Pflügers Archiv

, Volume 369, Issue 2, pp 99–109

A naturally occurring delta-EEG enhancing nonapeptide in rabbits

X. Final isolation, characterization and activity test
  • Guido A. Schoenenberger
  • P. F. Maier
  • H. J. Tobler
  • M. Monnier
Article

Summary

By extracorporeal dialysis of blood from the sagittal venous sinus in rabbit donors during electrical stimulation of the ventromedial intralaminary thalamus, a hemodialysate was obtained. This dialysate, or its purified fractions, infused into the mesodiencephalic ventricular system of recipient rabbits, induced behavioral and electroencephalographic (EEG) changes, i.e., enhanced delta-EEG. These effects of the dialysate and of its subsequent fractions were quantified by EEG analysis of the cortical delta activity (2–3 Hz), using a calibrated automatic wave analyzer. The activity values of the last pure fractions were furthermore processed on an 1108 computer system. It was thus possible to express the delta enhancement in RMS μV and the corresponding time integral referred to the preinfusion values as well as to the values of the control rabbits.

Ultrafiltration through a UM-05 Diaflo membrane and gel-filtration over Sephadex G-15 yielded an active fraction showing a symmetrical ninhydrinpositive peak, with a corresponding absorption peak at 280 nm and a mol.wt. between 355 and 1000. Preparative TLC and high voltage paper electrophoresis, followed by a second gel-filtration over Sephadex G-15 and quantitative TL-electrophoresis, yielded a progressively purified ninhydrin-positive EEG active compound, with an absorption maximum at 280 nm. A final compound was analyzed and quantified by amino acid analysis, UV and fluorescence spectroscopy. The delta-EEG enhancing compound was shown to be a small peptide, comprising 9 amino acid residues: 1 Trp-(2 Ala, 1 Asp, 1 Glu, 3 Gly, 1 Ser), with a mol.wt. of 848.98. The minimal effective dose for delta-EEG enhancement has been estimated at 6×10−9 mol/kg B.W. when dissolved in 0.05 ml c.s.f.-like solution and infused intraventricularly over a period of 3.5 min. The specific delta activity due to the peptide was compared to that in control rabbits infused not only with a c.s.f.-like solution, but also with a c.s.f. solution to which a similar synthetic nonapeptide (mol. wt. 996.14) had been added. The purpose of this comparison was to test the effect of the specific delta-EEG enhancing peptide against a corresponding unspecific nonapeptide-“analogue”. RMS μV and time integrals of the delta values of the two control groups showed no difference. By contrast, the RMS μV and time integral of the delta-EEG enhancement due to the “delta-sleep-inducing-peptide” (DSIP), when compared to the values of one of the two or both control groups, were significant: The nonapeptide increased the delta-EEG-activity by +45.8±6.65 RMS μV or 43.1±6.25% as compared to the controls.

Key words

Brain Sleep Peptide EEG Rabbit 

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References

  1. Diezel, W., Kopperschläger, G., Hofmann, E.: An improved procedure for protein staining in polyacrylamide gels with a new type of coomassie brilliant blue. Analyt. Biochem.48, 617–620 (1972)Google Scholar
  2. Edvinsson, L., Håkanson, R., Sundler, F.: Separation of tryptophyl-peptides by thin-layer chromatography and microspectro-fluorometric characterization of their formaldehyde-induced fluorophores. Analyt. Biochem.46, 473–481 (1972)Google Scholar
  3. Fencl, V., Koski, G., Pappenheimer, J. R.: Factors in cerebrospinal fluid from goats that affect sleep and activity in rats. J. Physiol. (Lond.)216, 565–589 (1971)Google Scholar
  4. Hess, W. R.: Das Schlafsyndrom als Folge diencephaler Reizung. Helv. physiol. pharmacol. Acta2, 305–344 (1944)Google Scholar
  5. Jouvet, M.: Etude de la dualité des états de sommeil et des mécanismes de la phase paradoxale. In: Aspects anatomo-fonctionnels de la physiologie du sommeil (M. Jouvet, ed.), pp. 397–400. Centre National de la Recherche Scientifique. Paris 1965Google Scholar
  6. Legendre, R., Piéron, H.: Des résultats histo-physiologiques de l'injeetion intraoccipito-atlantoidienne des liquides insomniques. C.R. Soc. Biol. (Paris)68, 1108–1109 (1910)Google Scholar
  7. Legendre, R., Piéron, H.: Recherches sur le besoin de sommeil consécutif à une veille prolongée. Z. allg. Physiol.14, 235–262 (1913)Google Scholar
  8. Monnier, J., Koller, Th., Graber, S.: Humoral influences of induced sleep and arousal upon electrical brain activity of animals with crossed circulation. Exp. Neurol8, 264–277 (1963)Google Scholar
  9. Monnier, M., Hösli, L.: Dialysis of sleep and waking factors in blood of rabbit. Sciences146, 796–798 (1964)Google Scholar
  10. Monnier, M., Hösli, L.: Humoral transmission of sleep and wake-fulness. II. Hemodialysis of a sleep inducing humor during stimulation of the thalamic somnogenic area. Pflügers Arch. ges. Physiol.282, 60–75 (1965)Google Scholar
  11. Monnier, M., Hatt, A. M.: Intraventricular infusions in acute and chronic rabbits. Application of the stereotaxie method of Monnier and Gangloff. Pflügers Arch.317, 268–277 (1970)Google Scholar
  12. Monnier, M., Hatt, A. M., Cueni, L. B., Schoenenberger, G. A.: Humoral transmission of sleep. VI. Purification and assessment of a hypnogenic fraction of “sleep dialysate” (factor delta). Pflügers Arch331, 257–265 (1972)Google Scholar
  13. Monnier, M., Dudler, L., Schoenenberger, G. A.: Humoral transmission of sleep. VIII. Effects of the “sleep factor delta” on cerebral, motor and visceral activities. Pflügers Arch345, 23–35 (1973)Google Scholar
  14. Monnier, M., Hatt, A. M., Dudler, L., Cueni L. B., Schoenenberger, G. A.: Humoral transmission of sleep. Cerebral, behavioral and visceral effects of a purified sleep factor delta. In: Sleep, physiology, biochemistry, psychology, pharmacology, clinical implications (W. P. Koella and P. Levin, eds.), pp. 258–263. Basel: Karger 1973Google Scholar
  15. Monnier, M., Schoenenberger, G. A., Dudler, L., Herkert, B.: Production, isolation and further characterization of the “sleep peptide delta”. Biological aspects. In: Sleep 1974 (P. Levin and W. P. Koella, eds.), pp. 41–46. Basel: Karger 1975Google Scholar
  16. Monnier, M., Dudler, L., Gächter, R., Schoenenberger, G. A.: Humoral transmission of sleep. IX. Activity and concentration of the sleep peptide delta in cerebral and systemic blood fractions. Pflügers Arch.360, 225–242 (1975)Google Scholar
  17. Nagasaki, H., Iriki, M., Inoué, S., Uchinozo, K.: The presence of a sleep-promoting material in the brain of sleep-deprived rats. Proc. Jap. Acad.50, 241–246 (1974)Google Scholar
  18. Pappenheimer, J. R., Miller, T. B., Goodrich, C. A.: Sleep-promoting effects of cerebrospinal fluid from sleep-deprived goats. Proc. nat. Acad. Sci. (Wash.)58, 513–517 (1967)Google Scholar
  19. Pappenheimer, J. R., Fencl, V., Karnovsky, M. L., Koski, G.: Peptides in cerebrospinal fluid and their relation to sleep and activity. Brain dysfunction in metabolic disorders. Res. Publ. Ass. nerv. ment. Dis.53, 201–210 (1974)Google Scholar
  20. Pappenheimer J. R., Koski, G., Fencl, V., Karnovsky, M. L., Krueger, J.: Extraction of sleep-promoting factor S from cerebrospinal fluid and from brains of sleep-deprived animals. J. Neurophysiol.38, 1299–1311 (1975)Google Scholar
  21. Rosenblatt, M. S., Margolies, M. N., Cannon, L. E., Haber, E.: Peptides: an analytical method for their resolution by polyacrylamide gel electrophoresis applicable to a wide range of sizes and solubilities. Analyt. Biochem.65, 321–330 (1975)Google Scholar
  22. Schoenenberger, G. A., Cueni, L. B., Monnier, M., Hatt, A. M.: Humoral transmission of sleep. VII. Isolation and physicalchemical characterization of the “sleep inducing factor delta”. Pflügers Arch.338, 1–17 (1972)Google Scholar
  23. Schoenenberger, G. A., Cueni, L. B., Hatt, A. M., Monnier, M.: Humoral transmission of sleep. Isolation and characterization of a sleep factor “delta”. In: Sleep, physiology, biochemistry, psychology, pharmacology, clinical implications (W. P. Koella and P. Levin, eds.), pp. 263–267. Basel: Karger 1973Google Scholar
  24. Schoenenberger, G. A., Monnier, M.: Isolation, partial characterization and activity of a humoral “delta sleep” transmitting factor. In: Brain and sleep (H. M. Van Praag and M. Meinardi, eds.), pp. 39–69. Amsterdam: De Erven Bohn BV 1974Google Scholar
  25. Schoenenberger, G. A., Monnier, M.: Isolation and partial characterization of a humoral delta sleep transmitting factor. Biochemical approach. In: Sleep 1974 (P. Levin and W. P. Koella, eds.), pp. 46–55 Basel: Karger 1975Google Scholar

Copyright information

© Springer-Verlag 1977

Authors and Affiliations

  • Guido A. Schoenenberger
    • 1
  • P. F. Maier
    • 1
  • H. J. Tobler
    • 2
  • M. Monnier
    • 3
  1. 1.Research Division, Department of Surgery, KantonsspitalUniversity BaselBaselSwitzerland
  2. 2.Division of Application Research and Development Sandoz Ltd.BaselSwitzerland
  3. 3.Physiological InstituteUniversity BaselBaselSwitzerland

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