Physiol. 5. Vasin, R. & Larsen, M. (1983) Neurosci. Lett. 70, 145-149. (London) 339, 325-337. 6. Monnier, M. & Gallard, J. M. (1979) Neurosci. Lett. 13, 26. Antunes-Rodrigues, J. & McCann, S. M. (1970) Proc. Soc. 169-172. Exp. Biol. Med. 133, 1464-1470. 7. Nagasaki, M., Kitahama, K., Valatx, J. L. & Jouvet, M. (1980) 27. Harms, P. G. & Ojeda, S. R. (1977) J. Appl. Physiol. 36, Brain Res. 192, 276-280. 391-393. 8. Kastin, A. J., Nissen, C., Schally, A. V. & Coy, D. H. (1978) 28. Ibuka, N., Inouye, S. T. & Kawamura, H. (1977) Brain Res. Brain Res. Bull. 3, 691-695. 122, 33-47. 9. Kastin, A. J., Nissen, C. & Coy, D. H. (1981) Brain Res. Bull. 29. Kastin, A. J., Nissen, C., Schally, A. V. & Coy, D. H. (1979) 7, 687-690. Pharmacol. Biochem. Behav. 11, 717-719. Downloaded by guest on September 24, 2021