Evidence of the duality of the states of sleep
(e) Structural findings
(i) Lesions of tlte pontine reticular formation
We have reported elsewhere the results of coagulation of the pontine reticular formation at the level of the dorsal and lateral part of the nucleus reticularis pontis oralis et caudalis (M. Jouvet, 1962a). There is no extinction of the nuchal EMG in these animals nor periods of rapid activity during behavioural sleep. On the other hand, typical EEG patterns of wakefulness and slow sleep persist. When deprived of P.S. these animals show periodical behavioural disturbances resembling hallucinations that occur most commonly after a phase of slow sleep. The percentage of slow sleep (60% of the total time) was usually normal in these animals (Fig. 10). These results speak in favour of a duality of the nervous structures responsible for triggering off the two states of sleep. Study of the waking-sleeping rhythm of chronic pontile cats strengthens this theory.
(ii) Sleep in the chronic pontile cat
The experimental evidence by which the periodic phases of atony in chronic mesencephalic or pontile animals can be identified with the phase of fast cortical activity in intact animals during sleep, has already been reported (M. Jouvet, 1961, 1962a). It is on these results and the results of coagulation of the pontine reticular formation that the concept of rhombencephalic sleep is based. But the periodic atonic state of pontile animals cannot be unreservedly identified with P.S. (Moruzzi, 1964).
Our initial results had been obtained in animals which did not survive more than 10 days, and we therefore felt it necessary to obtain new data from animals with a much longer period of survival. Better knowledge of their requirements enabled us to study some 20 posterior mesencephalic or pontile animals for over 2 months. For such periods it is much easier to observe the different states of wakefulness. In a number of animals it was further possible to study more extensively the electrical activity of the brain stem.
Our technique was very similar to that developed by Bard and Macht (1958). After total section of the brain stem in front of the tentorium with a leukotomy knife, the cerebral hemispheres and the thalamus are removed by aspiration. A hypothalamic island is left in situ, the dorsal surface of which is flush with the Horsley-Clark plane zero. The brain stem section between the pons and the remaining hypothalamic island is then completed by aspiration and an acrylic strip 1-2 mm thick and 15 mm wide, fixed dorsally to the tentorium cerebelli, is wedged between the brain stem section surface and the remaining hypothalamic island in such a manner as to prevent any neurocrine connections between the hypothalamus and the brain stem (Fig. 11). These poikilothermic preparations are then placed in an incubator and observed through a perspex window. A thermo-electric probe in the rectum of the animal conveys its temperature to a regulator (Fig. 12, B). The animal's temperature is thus maintained constant at any desired level by altering the heating element of the incubator. The urine is collected (K). The EEG activity of the brain stem (pons and medulla oblongata) and the EMG activity of the neck, are registered by permanently implanted electrodes and transmitted by cable I to the amplifiers (C) of the EEG apparatus (D). After amplification the muscular activity is integrated in an Oneirograph (F). During P.S. the absence of EMG activity is conveyed to a system of relays (G) so that either the EEG motor (E) is automatically set in operation to record P.S. during the night, or the periodicity of P.S. is registered by a signal on a slow-running apparatus (H). The sleeping-waking rhythm of the animals can thus be continuously recorded. More than 10 000 periods of P.S. have thus been observed in 20 chronic pontile cats (Fig. 13).
Immediately following the postoperative phase (after 12-36 h) the animals alternately presented wakefulness and P.S. without slow sleep intervening.
Wakefulness is characterized by muscular hypertonia with regular respiration - a complete absence of movements over the first few days. This state is thus difficult to identify since behaviourally it can resemble calm sleep during the first week. After about 10 days, however, this state can be regarded as a state of wakefulness. Theanimal then responds to acoustic stimuli of high intensity by turning its head towards the stimulus. Furthermore, as observed by Bard and Macht (1958), the animals are able to remain in a crouching position, with the head raised, supporting themselves on their forelegs (Sphinx position). In this state the EEG of the brain stem shows continuous, fast, microvoltage activity which is not affected by any sensory stimuli.
The waking state is periodically and regularly interrupted (Fig. 13) by a state of which all the characteristics are similar with those of P.S. in intact cats. This state consists of complete muscular atony, during which the head falls abruptly and eye movements appear. These movements are lateral (i.e. dependent on N. VI) external, rapid, returning slowly to the median line with a frequency of 20-30 per min. Their oculographic appearance is monotonous, in contrast to those of intact cats where several types of bursts of varying complexity can be recognized. During this state respiration becomes more rapid and irregular, while the heart rate increases in most cases. At the same time a characteristic electrical activity appears in the pons (Fig. 14): either isolated monophasic spikes or groups of 'pseudo spindles', 3-5 per sec, repeated at a frequency of 10-15 per min, sometimes rising in amplitude, seldom rising and falling. In the majority of cases these spikes accompany the rapid eye movements, but not infrequently, especially in the first few days, there are isolated spikes without eye movements. The topographical distribution of these elements, and their electrical appearance, is similar to that in the pontine reticular formation during P.S. (M. Jouvet, 1962a; Brooks and Bizzi, 1963).
(1) P.S. occurs immediately after the waking state. It has, in fact never been possible to demonstrate EEG or behavioural criteria of slow sleep in pontile animals.
Whereas in intact cats the electrodes located in the pontine reticular formation regularly receive spindle activity and/or slow waves during the slow sleep preceding the appearance of P.S., no slow activity or spindles have ever been recorded during the minutes preceding P.S. in pontile animals. Furthermore, no behavioural criteria have been observed for a stage intermediate between wakefulness and P.S. The state of the pupils and the nictitating membranes remains constant in these animals. In certain cases - mesencephalic animals with intact oculomotor nucleus - a tonic ocular sleep syndrome (in which the eyeballs rock in and down) can appear. This phenomenon immediately precedes, by a few seconds, the rapid eye movements of P.S. It has never been observed alone during long periods without P.S. Furthermore in 90% of such animals muscular activity remains constant during the waking phases and begins to diminish only 30-40 sec before the inception of P.S., when the first monophasic spikes appear in the pons. In the normal animal, on the other hand, the nuchal EMG activity diminishes considerably in about 60% of cases during the phase of slow sleep preceding P.S. Thus, the muscular criterion of slow sleep (which even in intact animals is not absolute and seems to depend on the animal's posture and thus often on the environmental temperature) does not show the hypotonic phases which would precede P.S. and would provide evidence of an intermediary behavioural sleep between wakefulness and P.S.
(2) Periodicity of P.S. The mean duration of P.S. in pontile and intact cats is the same (6 min and 6 min 20 sec respectively). Thus the two phenomena, which are identical from the point of view of subcortical electrical activity and of behaviour, are also identical from the point of view of time and can be entirely equated one with the other. The only difference is found in their periodicity. In pontile cats it is very regular and P.S. accounts for 10% of the total time (there is no distinction between day and night). In normal animals, on the other hand, the innumerable environmental influences (to which the pontile cat is almost totally insensitive) make it difficult to establish a regular periodicity of P.S. Furthermore, P.S. occurs only after periods of slow sleep when it accounts for approximately 25% of behavioural sleep, or 15% of the total time (since intact cats slept for an average of 70% of the time under the conditions of our trial, i.e. in sound-proof cages to which they have become 'accustomed').
Summarizing, no EEG or behavioural phenomenon indicates the existence of slow sleep in pontile animals. On the other hand, the periods of muscular atonia occurring in such animals immediately after awakening can be completely equated with the P.S. of intact animals. Their average duration is the same as the latter, while the percentage of the total time they represent is slightly lower. P.S. thus appears to be absolutely independent of slow sleep.
After having discussed in this first section the autonomy of P.S. with respect to slow sleep we shall pass in the second to some experimental results which make it possible to define some of the mechanisms at work. Most of these results have been obtained in pontile animals, in which P.S. appears as a veritable 'biological clock', the periodicity of which is subject to fewer factors than in the intact animal.
The possibility of triggering off P.S. as a reflex suggests that peripheral mechanisms may be involved, but not exclusively, as the results of different deafferentations have shown.
Experiments with automatic deprivation of P.S. in pontile animals suggest the existence of an active mechanism in the lower brain stem.
Finally, the effects of temperature and certain drugs, and the relationship of P.S. with the internal milieu permit the hypothesis of a periodic function in which the neuroglia is perhaps implicated.