Insomnia Following Selective Decrease of Cerebral Serotonin

Pharmacologically induced decrease. It has been shown that p-chlorophenylalanine selectively decreases the concentration of serotonin in the brain without altering the concentration of either noradrenalin or dopamine. This selective action is probably effected by inhibition of the enzyme tryptophan hydroxylase (41). In cats (42, 43), rats (44), and monkeys (45), the action of this drug has been recently studied by means of electrodes permanently implanted in the brain. After a single injection of p-chlorophenylalanine (400 milligrams per kilogram) in the cat (42) no apparent variation of behavior or of the electroencephalographic record is observed during the first 24 hours. This fact demonstrates that the drug in itself has no direct pharmacological action upon the brain. Following this period there is an abrupt decrease in the in cidence of both sleep states, and after 30 hours there is almost total insomnia, as shown by continuous quiet waking behavior, mild mydriasis, and a permanent rapid low-voltage cortical activity. The reoccurrence of sleep begins after the 60th hour and is accompanied by the appearance of continuous phasic waves in the lateral geniculate body and occipItal. cortex (similar to the PGO wave activity usually observed during the slow-wave sleep that immediately precedes paradoxical sleep and during paradoxical sleep). Discrete episodes of paradoxical sleep occur, following short periods of slow-wave sleep and even immediately following waking. Slow-wave-sleep episodes of longer duration gradually reappear at shorter intervals. Normal patterns of sleep reoccur after about 200 hours. A significant correlation has been found to exist between the decrease in slow-wave sleep and the decrease in cerebral concentrations of serotonin (at the level of serotonin-containing terminals in the rat). There was no significant alteration in the cerebral concentrations of noradrenalin or dopa mine (44) (Fig. 2).

Since p-chlorophenylalanine inhibits only the first step in the synthesis of serotonin, it is possible to bypass its blocking action and thereby reestablish the concentration of serotonin by injecting the direct precursor of serotonin. 'This has been accomplished by the injection of 5-hydroxytryptophan (which readily crosses the blood-brain barrier) following the injection of p-chlorophenylalanine. In this way it has been shown that the sleep state of the animal can be readily manipulated (42). A single injection (intravenous or peritoneal) of a very small dose of 5-hydroxytryptophan (2 to 5 milligrams per kilogram) administered when the decrease in sleep has reached its maximum (30 hours following injection of p chlorophenylalanine) results in restoration of a normal pattern of both states of sleep (Fig. 3). This normal pattern may last for a period of 6 to 10 hours, after which there is a rapid return to insomnia. In another series of experiments a control cat receiving daily doses of p-chlorophenylalanine experiences a severe and long-lasting insomnia as sociated with behavioral disturbances (anorexia, inability to walk), whereas an animal receiving balanced doses of 5-hydroxytryptophan and p-chlorophenylalanine sleeps a normal amount, or even more than normal, for several days, with no behavioral disturbances. These experiments show rather conclusively that one can manipulate sleep mechanisms at will merely by interfering with the synthesis of serotonin.

The results of this study (which are in agreement with the previous findings that serotonin injected after the injection of reserpine restores slow-wave sleep) led to the hypothesis that slow-wave sleep requires the presence of serotonin at the terminals of serotonin-containing neurons. Since other pharmacological data demonstrate that paradoxical sleep is eliminated for a long period by in jection of monoamine oxidase inhibitors (36), it is suggested that a still unknown deaminated metabolite of serotonin may be responsible for the triggering of para doxical sleep (30). The results of this pharmacological study must be critically evaluated and cannot be accepted as conclusive evidence for the existence of a serotonergic mechanism of slow-wave sleep. It must be recognized that not only the metabolism of serotonin in the brain but also that in the total periphery is altered by p-chlorophenylalanine. It is therefore difflcult to rule out a possible extracerebral factor in the dramatic insomnia which follows injection of this drug.

For this reason it is necessary to make a neurophysiological study of the role of serotonin as well as a pharmacological study. Because of the topographical localization of the serotonin-containing neurons, it was found possible to selectively destroy most of these neurons and to thus diminish the level of serotonin in the brain.

Decrease by neurophysiological techniques. By means of stereotaxic methods, the serotonin-containing neurons of the raphe system were destroyed study must be critically evaluated and in cats having electrodes permanently implanted in the brain (Fig. 4). The animals' brain waves were continuously recorded for 10 to 13 days after the operation, this being the critical period for the voiding of the serotonin-containing terminals (37). At the end of this period the cats were killed (at a fixed hour so as to avoid differences due to circadian alteration of serotonin concentration) for histological evaluation of the lesioned area of the brain and for biochemical analysis of the intact regions of the brain (telencephalon, diencephalon, mesencephalon, and spinal cord). By this method, the following information was obtained: a valid quantitative measure of the sleep states (obtained from the mean percentages of slow-wave sleep and paradoxical sleep during the 10 to 13 days of recording); a measure of the extent of the lesion (the percentage of the total raphe system destroyed), obtained by topographical analysis; and an estimate of the percentages of serotonin and noradrenalin in the brains of cats with brain lesions relative to the percentages in the brains of cats which had undergone sham operations (46).

After destruction of a large part (80 to 90 percent) of the raphe system, a continuous state of insomnia, as evidenced by the cat's behavior and by the encephalographic record, is observed during the first 3 to 4 days. In the period that follows, some slow-wave sleep is observed, but the total duration of the periods of slow-wave sleep never exceeds 10 percent of the recording time. Paradoxical sleep is never observed in these cats. Partial lesions of the raphe system (in the anterior or posterior regions) result in an insomnia which is less pronounced (due to a gradual recuperation after the first 2 days). In cats with such lesions paradoxical sleep is found to occur in association with slow wave sleep when the daily percentage of slow-wave sleep exceeds 15 percent; when the percentage is less than this, no paradoxical sleep occurs (Fig. 5). Lesioning of the rostral nuclei often results in "narcolepsy," with paradoxical sleep following directly upon the waking state. Destructions involving less than 15 percent of the raphe system provoke no significant change in the sleep states. A significant correlation between the volume of the raphe system destroyed and the percentage of sleep was thus demonstrated (47) (Fig. 6).

Biochemical analysis of cat brains in which there had been significant de struction of the raphe system revealed a significant decrease in cerebral serotonin with no variation in the concentration of noradrenalin. Thus, a significant correlation between the volume of the raphe system destroyed, the intensity of the resulting insomnia, and the decrease in cerebral serotonin was demonstrated (Fig. 6) (Table 1). In view of the neuropharmacological findings which showed that inhibition of the synthesis of serotonin (by p-chlorophenylalanine) produces total insomnia and that administration of 5-hydroxytryptophan, the precursor of serotonin, reverses this insomnia, the evidenee favoring the involvement of serotonin in sleep is exeedingly strong.

However, the intrieate and intimate bioehemieal mechanisms underlying the role of serotonin in the sleep process are as yet not well defined. Analysis has failed to reveal a significant change in the concentration of endogenous sero tonin during total deprivation of sleep (48). Nevertheless it has recently been reported that a small but significant fall in serotonin concentration accompanied by a rise in 5-hydroxyindoleacctic acid concentration occurs in the rat hrain during deprivation of paradoxical sleep (49). It has also been demonstrated that the brain concentration of serotonin is higher during the day (when the rat sleeps most of the time) than during the night (22). Only through in vivo study of the tunrnover of serotonin can understanding be achieved of the intricate processes underlying the serotonergic mechanism of sleep.

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