The experimental studies on sleep deprivation were initiated by the Russian physician and scientist, Marie de Manacéine, who studied sleep-deprived puppies kept in constant activity. She reported in 1894 that the complete absence of sleep was fatal in a few days, pointing out that the most severe lesions occurred in the brain. In 1898, the Italian physiologists Lamberto Daddi and Giulio Tarozzi also kept dogs awake by walking them; the animals died after 9-17 days, and their survival was unrelated to food consumption. In the histological study performed by Daddi, degenerative alterations, mainly represented by chromatolytic changes, were observed in neurons of the spinal ganglia, Purkinje cells of the cerebellum, and neurons of the frontal cortex. Daddi ascribed these changes to a state of autointoxication of the brain during insomnia. In 1898, the psychiatrist Cesare Agostini, interested in the psychic phenomena caused by prolonged insomnia in humans, sleep deprived dogs by keeping them in a metallic cage in order to avoid fatigue. The dogs survived about 2 weeks, and degenerative changes were observed in their brains. In these experimental paradigms, the effect of sleep loss was confounded by motor exhaustion and/or intense sensory stimulation. In spite of the absence of adequate controls, the pioneering studies performed at the end of the 19th century represented the first experimental attempts to relate sleep with neural centers and suggested that sleep is a vital function and that the brain may be affected by insomnia.