Extraction of sleep promoting factor S from cerebrospinal fluid and from brains of sleep deprived animals

Abstract

Sleep promoting factor (factor S) was extracted, partially purified, and concentrated from cerebrospinal fluid and from acid acetone extracts of brain stem and cortex of sleep deprived goats and sheep. Solutes > 500 daltons were largely removed by serial ultrafiltrations through molecular sieves (Amicon membranes UM10 and UM05); solutes < 350 daltons were largely eliminated by gel filtration through Sephadex G10 columns. Sleep promoting activity was found in a fraction eluted prior to [14C] sucrose marker. Concentrated fractions were infused intraventricularly in rats (0.1 ml in 30 min just prior to 12 h dark cycle) and in rabbits (0.3 ml in 90 min in morning). Sleep promoting activity was assayed by decrease in nocturnal locomotor activity of rats and by duration and amplitude of slow wave cortical EEG in rabbits. Twentyfold concentrates of CSF or material from 1 g cortex or brain stem of sleep deprived goats or sheep depressed locomotor activity of rats to about 50% of normal during first 6 h and to about 65% of normal during second 6 h, with complete recovery the following night. In rabbits, the equivalent of 3 g brain tissue increased the duration of slow wave sleep for about 7 h subsequent to infusion, with maximum effect between 3 and 5 h. The active fraction contained material which reacted with fluorescamine; the ratio of fluorescence measured at pH 7 and pH 9 was in the range 0.4-0.6. Sleep promoting activity was observed following infusion of less than 1 nmol primary amine equivalents referred to Gly Gly peptide standard. Sleep promoting activity was destroyed by incubation with Pronase (10 μg ml-1, 37°C, 1 h). Sleep induced by exogenous factor S in rabbits was characterized by slow wave EEG, which exceeded in amplitude and mean integrated voltage the maximum values recorded in the same animals during normal sleep. Similar high amplitude slow wave EEG was observed in the same rabbits during the first hours of sleep following 24 h of sleep deprivation. The results indicate that induction, duration, and electrical concomitants of slow wave sleep are related to factor S, whether this be administered intraventricularly from exogenous sources or whether it is allowed to accumulate in brain during sleep deprivation. Properties of factor S appear to differ in important respects from those of 'factor delta' described by Monnier and Schoenenberger. However, factor S may be related to the sleep promoting factor extracted from the brain stems of sleep deprived rats described by Nagasaki et al.