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A pharmacological approach to paradoxical sleep
Physiology and Behavior. Vol. 3, pp. 91-97. Pergamon Press, 1968 Printed in Great Britain
A Pharmacological Approach to Paradoxical Sleep' A. L O I Z Z O A N D V. G. L O N G O
Instituto Superiore di SanitY, Rome, and Centro di Neurofarmacologia del CNR, Department of Pharmacology, University of Sassari, Sassari, Italy (Received 3 June 1967) Lolzzo, A. AND V. G. LOhv,o. A pharmacological approach to paradoxical sleep. PHYSIOL.BEHAV.3 (1) 91--97, 1968.-A series of experiments were performed in order to produce paradoxical sleep (PS) by means of drugs. This paper reports on the EEG and behavioral effects of compounds possessing myrelaxant properties, and in particular: mephenesin, zoxazolamine, phenyrarnidol, several 2-amino benzothiazole derivatives, chlordiazepoxide, diazepam. Two series of experiments were carried out in the rabbit. In one group of animals, bearing chronically implanted electrodes, the drugs were injected intravenously; in the second group, consisting of acute preparations, the drugs were administered into the vertebral artery. In the first group a clear-cut PS pattern could not be related to drug administration. With intravertebral injection it was possible to obtain in some cases a picture similar to PS. Diazepam and 2-amino-4chlorobenzothiazole, at doses of 0.5-2 mg were most active in eliciting a PS-like state. Paradoxical sleep
Sleep
Paradoxical sleep and drugs
IN 1958 Dement observed, in the course of eleetroencephalographic registrations on cats bearing chronically implanted electrodes, the presence of a period of behavioral sleep during which the tracing desynchronized. This was accompanied by a reduction in muscular tone, especially of the neck muscles, and by nystagrniform movements of the eyeballs. This complex was called "Activated Sleep" by Dement. During the following years, a great deal of research was conducted with the purpose of clarifying the various aspects of activated sleep (also designated: desynchronized sleep, paradoxical sleep, rapid eye movement (REM) sleep, romboencephalic sleep, para-sleep). From all the investigations on this phenomenon a clear-cut physiological entity has evolved.
Sleep and EEG
EEG
cedes certain motor phenomena, consisting of muscular relaxation, particularly of the neck muscles, conjugated rapid eye movements, both in lateral and vertical directions, at times nystagmiform, at other times having a rotatory component. Twitchings of vibrissae, of the ears and of the legs muscles sometimes occur. The pupil is myotic, cardiac rate is altered, respiration becomes irregular, rapid and superficial. Rectal temperature is sometimes lowered by 0.1-0.2°C, while that of the internal organs (uterus, brain, liver) at times increases as much as 0.7°C. PS is usually preceded by a period of slow sleep; however, under some experimental conditions, i.e., following a period of PS deprivation [5], or in pathologic conditions, such as during attacks of narcolepsy, PS can immediately follow a phase of wakefulness [9, 24]. In studies performed in animals with chronically implanted electrodes, the average duration of the PS periods was calculated to be about 30-40 per cent of the behavioral sleep and approximately 20-25 per cent of the total registrations. The appearance of PS is percentwise greater in young animals [6].
Characteristics of Paradoxical Sleep (PS) Slow wave sleep is characteristic of vertebrates; PS, on the contrary, appears in the zoological scale in a rudimentary way in birds and acquires well defined patterns in mammals, perhaps suggesting a parallel phylogenic and ontogenic evolution of the two sleep states. The following E E G signs are considered characteristic of I S : low voltage rapid activity (20-30 cp/s, 30-40 ~tV) in the cortex, diencephalon and mesencephalon, similar to the tracing recorded during wakefulness. The hippocampus presents regular waves, at a frequency of about 5 cp/s and at a larger amplitude (50-70 tLV); a similar picture is observed in the periaqueductai gray substance, the interperpendicular nucleus posterior hypothalamus, septurn and gyrus cinguli. The lateral geniculate nucleus presents spike-like activity, synchronous with the ocular movements, to be later described. This picture coincides or shortly pre-
Methods Employed to Elicit PS, with Particular Attention to Drug-induced PS Many methods have been employed to cause the artificial appearance of PS. Low frequency electrical stimulation of the hypothalamus, of various regions of the llmbic system, of the posterior and median part of the mesencephalic teg-
mentum (nuclei reticularis pontis oralis and reticularis pontis caudalis) have elicited PS [3, 5, 28] obtained PS by electrical stimulation of various neocortical areas. In animals cooled to rectal temperature of 31-34°C an increase in PS periods
tAn abstract of this paper was presented at the Annual Meeting of the Italian Physiological Society (Loizzo and Longo, 1966). 91
92 is obtained; however, these disappear when the temperature is further lowered. Sawyer and co-workers [11, 29] have studied the relationships between sexual stimulation and PS in the female rabbit, describing PS as an "after reaction" to coitus. The possibility of inducing PS with drugs has also been studied. Based on the observations of Laborit e t aL [15] on the hypnotic properties of sodium 4-hydroxybutyrate and of 4-butyrolactone, Jouvet et al. [7] found that 50-100 mg/kg (i.v.) of these two drugs elicited in the cat the appearance of PS 5-10 min after the injection. Matsuzaki e t al. [22] confirmed the results of Jouvet e t al. [7] and described the same effect for other short-chain fatty acids. Tokizane [30] obtained results similar to those of Matsuzaki et al. [22] and of Jouvet et al. [7], using both chronic intact cats and acute preparations (mesencephalic, pontine and bulbar). In addition, he demonstrated that, in the acute prebulbar cat, sodium nbutyrate (i.v.) produces a marked muscle relaxation. The same author described the appearance of PS, in the acutely cerebellectomized cat, within 2-3 sec following intravertebral injection of 0.5 mM/kg of sodium n-butyrate. This effect was not obtained when the drug was injected into the lingual or carotid artery. Winters and Spooner [31] on the other hand, performed a detailed analysis of the EEG effects of sodium 4-hydroxybutyrate in the cat. In addition to observing an increase on the PS periods, these authors noted the appearance of EEG and motor convulsive manifestations. Rosina and Mancia [26], in a series of experiments carried out in cats with chronically implanted electrodes and with the basilar artery ligated at the median or caudal pontine level, found that the intravertebral injection of Thiopental (0.4--0.6 mg) during synchronized sleep caused the appearance of PS within a few seconds. Thiopental was without effect if the animal was awake; if a state of PS was already existing, the animal awoke. In animals with rostro-pontine ligature of the basilar artery, the barbiturate constantly caused an awakening response with an increase in muscular tone, thus confirming the results of Magni et al. [19]. The same arousing effect is provoked by the injection of barbiturates into the vertebral circulation of the rabbit, without any damping of the basilar artery [25]. Yamamoto and Domino [32] obtained an increase of the PS periods using small doses (0.005--0.01 mg/kg, i.v:) of nicotine in chronically implanted cats. Ledebur and Tissot [16] brought about PS in the rabbit by means of microinjections of 5-hydroxytryptophan in the pontine reticular formation. George e t al. [4], also using the microinjection technique, provoked PS using compounds possessing muscarinic action oxotremorine, carbachol) deposited at the level of the mesencephalic reticular formation of the cat; the PS lasted 40-50 min and was antagonized by atropine injected in situ or systemically. According to these authors, in that region of the reticular formation there is a cholinergic center, activated by the drugs, responsible for the appearance of PS. Khazan and Sawyer [12] demonstrated that atropine elevates the threshold for the production of PS by electrical stimulation of the pontine reticular formation. The same effect was obtained with pentobarbital, morphine, chlorpromazine. These authors also reported the disappearance of PS after treatment with LSD-25 and amphetamine. Chlorpromazine, in doses of 5-10 mg/kg, s.c., does not have any influence on the periodicity and characteristics of the behavioral PS, while it inhibits the electrocortical manifestations (EEG activation) [5]. Inhibition of PS has been described after treatment with atropine [5] and imipramine
LOIZZO AND LONGO [l 3]. According to Khazan and Sawyer [12], small doses of reserpine (0.1 mg/kg) potentiate PS, while larger doses (0.5 mg/kg) inhibit its appearance for a few days [21]. Some years ago we became interested in this problem, our working hypothesis was based on the premise that PS, like other physiological states, such as sleep and wakefulness, could be duplicated by the administration of drugs. In particular, attempts were made to obtain PS as the immediate consequence of drug administration so that the pattern so obtained could be definitely ascribed to the treatment. This paper reports in detail the results obtained with compounds possessing to a greater or lesser extent myorelaxant properties. Investigations on these substances was prompted by the observations of Jouvet [5], who noted in decerebrated cats, for the whole duration of PS, a complete muscular relaxation in the place of the rigidity in this type of preparation. The following drugs were studied: three aminobenzothiazole derivatives, Chlordiazepoxide, Diazepam, Mephenesin, Pheniramidol, Zoxazolamine (see Table 1). These compounds were selected because of their centrally-mediated myorelaxant action. For comparative purposes, drugs belonging to other pharmacological categories were also employed. TABLE 1 THE TABLELlSl'S THE DRUGS AND DOSAGESADMINISTERED INTRAVENOUSLY TO RABBITS BEARING CHRONICALLY IMPLANTEDELECTRODES Mephenesin Zoxazolamine~a Phenyramidol Chlordiazepoxide Diazepam (b) SKF 1448 A (2-amino-4-chloro benzothiazole) (e) SKF 1407 A (2-amino-4-methylbenzothiazole) SKF 1153 A (2-amino benzothiazole)
10--120 mg/kg 2-10 mg/kg 2-20 mg/kg 1-10 mg/kg 0.5-5 mg/kg 1-10 mg/kg 1-10 mg/kg 1-10 mg/kg
ta)10 per cent in polyethylenglycol. (b)The commercially available ampoules of Valium (Roche) were used, containing a solution of 0.5 per cent of Diazepam in organic solvents. (e)l per cent in ethanol 50 per cent. MATERIALSAND METHODS TWO series of experiments were run, using unanesthetized rabbits. One series of experiments utilized six animals with electrodes implanted chronically in various cortical and subcortical zones. Silver wires were passed through the neck muscles and through the skin of the outer canthi of the eyes, so as to respectively register muscular activity and eye movements. The drugs were injected by remote control using a long polythene tube connected to a cannula inserted permanently into the jugular vein. During the experiments the animal was kept in a shielded cage (ll × 1 × 0.7 m); the electrodes were connected with the EEG apparatus by means of long wires, thereby insuring complete freedom of movement. The other series of experiments was carried out in 72 rabbits prepared acutely for registration. In these animals the drugs were injected into the vertebral artery. After exposure of the subclavian artery, and tying-off of the collaterals (transversa
PHARMACOLOGICAL APPROACH TO PARADOXICAL SLEEP
colli, thyroidea inferior and mammari ainterna), thereby leaving open only the vertebral artery, a thin polythene canula was introduced in the subclavian (for detail of the technique, see also Calma and Wright [1], Mantegazzini et al. [20]. To insure direct access and maximum concentration of the drug at the ponto-mesencephalic area, the omolateral carotid was also tied. Electrodes were then fixed in various cortical and subcortical zones (cf. for technique Longo [18]). The eye movements were registered by means of two screw electrodes fixed in the orbital crest. The activity of the neck muscles was led by means of silver wires threaded separately through the left and right muscular bodies so that the E M G ' s of the two sides could be obtained. Registration was started at least one hour after the preparation. Except when otherwise indicated, aqueous solutions were used, in amounts of 0.1-0.5 ml; repeptitive controls were performed using saline or the diluting media. At the end of the experiment the patency of the pathway was controlled by injection of indian ink through the cannula into the vertebro-basilar circulation. The positions of the deep electrodes were histologically checked using electrolytic deposition of iron. RESULTS
Experiments in Chronically Implanted Animals In the animal bearing implanted electrodes, all the myorelaxant drugs (see Table 1), administered intravenously, provoke, at a certain dosage levels, muscular relaxation, accompanied by a synchronization of the tracing. When the drugs were injected rapidly, a short period of spasticity preceeded the muscular relaxation; sometimes nystagmus appeared. In the period following the acute phase of drug action, the PS episodes were not more frequent than those observed in the non-treated animals.
Intravertebral Drug Administration in the Acute Preparation In the course of the experiments it was observed that some of the myorelaxant drugs injected intravertebrally caused a series of symptoms, both behavioral and EEG, which recalled in some aspects the PS polygraphic patterns. These signs appeared within a few seconds following drug administration and consisted of muscular relaxation, particularly of the ears and the neck, eye movements, quivering of the vibrissae and
93
activation of the EEG. Stimuli of various kinds (touching of the fur, pinching of the tail, noise) were not able to alter the course of this period of PS-like state, which lasted, however, for a very short time; within a few minutes the E E G and muscular tone returned to normal. When repeated injections were performed at suitable intervals, the same manifestations were not always provoked, although particular care was taken to inject the drug only during the sleeping state of the animal, as shown by the EEG. In fact in some cases an entirely different reaction appeared, consisting of controlateral head torsion, struggling and hypernea. It was not possible to foresee the occurrence of such a reaction, which seemed to be unrelated either to the dose or to the basic state of the animal. The impression was gathered that the first injection of each drug in each experiment had a more typical and lasting effect than subsequent administrations. Table 2 lists the doses of the drugs and the occurrence of a PS-like polygraphic picture. The 2-amino benzothiazole derivative, S K F 1448A, proved to be the most effective compound producing this picture. Out of a total of 16 administrations, performed in five animals, S K F 1448A caused in seven cases the PS-like polygraphic picture, while in four cases, together with E E G activation and eye movements, muscular neck relaxation was noted only omolaterally to the side of injection. In the five remaining cases an atypical reaction was observed; this consisted of the appearance of 3-4 cp/s high voltage wave in the superficial and deep leads, chewing movements, head twitchings, contraction of the neck muscles, and struggling. The doses ranged from 0.1 to 4 mg in tote; the PS-like picture appeared with lower doses, from 0.2 to 2 (Fig. 1). Similar results have been obtained with two other 2-amino benzothiazole derivatives (see Table 2) which were active, however, at higher dosages. Diazepam, in doses of 0.2-2 mg, provoked a polygraphic tracing similar to PS in 4 out of 15 cases; in four other cases the neck muscles relaxation was more pronounced on one side; the remaining seven cases demonstrated the atypical reaction already described (Fig. 2). Eight trials were performed with Chlordiazepoxide, in doses ranging from 1-20 mg: activation of the tracing accompanied by spasm of the neck muscles was the prevailing picture observed. Mephenesin was tested in 12 trials (2-30 nag): the response consisted of muscular relaxation and nystagmus,
TABLE 2 EEG AND BEHAVIORAL
EFFECTS OF THE MYORELAXANT DRUGS INJECTED INTRAVERTEBRALLY IN THE RABBIT
Total trials
Doses in mg
Appearance of PS-like patterns
Diazepam SKF 1448 A
15 16
0.2-2 0.1-4
8 11
SKF 1407 A SKF 1153 A Mephenesin
5 6 12
2-4 2-4 2-30
Chiordiazepoxide Zoxazolamine
8 9
1-20 0.5-15
Phenyramidol
4
1-10
2 2 -m
Remarks the other injections provoked EEG activation and spasm of the neck. the other injections provoked the appearance of 3 c/see waves in the EEG, head twitchings, struggling. see above. see above. the picture observed with doses higher than 10 nag was neck muscles relaxation, nystagmus, slow waves in the EEG. the picture observed was EEG activation and spasm of the neck. in two cases, 15 mg of the drug gave rise to EEG activation and muscle relaxation, no nystagmic movements were present. the picture observed with doses higher than 4 nag. was EEG activation and spasm of the neck.
94
LOIZZO A N D LONGO controllo
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FIG. 1. Occurrence of a PS-like polygraphic picture after intravertebral administration of 2-amino-4 chlorobenzothiazole (SKF 1448 A) in the rabbit. A control tracing is shown in the upper part of the figure; the lower tracing, registered 30 see after 2 mg of SKF 1444 A, shows activation of the electrocorticogram, scattered eye movements, considerable reduction of neck EMG activity of both sides. Leads--FPD: right fronto-parietal cortex; POD: right parietooccipital cortex; ORB: orbital electrodes for the registration of eye movements; MD: right nuchal muscles; MS: left nuchal muscles.
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FIG. 2. Development of a PSllike polygraphic picture upon intravertebral administration of Diazepam in the rabbit. The three tracings are continuous; at the arrow 0.4 mg of the drug is injected. After a brief struggling, relaxation of the nuchal muscles ensues, more evident on the side corresponding to the injection (MD). Burst of jerky movements of the eyes and of the auricular muscles are also noticed. Leads--FD: right frontal cortex; PD right parietal cortex; HW: left dorsal hippocampus; ORB: orbital electrodes for the registration of eye movements; MS: left nuchai muscles; MD: right nuchal muscles.
PHARMACOLOGICAL APPROACH TO PARADOXICAL SLEEP which occurred simultaneously to the appearance of slow waves in the EEG. Only in 3 trials was activation of the E E G noticed, however, it was short duration and could also be attributed to causes other than the drug. Zoxazolamine and Phenyramidol, injected in doses of 0.5-15 mg did not cause the appearance of a PS-similar picture.
Effect of other Compounds Administered h: the Vertebral Artery on the EEG and Behavior So that comparison may be made with other reported results, a series of additional experiments were run to determine the activity of various compounds to which a PStriggering effect had been attributed. The administration of eserine (0.05--0.1 mg) caused a PS-similar picture: however, the neck muscle hypotonia was covered somewhat by muscle fasciculations, that should be attributed to a peripheral origin. These were evident above all in the muscular masses homolateral to the injected side. Acetylcholine (0.001-1.01 mg) and arecoline (0.1--0.5 mg) were responsible for desynchronization of the tracing, accompanied by an increase of the amplitude of the electromyographic recording of the neck muscles. Marked salivation followed arecoline administration. Pentobarbital and Thiopental, injected in doses of 0.5-15 mg, provoked activation of the EEG, spasm of the neck muscles and prolonged oculogyric crises; higher doses caused a late synchronisation of the tracing. Analogous results were obtained with Nitrazepam (2-10 mg). Sodium n-butyrate (1-30mg) did not provoke the PS-like picture; the most frequently observed reaction was a desynchronization of the tracing and an increase of the E E G of the neck muscles. The data obtained with the other drugs are summarized in Table 3.
95
Experiments Performed on Animals with Ligature of the Basilar Artery The two drugs (SKF 1448A and Diazcpam) that were found most active in producing the PS-like picture were administered intravertehrally to five animals in which the basilar artery was acutely tied-off at the caudo- or medio-pontine level. Under these conditions the two drugs produced symptoms which were not previously seen. A t doses as low as 0.5 mg there was immediate respiratory arrest (reversible after 5-15 min of artificial respiration) and disappemance of the corneal reflex. Thiopental (0.5-1 nag), in comparative trials, demonstrated the same effects; in only one case out of five did it cause a PS-similar picture.
DISCUssION
These results demonstrate that it is possible to obtain an E E G and behavioral picture similar in many aspects to that of PS by means of injection of som¢ myorelaxant drugs into the vertebral artery. This PS-like picture enstms immediately after the administration and is independent of the basal state of the animal, in that it appears both when the drug is given during synchronization or during desynchronization of the E E G tracing. This effect is typical of an "acute" pharmacological action, and can be compared to the narcotic state induced by intravenous administration of barbiturates or to the state of wakefulness following intravenous injection of amphetamine. The compounds which in our experiments were able to induce this PS-like state upon systematic administration cause a generalized muscular relaxation that is accompanied by an E E G picture of synchronization. The intravertebral approach, in all probability, induces this particular picture
TABLE 3 THE TABLELISTSALL THE DRUGSTRIED IN THE COURSEOF THE INVESTIGATION.THE DATA SHOULDBE CONSIDEREDONLY ORIENTATIVE.
Total trials
Doses in mg
Acetylcholine Arecoline Eserine
3 3 5
0.001-0.01 0.1-0.5 0.02-0.1
Sodium n-butyrate
5
1-30
Pentobarbital
8
l-15
Thiopental Hydroxydione sodium Nitrazepam Ethyl urethan
6 2 4 7
0.5-2 1-2 0.5-2 10--60
Apomorphine Chlorpromazine Trihexyphenydil
4 2 4
0.5-2 0.5-1 0.5-2
Tryptamine Dihydroxyphenylalanine (DOPA)
2 2
1-2 2-5
Remarks EEG desynchronization, spasm of the neck. see above, plus salivation. EEG desynchronization, nystagmus, fasciculations of the neck muscles, particularly of the side of injection. EEG desynchronization, spasm of the neck muscles, with highest doses spikes appear in the EEG. EEG desynchronization, spasm of the neck, ocuiogyric crises, mydriasis. see above. see above. see above, no octdogyric crises were noticed. desynehronization of the EEG and relaxation of the neck muscles, accompanied by tachypnea, was noticed in two cases. EEG synchronization, relaxation of the neck muscles. EEG synchronization, spasm of the neck muscles. EEG desynchronization with 0.5 rag, grand mal EEG patterns with 2, no changes in the EMG of the neck. EEG desynchronization, head tremors. no changes.
96
LOIZZO AND LONGO
since it selectively concentrates the action of these drugs in the mesencephalon, to which has been attributed a role in the origin of the E E G and motor phenomena characteristic of PS [5]. A PS-like picture appearing acutely following intravertebral injection of sodium n-butyrate has been described by Tokizane [30] in the decerebellated cat. Under our experimental conditions it was not possible to reproduce these results. In fact this compound, in the dose indicated by this author, injected intravertebrally in the rabbit, always gave rise, rogether with an E E G activation, to an increase in the neck muscular tone. Rosina and Mancia [26] observed the appearance of PS following intravertebral administration of 0.1-0.4 mg of Thiopental in the chronic cat with ligature of the basilar artery at the medio- or caudo-pontine level. Our results indicated that ligature of the basilar artery at this level primarily cause a marked increase of the toxicity of this drug; disappearance of the corneal reflex (noted also by the above-mentioned authors) and respiratory arrest. The inconstancy of results upon intravertebral administration of drugs noted in our experiments has been reported by others who have used the same technique. Rosina and Mancia [26] pointed out that following Thiopental injection sometimes ataxia and spasms appeared instead of the PS-like picture. Rosadini et aL [25] and Tokizane [30] also insist on the variability of the EEG, and motor responses following drug administration by the same route; Rosadini et aL [25] attribute this phenomenon to spasms of the vertebral artery. Other possible mechanisms are: air emboli, speed of injection and distribution in the involved areas. In the course of the present investigation, for example, the posterior cerebral artery was not always injected with the indian ink administered at the end of the experiments to control the patency of the vertebro-basilar vessels. In regards to the mechanisms of the E E G and behavioral patterns provoked by some of
the myorelaxant drugs injected intravertebrally, one must consider them in relation to the mechanism underlying PS. Jouvet [5] indicates the localization of the PS-triggering centers in the pens. The nucleus caudalis reticularis pontis seems to play a prominent role in the production of the E E G and motor manifestations of the PS: electrical stimulation of this nucleus [5] provokes PS under certain experimental conditions, while in other cases excitement and muscular spasms were seen. Therefore, also upon stimulation, a certain variability of the response has been reported, similar to that following the intravertebral administration of drugs. The negative results obtained with other drugs and with the control injections of saline and of the solvents permits the exclusion of an aspecific effect. The drugs which caused this reaction in our experiments all have in common an effect on the central internucial neurons system. This effect has been demonstrated at various levels of the neuraxis: blockade of the spinal and supraspinal polysynaptic reflexes and of the bulbomesencephalic inhibitory and facilitatory system [14, 23]. A possible explanation is that these drugs provoke the PS-like syndrome because of the high concentration reached, due to the route of the administration, at the level of the bulbo-mesencephalic centers. The E E G desynchronization could be explained through inactivation of the brain stem centers having a tonic facilitating influence on E E G synchronization. A similar interpretation has been given to the desynchronization obtained in the intact animal with intravertebral administration of barbiturates [27]; these drugs, however, concomitantly produce behavioral signs of excitation [25]. Available data do not permit us to state whether the relaxation of the neck muscles and the nystagmic movements are due to paralysing or irritating effect; the possibility exists that both are operating simultaneously on the different neuronal systems present at the bulbo-mesencephalic level.
REFERENCES 1. Calma, I. and S. Wright. Action of acetylcholine, atropine and eserine on the central nervous system of the decerebrated cat. J. PhysioL, Lend. 103: 93-102, 1944. 2. Dement, W. The occurrence of low voltage, fast, electroeencephalogram patterns during behavioral sleep in the cat. Electroenceph. clin. NeurophysioL 10: 291-296, 1958. 3. Di Paola, M., G. F. Rossi and J. Zattoni. Induction of EEG desynchronized sleep by electrical stimulation of the neocortex. Archs. ital. BioL 103: 812-831, 1965. 4. George, R., W. L. Haslett and D. J. Jenden. A cholinergic mechanism in the brainstem reticular formation: induction of paradoxical sleep. Int. J. Neuropharm. 3: 541-552, 1964. 5. Jouvet, M. Reeberches sur les structures nerveuses et les m6canismes responsables des differents phases du sommeil physiologique. Archs ital. BioL I00: 125-206, 1962. 6. Jouvet, M. Paradoxical sleep: A study of its nature and mechanism. In: Progress in Brain Research, Vol 18, edited by Elsevier, Amsterdam, 1965, pp. 20-62. 7. Jouvet, M., A. Cier, D. Mounier and J. L. Valatx. Effets du 4 butyzolactoue et due 4 hydroxybutyrate de sodium sur rEEG et le comportement du chat. C.R. See. Biol. (Paris) 155: 1313-1316, 1961. 8. Jouvet, D., P. Vimont, J. F. Delorme and M. Jouvet. Etude de la privation de phase paradoxale du sommeil ehez le chat. C.R. Soc. Biol. (Paris). 158: 756-759, 1964. 9. Hishikawa, Y. and Z. Kaneko. EEG study on narcolepsy. Electroenceph. clin. Neurophysiol 18: 249-259, 1965. 10. Kawakami, M., and C. H. Sawyer. Induction of behavioral and EEG changes in the rabbit by hormone administration or brain stimulation. Endocrinology, 65: 631-643, 1959.
11. Kawakami, M. and C. 1-I. Sawyer. Neuroendocrine correlates of changes in brain activity thresholds by sex steroids and pituitary hormones. Endocrinology 65: 652-668, 1959. 12. Khazan, N. and C. H. Sawyer. Mechanism of paradoxical sleep as revealed by neurophysiologic and pharmacologic approaches in the rabbit. Psychopharmacologia 5: 457-466, 1964. 13. Khazan, N. and F. G. Stdman. Effect of imipramine on paradoxical sleep in animals with reference to dreaming and enuresis. Psychopharmacologia 10: 89-95, 1966. 14. King, E. E. and K. R. Unna. The action of mephenesin and other interneuron depressants on the brain stem. J. Pharmacol. 111: 293-301, 1954. 15. Laborit, H., J. M. Jouany, J. Gerard and F. Fabiani. Sur un substrat m6tabolique /t action centrale inhibitrice, le 4hydroxybutyrate de No. Presse Mdd. 68: 1867-1869, 1960. 16. Ledebur, I. and R. Tissot. Modification de l'activit6 61ectrique c6r6brale du lapin sous l'effet de microinjections de pr6curseurs des monoamines dons les structures somnog6nes bulbaires et pontiques. Electroenceph. clin. NeurophysioL 20: 370-381, 1966. 17. Loizzo, A. and V. G. Longo. Possibilit/~ di provocare il sonno paradosso mediante somministrazione di farmaci. Boll. See. ital. Biol. Sper. 42:213 (abstract), 1966. 18. Longo, V. G. ElectroencephalographicAtlas for Pharmacological Research. Rabbit Brain research, eel. II. Elsevier, Amsterdam, 1962.
PHARMACOLOGICAL APPROACH TO PARADOXICAL SLEEP 19. Magni, F., G. Moruzzi, G. F. Rossi and A. Zanchetti. EEG arousal following inactivation of the lower brain stem by selective injection of barbiturate into the cerebral circulation. Archs ital. BioL 97: 33-46, 1959. 20. Mantegazzini, P., K. Poeck and M. G. Santibafiez. The action of adrenaline and noradrenaline on the cortical activity of the "enc6phale isolC' cat. Archs ital. BioL 97: 222-242, 1959. 21. Matsumoto, J. and M. Jouvet. The effect of reserpine, 5-HTP and DOPA on the two phases of sleep. 23 Int. Congress of PhysioL Sci. Tokyo, 1965~Abstracts of Papers, p. 454. 22. Matsusaki, M., H. Takagi and T. Tokizane. Paradoxical phase of sleep: its artificial induction in the cat by sodium n-butyrate. Science, N.Y., 146: 1329-1323, 1964. 23. Randall, L. O., G. A. Heise, W. Schallek, R. E. Bagdon, R. Banziger, A. Boris, R. A. Moe and W. B. Abrams. Pharmacological and clinical studies on Valium, a new psychotherapeutic agent of the benzodiazepine class. Curt. ther. Res. 3: 405-425, 1961. 24. Rechtshaffen, A., E. A. Wolpert, W. C. Dement, S. A. Mitchell and C. Fisher. Nocturnal sleep of narcoleptics. Electroenceph. clin. Neurophysiol. 15: 599-609, 1963. 25. Rosadini, G., G. Gentilomo, G. Alem~t and G. F. Rossi. Effetti neurologici ed elettroencefalograci dell'iniezione diretta di un barbiturico nel circolo vertebro-basilare del coniglio. Boll. Soc. itaL Biol. sper. 40: 838-840, 1964.
97
26. Rosina, A. and M. Mancia. Electrophysiologicai and behavioral changes following selective and reversible inactivation of lower brain-stem structures in chronic cats. Electroenceph. clin. Neurophysiol. 21: 156-167, 1966. 27. Rossi, G. Brain stem facilitating influences on EEG synchronization. Experimental findings and observations in man. Acta neurochir. 13: 257-288, 1965. 28. Rossi, G. F., E. Favale, T. Hara, A. Giussani and G. Sacco. Research on the nervous mechanisms underlying deep sleep in the cat. Archs ital. Biol. 99: 270-292, 1961. 29. Sawyer, C. H. and M. Kawakami. Characteristics of behavioral and EEGraphic stimulation in the female rabbit. Endocrinology 65: 622-630, 1959. 30. Tokizane, T. Studies on the paradoxical phase of sleep in the cat. In: Progress in Brain Research, vol. 21, edited by Elsevier, Amsterdam, 1966, pp. 230-268. 31. Winters, W. D. and C. E. Spooner. Various seizure activities following gamma-hydroxybutyrate. Int. J. Neuropharm. 4: 197-200, 1965. 32. Yamamoto, K. and E. F. Domino. Nicotine induced EEG and behavioral arousal. Int. J. Neuropharm. 4: 359-373, 1965.
A Pharmacological Approach to Paradoxical Sleep' A. L O I Z Z O A N D V. G. L O N G O
Instituto Superiore di SanitY, Rome, and Centro di Neurofarmacologia del CNR, Department of Pharmacology, University of Sassari, Sassari, Italy (Received 3 June 1967) Lolzzo, A. AND V. G. LOhv,o. A pharmacological approach to paradoxical sleep. PHYSIOL.BEHAV.3 (1) 91--97, 1968.-A series of experiments were performed in order to produce paradoxical sleep (PS) by means of drugs. This paper reports on the EEG and behavioral effects of compounds possessing myrelaxant properties, and in particular: mephenesin, zoxazolamine, phenyrarnidol, several 2-amino benzothiazole derivatives, chlordiazepoxide, diazepam. Two series of experiments were carried out in the rabbit. In one group of animals, bearing chronically implanted electrodes, the drugs were injected intravenously; in the second group, consisting of acute preparations, the drugs were administered into the vertebral artery. In the first group a clear-cut PS pattern could not be related to drug administration. With intravertebral injection it was possible to obtain in some cases a picture similar to PS. Diazepam and 2-amino-4chlorobenzothiazole, at doses of 0.5-2 mg were most active in eliciting a PS-like state. Paradoxical sleep
Sleep
Paradoxical sleep and drugs
IN 1958 Dement observed, in the course of eleetroencephalographic registrations on cats bearing chronically implanted electrodes, the presence of a period of behavioral sleep during which the tracing desynchronized. This was accompanied by a reduction in muscular tone, especially of the neck muscles, and by nystagrniform movements of the eyeballs. This complex was called "Activated Sleep" by Dement. During the following years, a great deal of research was conducted with the purpose of clarifying the various aspects of activated sleep (also designated: desynchronized sleep, paradoxical sleep, rapid eye movement (REM) sleep, romboencephalic sleep, para-sleep). From all the investigations on this phenomenon a clear-cut physiological entity has evolved.
Sleep and EEG
EEG
cedes certain motor phenomena, consisting of muscular relaxation, particularly of the neck muscles, conjugated rapid eye movements, both in lateral and vertical directions, at times nystagmiform, at other times having a rotatory component. Twitchings of vibrissae, of the ears and of the legs muscles sometimes occur. The pupil is myotic, cardiac rate is altered, respiration becomes irregular, rapid and superficial. Rectal temperature is sometimes lowered by 0.1-0.2°C, while that of the internal organs (uterus, brain, liver) at times increases as much as 0.7°C. PS is usually preceded by a period of slow sleep; however, under some experimental conditions, i.e., following a period of PS deprivation [5], or in pathologic conditions, such as during attacks of narcolepsy, PS can immediately follow a phase of wakefulness [9, 24]. In studies performed in animals with chronically implanted electrodes, the average duration of the PS periods was calculated to be about 30-40 per cent of the behavioral sleep and approximately 20-25 per cent of the total registrations. The appearance of PS is percentwise greater in young animals [6].
Characteristics of Paradoxical Sleep (PS) Slow wave sleep is characteristic of vertebrates; PS, on the contrary, appears in the zoological scale in a rudimentary way in birds and acquires well defined patterns in mammals, perhaps suggesting a parallel phylogenic and ontogenic evolution of the two sleep states. The following E E G signs are considered characteristic of I S : low voltage rapid activity (20-30 cp/s, 30-40 ~tV) in the cortex, diencephalon and mesencephalon, similar to the tracing recorded during wakefulness. The hippocampus presents regular waves, at a frequency of about 5 cp/s and at a larger amplitude (50-70 tLV); a similar picture is observed in the periaqueductai gray substance, the interperpendicular nucleus posterior hypothalamus, septurn and gyrus cinguli. The lateral geniculate nucleus presents spike-like activity, synchronous with the ocular movements, to be later described. This picture coincides or shortly pre-
Methods Employed to Elicit PS, with Particular Attention to Drug-induced PS Many methods have been employed to cause the artificial appearance of PS. Low frequency electrical stimulation of the hypothalamus, of various regions of the llmbic system, of the posterior and median part of the mesencephalic teg-
mentum (nuclei reticularis pontis oralis and reticularis pontis caudalis) have elicited PS [3, 5, 28] obtained PS by electrical stimulation of various neocortical areas. In animals cooled to rectal temperature of 31-34°C an increase in PS periods
tAn abstract of this paper was presented at the Annual Meeting of the Italian Physiological Society (Loizzo and Longo, 1966). 91
92 is obtained; however, these disappear when the temperature is further lowered. Sawyer and co-workers [11, 29] have studied the relationships between sexual stimulation and PS in the female rabbit, describing PS as an "after reaction" to coitus. The possibility of inducing PS with drugs has also been studied. Based on the observations of Laborit e t aL [15] on the hypnotic properties of sodium 4-hydroxybutyrate and of 4-butyrolactone, Jouvet et al. [7] found that 50-100 mg/kg (i.v.) of these two drugs elicited in the cat the appearance of PS 5-10 min after the injection. Matsuzaki e t al. [22] confirmed the results of Jouvet e t al. [7] and described the same effect for other short-chain fatty acids. Tokizane [30] obtained results similar to those of Matsuzaki et al. [22] and of Jouvet et al. [7], using both chronic intact cats and acute preparations (mesencephalic, pontine and bulbar). In addition, he demonstrated that, in the acute prebulbar cat, sodium nbutyrate (i.v.) produces a marked muscle relaxation. The same author described the appearance of PS, in the acutely cerebellectomized cat, within 2-3 sec following intravertebral injection of 0.5 mM/kg of sodium n-butyrate. This effect was not obtained when the drug was injected into the lingual or carotid artery. Winters and Spooner [31] on the other hand, performed a detailed analysis of the EEG effects of sodium 4-hydroxybutyrate in the cat. In addition to observing an increase on the PS periods, these authors noted the appearance of EEG and motor convulsive manifestations. Rosina and Mancia [26], in a series of experiments carried out in cats with chronically implanted electrodes and with the basilar artery ligated at the median or caudal pontine level, found that the intravertebral injection of Thiopental (0.4--0.6 mg) during synchronized sleep caused the appearance of PS within a few seconds. Thiopental was without effect if the animal was awake; if a state of PS was already existing, the animal awoke. In animals with rostro-pontine ligature of the basilar artery, the barbiturate constantly caused an awakening response with an increase in muscular tone, thus confirming the results of Magni et al. [19]. The same arousing effect is provoked by the injection of barbiturates into the vertebral circulation of the rabbit, without any damping of the basilar artery [25]. Yamamoto and Domino [32] obtained an increase of the PS periods using small doses (0.005--0.01 mg/kg, i.v:) of nicotine in chronically implanted cats. Ledebur and Tissot [16] brought about PS in the rabbit by means of microinjections of 5-hydroxytryptophan in the pontine reticular formation. George e t al. [4], also using the microinjection technique, provoked PS using compounds possessing muscarinic action oxotremorine, carbachol) deposited at the level of the mesencephalic reticular formation of the cat; the PS lasted 40-50 min and was antagonized by atropine injected in situ or systemically. According to these authors, in that region of the reticular formation there is a cholinergic center, activated by the drugs, responsible for the appearance of PS. Khazan and Sawyer [12] demonstrated that atropine elevates the threshold for the production of PS by electrical stimulation of the pontine reticular formation. The same effect was obtained with pentobarbital, morphine, chlorpromazine. These authors also reported the disappearance of PS after treatment with LSD-25 and amphetamine. Chlorpromazine, in doses of 5-10 mg/kg, s.c., does not have any influence on the periodicity and characteristics of the behavioral PS, while it inhibits the electrocortical manifestations (EEG activation) [5]. Inhibition of PS has been described after treatment with atropine [5] and imipramine
LOIZZO AND LONGO [l 3]. According to Khazan and Sawyer [12], small doses of reserpine (0.1 mg/kg) potentiate PS, while larger doses (0.5 mg/kg) inhibit its appearance for a few days [21]. Some years ago we became interested in this problem, our working hypothesis was based on the premise that PS, like other physiological states, such as sleep and wakefulness, could be duplicated by the administration of drugs. In particular, attempts were made to obtain PS as the immediate consequence of drug administration so that the pattern so obtained could be definitely ascribed to the treatment. This paper reports in detail the results obtained with compounds possessing to a greater or lesser extent myorelaxant properties. Investigations on these substances was prompted by the observations of Jouvet [5], who noted in decerebrated cats, for the whole duration of PS, a complete muscular relaxation in the place of the rigidity in this type of preparation. The following drugs were studied: three aminobenzothiazole derivatives, Chlordiazepoxide, Diazepam, Mephenesin, Pheniramidol, Zoxazolamine (see Table 1). These compounds were selected because of their centrally-mediated myorelaxant action. For comparative purposes, drugs belonging to other pharmacological categories were also employed. TABLE 1 THE TABLELlSl'S THE DRUGS AND DOSAGESADMINISTERED INTRAVENOUSLY TO RABBITS BEARING CHRONICALLY IMPLANTEDELECTRODES Mephenesin Zoxazolamine~a Phenyramidol Chlordiazepoxide Diazepam (b) SKF 1448 A (2-amino-4-chloro benzothiazole) (e) SKF 1407 A (2-amino-4-methylbenzothiazole) SKF 1153 A (2-amino benzothiazole)
10--120 mg/kg 2-10 mg/kg 2-20 mg/kg 1-10 mg/kg 0.5-5 mg/kg 1-10 mg/kg 1-10 mg/kg 1-10 mg/kg
ta)10 per cent in polyethylenglycol. (b)The commercially available ampoules of Valium (Roche) were used, containing a solution of 0.5 per cent of Diazepam in organic solvents. (e)l per cent in ethanol 50 per cent. MATERIALSAND METHODS TWO series of experiments were run, using unanesthetized rabbits. One series of experiments utilized six animals with electrodes implanted chronically in various cortical and subcortical zones. Silver wires were passed through the neck muscles and through the skin of the outer canthi of the eyes, so as to respectively register muscular activity and eye movements. The drugs were injected by remote control using a long polythene tube connected to a cannula inserted permanently into the jugular vein. During the experiments the animal was kept in a shielded cage (ll × 1 × 0.7 m); the electrodes were connected with the EEG apparatus by means of long wires, thereby insuring complete freedom of movement. The other series of experiments was carried out in 72 rabbits prepared acutely for registration. In these animals the drugs were injected into the vertebral artery. After exposure of the subclavian artery, and tying-off of the collaterals (transversa
PHARMACOLOGICAL APPROACH TO PARADOXICAL SLEEP
colli, thyroidea inferior and mammari ainterna), thereby leaving open only the vertebral artery, a thin polythene canula was introduced in the subclavian (for detail of the technique, see also Calma and Wright [1], Mantegazzini et al. [20]. To insure direct access and maximum concentration of the drug at the ponto-mesencephalic area, the omolateral carotid was also tied. Electrodes were then fixed in various cortical and subcortical zones (cf. for technique Longo [18]). The eye movements were registered by means of two screw electrodes fixed in the orbital crest. The activity of the neck muscles was led by means of silver wires threaded separately through the left and right muscular bodies so that the E M G ' s of the two sides could be obtained. Registration was started at least one hour after the preparation. Except when otherwise indicated, aqueous solutions were used, in amounts of 0.1-0.5 ml; repeptitive controls were performed using saline or the diluting media. At the end of the experiment the patency of the pathway was controlled by injection of indian ink through the cannula into the vertebro-basilar circulation. The positions of the deep electrodes were histologically checked using electrolytic deposition of iron. RESULTS
Experiments in Chronically Implanted Animals In the animal bearing implanted electrodes, all the myorelaxant drugs (see Table 1), administered intravenously, provoke, at a certain dosage levels, muscular relaxation, accompanied by a synchronization of the tracing. When the drugs were injected rapidly, a short period of spasticity preceeded the muscular relaxation; sometimes nystagmus appeared. In the period following the acute phase of drug action, the PS episodes were not more frequent than those observed in the non-treated animals.
Intravertebral Drug Administration in the Acute Preparation In the course of the experiments it was observed that some of the myorelaxant drugs injected intravertebrally caused a series of symptoms, both behavioral and EEG, which recalled in some aspects the PS polygraphic patterns. These signs appeared within a few seconds following drug administration and consisted of muscular relaxation, particularly of the ears and the neck, eye movements, quivering of the vibrissae and
93
activation of the EEG. Stimuli of various kinds (touching of the fur, pinching of the tail, noise) were not able to alter the course of this period of PS-like state, which lasted, however, for a very short time; within a few minutes the E E G and muscular tone returned to normal. When repeated injections were performed at suitable intervals, the same manifestations were not always provoked, although particular care was taken to inject the drug only during the sleeping state of the animal, as shown by the EEG. In fact in some cases an entirely different reaction appeared, consisting of controlateral head torsion, struggling and hypernea. It was not possible to foresee the occurrence of such a reaction, which seemed to be unrelated either to the dose or to the basic state of the animal. The impression was gathered that the first injection of each drug in each experiment had a more typical and lasting effect than subsequent administrations. Table 2 lists the doses of the drugs and the occurrence of a PS-like polygraphic picture. The 2-amino benzothiazole derivative, S K F 1448A, proved to be the most effective compound producing this picture. Out of a total of 16 administrations, performed in five animals, S K F 1448A caused in seven cases the PS-like polygraphic picture, while in four cases, together with E E G activation and eye movements, muscular neck relaxation was noted only omolaterally to the side of injection. In the five remaining cases an atypical reaction was observed; this consisted of the appearance of 3-4 cp/s high voltage wave in the superficial and deep leads, chewing movements, head twitchings, contraction of the neck muscles, and struggling. The doses ranged from 0.1 to 4 mg in tote; the PS-like picture appeared with lower doses, from 0.2 to 2 (Fig. 1). Similar results have been obtained with two other 2-amino benzothiazole derivatives (see Table 2) which were active, however, at higher dosages. Diazepam, in doses of 0.2-2 mg, provoked a polygraphic tracing similar to PS in 4 out of 15 cases; in four other cases the neck muscles relaxation was more pronounced on one side; the remaining seven cases demonstrated the atypical reaction already described (Fig. 2). Eight trials were performed with Chlordiazepoxide, in doses ranging from 1-20 mg: activation of the tracing accompanied by spasm of the neck muscles was the prevailing picture observed. Mephenesin was tested in 12 trials (2-30 nag): the response consisted of muscular relaxation and nystagmus,
TABLE 2 EEG AND BEHAVIORAL
EFFECTS OF THE MYORELAXANT DRUGS INJECTED INTRAVERTEBRALLY IN THE RABBIT
Total trials
Doses in mg
Appearance of PS-like patterns
Diazepam SKF 1448 A
15 16
0.2-2 0.1-4
8 11
SKF 1407 A SKF 1153 A Mephenesin
5 6 12
2-4 2-4 2-30
Chiordiazepoxide Zoxazolamine
8 9
1-20 0.5-15
Phenyramidol
4
1-10
2 2 -m
Remarks the other injections provoked EEG activation and spasm of the neck. the other injections provoked the appearance of 3 c/see waves in the EEG, head twitchings, struggling. see above. see above. the picture observed with doses higher than 10 nag was neck muscles relaxation, nystagmus, slow waves in the EEG. the picture observed was EEG activation and spasm of the neck. in two cases, 15 mg of the drug gave rise to EEG activation and muscle relaxation, no nystagmic movements were present. the picture observed with doses higher than 4 nag. was EEG activation and spasm of the neck.
94
LOIZZO A N D LONGO controllo
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FIG. 1. Occurrence of a PS-like polygraphic picture after intravertebral administration of 2-amino-4 chlorobenzothiazole (SKF 1448 A) in the rabbit. A control tracing is shown in the upper part of the figure; the lower tracing, registered 30 see after 2 mg of SKF 1444 A, shows activation of the electrocorticogram, scattered eye movements, considerable reduction of neck EMG activity of both sides. Leads--FPD: right fronto-parietal cortex; POD: right parietooccipital cortex; ORB: orbital electrodes for the registration of eye movements; MD: right nuchal muscles; MS: left nuchal muscles.
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FIG. 2. Development of a PSllike polygraphic picture upon intravertebral administration of Diazepam in the rabbit. The three tracings are continuous; at the arrow 0.4 mg of the drug is injected. After a brief struggling, relaxation of the nuchal muscles ensues, more evident on the side corresponding to the injection (MD). Burst of jerky movements of the eyes and of the auricular muscles are also noticed. Leads--FD: right frontal cortex; PD right parietal cortex; HW: left dorsal hippocampus; ORB: orbital electrodes for the registration of eye movements; MS: left nuchai muscles; MD: right nuchal muscles.
PHARMACOLOGICAL APPROACH TO PARADOXICAL SLEEP which occurred simultaneously to the appearance of slow waves in the EEG. Only in 3 trials was activation of the E E G noticed, however, it was short duration and could also be attributed to causes other than the drug. Zoxazolamine and Phenyramidol, injected in doses of 0.5-15 mg did not cause the appearance of a PS-similar picture.
Effect of other Compounds Administered h: the Vertebral Artery on the EEG and Behavior So that comparison may be made with other reported results, a series of additional experiments were run to determine the activity of various compounds to which a PStriggering effect had been attributed. The administration of eserine (0.05--0.1 mg) caused a PS-similar picture: however, the neck muscle hypotonia was covered somewhat by muscle fasciculations, that should be attributed to a peripheral origin. These were evident above all in the muscular masses homolateral to the injected side. Acetylcholine (0.001-1.01 mg) and arecoline (0.1--0.5 mg) were responsible for desynchronization of the tracing, accompanied by an increase of the amplitude of the electromyographic recording of the neck muscles. Marked salivation followed arecoline administration. Pentobarbital and Thiopental, injected in doses of 0.5-15 mg, provoked activation of the EEG, spasm of the neck muscles and prolonged oculogyric crises; higher doses caused a late synchronisation of the tracing. Analogous results were obtained with Nitrazepam (2-10 mg). Sodium n-butyrate (1-30mg) did not provoke the PS-like picture; the most frequently observed reaction was a desynchronization of the tracing and an increase of the E E G of the neck muscles. The data obtained with the other drugs are summarized in Table 3.
95
Experiments Performed on Animals with Ligature of the Basilar Artery The two drugs (SKF 1448A and Diazcpam) that were found most active in producing the PS-like picture were administered intravertehrally to five animals in which the basilar artery was acutely tied-off at the caudo- or medio-pontine level. Under these conditions the two drugs produced symptoms which were not previously seen. A t doses as low as 0.5 mg there was immediate respiratory arrest (reversible after 5-15 min of artificial respiration) and disappemance of the corneal reflex. Thiopental (0.5-1 nag), in comparative trials, demonstrated the same effects; in only one case out of five did it cause a PS-similar picture.
DISCUssION
These results demonstrate that it is possible to obtain an E E G and behavioral picture similar in many aspects to that of PS by means of injection of som¢ myorelaxant drugs into the vertebral artery. This PS-like picture enstms immediately after the administration and is independent of the basal state of the animal, in that it appears both when the drug is given during synchronization or during desynchronization of the E E G tracing. This effect is typical of an "acute" pharmacological action, and can be compared to the narcotic state induced by intravenous administration of barbiturates or to the state of wakefulness following intravenous injection of amphetamine. The compounds which in our experiments were able to induce this PS-like state upon systematic administration cause a generalized muscular relaxation that is accompanied by an E E G picture of synchronization. The intravertebral approach, in all probability, induces this particular picture
TABLE 3 THE TABLELISTSALL THE DRUGSTRIED IN THE COURSEOF THE INVESTIGATION.THE DATA SHOULDBE CONSIDEREDONLY ORIENTATIVE.
Total trials
Doses in mg
Acetylcholine Arecoline Eserine
3 3 5
0.001-0.01 0.1-0.5 0.02-0.1
Sodium n-butyrate
5
1-30
Pentobarbital
8
l-15
Thiopental Hydroxydione sodium Nitrazepam Ethyl urethan
6 2 4 7
0.5-2 1-2 0.5-2 10--60
Apomorphine Chlorpromazine Trihexyphenydil
4 2 4
0.5-2 0.5-1 0.5-2
Tryptamine Dihydroxyphenylalanine (DOPA)
2 2
1-2 2-5
Remarks EEG desynchronization, spasm of the neck. see above, plus salivation. EEG desynchronization, nystagmus, fasciculations of the neck muscles, particularly of the side of injection. EEG desynchronization, spasm of the neck muscles, with highest doses spikes appear in the EEG. EEG desynchronization, spasm of the neck, ocuiogyric crises, mydriasis. see above. see above. see above, no octdogyric crises were noticed. desynehronization of the EEG and relaxation of the neck muscles, accompanied by tachypnea, was noticed in two cases. EEG synchronization, relaxation of the neck muscles. EEG synchronization, spasm of the neck muscles. EEG desynchronization with 0.5 rag, grand mal EEG patterns with 2, no changes in the EMG of the neck. EEG desynchronization, head tremors. no changes.
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LOIZZO AND LONGO
since it selectively concentrates the action of these drugs in the mesencephalon, to which has been attributed a role in the origin of the E E G and motor phenomena characteristic of PS [5]. A PS-like picture appearing acutely following intravertebral injection of sodium n-butyrate has been described by Tokizane [30] in the decerebellated cat. Under our experimental conditions it was not possible to reproduce these results. In fact this compound, in the dose indicated by this author, injected intravertebrally in the rabbit, always gave rise, rogether with an E E G activation, to an increase in the neck muscular tone. Rosina and Mancia [26] observed the appearance of PS following intravertebral administration of 0.1-0.4 mg of Thiopental in the chronic cat with ligature of the basilar artery at the medio- or caudo-pontine level. Our results indicated that ligature of the basilar artery at this level primarily cause a marked increase of the toxicity of this drug; disappearance of the corneal reflex (noted also by the above-mentioned authors) and respiratory arrest. The inconstancy of results upon intravertebral administration of drugs noted in our experiments has been reported by others who have used the same technique. Rosina and Mancia [26] pointed out that following Thiopental injection sometimes ataxia and spasms appeared instead of the PS-like picture. Rosadini et aL [25] and Tokizane [30] also insist on the variability of the EEG, and motor responses following drug administration by the same route; Rosadini et aL [25] attribute this phenomenon to spasms of the vertebral artery. Other possible mechanisms are: air emboli, speed of injection and distribution in the involved areas. In the course of the present investigation, for example, the posterior cerebral artery was not always injected with the indian ink administered at the end of the experiments to control the patency of the vertebro-basilar vessels. In regards to the mechanisms of the E E G and behavioral patterns provoked by some of
the myorelaxant drugs injected intravertebrally, one must consider them in relation to the mechanism underlying PS. Jouvet [5] indicates the localization of the PS-triggering centers in the pens. The nucleus caudalis reticularis pontis seems to play a prominent role in the production of the E E G and motor manifestations of the PS: electrical stimulation of this nucleus [5] provokes PS under certain experimental conditions, while in other cases excitement and muscular spasms were seen. Therefore, also upon stimulation, a certain variability of the response has been reported, similar to that following the intravertebral administration of drugs. The negative results obtained with other drugs and with the control injections of saline and of the solvents permits the exclusion of an aspecific effect. The drugs which caused this reaction in our experiments all have in common an effect on the central internucial neurons system. This effect has been demonstrated at various levels of the neuraxis: blockade of the spinal and supraspinal polysynaptic reflexes and of the bulbomesencephalic inhibitory and facilitatory system [14, 23]. A possible explanation is that these drugs provoke the PS-like syndrome because of the high concentration reached, due to the route of the administration, at the level of the bulbo-mesencephalic centers. The E E G desynchronization could be explained through inactivation of the brain stem centers having a tonic facilitating influence on E E G synchronization. A similar interpretation has been given to the desynchronization obtained in the intact animal with intravertebral administration of barbiturates [27]; these drugs, however, concomitantly produce behavioral signs of excitation [25]. Available data do not permit us to state whether the relaxation of the neck muscles and the nystagmic movements are due to paralysing or irritating effect; the possibility exists that both are operating simultaneously on the different neuronal systems present at the bulbo-mesencephalic level.
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