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Electroencephalographic analysis of the central action of dihydroxyphenylalanine
Electrocncephalography and Clinical Neurophysiology Elsevier Publishing Company, Amsterdam - Printed in Th© N©th©rlands
259
ELECTROENCEPHALOGRAPHIC ANALYSIS OF THE ACTION OF DIHYDROXYPHENYLALANINE
CENTRAL
K. KADZIELAWA AND E. WIDY-TYSZKIEWlCZ
Department of Experimental Pharmacology, Academy of Medicine, Warsaw 64 (Poland) (Ac.c.cpted for publication: July 16, 1969)
The catecholamine precursor /~-(3~4-dihydroxyphenyl)-alanine (DOPA) enters the brain, where it is readily decarboxylated to dopamine (Glowinski and Baldessarini 1966; Hornykiewicz 1966 and Marley 1966 for references). However, it has been recently discovered that DOPA is mainly decarboxylated in the vascular endothelium of brain capillaries, which form an enzymic barrier restricting the penetration of this amino acid into the brain (Bertler et ai. 1964; Owman and Rosengren 1967). In EEG studies it was found that DOPA (20-40 mg/kg) infused i.e. caused desynchronization in rabbit cerveau isold preparations and in locally anaesthetized rabbits (Monnier and Tissot 1958; Monnier 1960). Costa et ai. 0960) found no consistent effects in curarized rabbits with DOPA injected into the common carotid artery. Mantegazzini and Gl~isser 0960) demonstrated an intense desynchronization in cerveau isold cats following intracarotid (8-20 rag) or intravenous (30-50 mg/kg) infusion of DOPA. This effect is not related to the peripheral autonomic actions of the amino acid (Mantegazzini and GlOsser 1960). Ledebur and Tissot (1966) demonstrated d esynchronization in the rabbit EEG following micro-injection of DOPA into the tractus solitarius, n. solitarius and n. pontis ventralis or into n. reticularis pontis caudalis, suggesting the inhibition of the former and activation of the latter centre. In the course of our experiments on the mechanism of action of tricyclic antidepressants and thei:" influence on the central effects of DOPA it was observed that DOPA had almost no influence on the EEG of gallamine-immobilized cats or those with chronically implanted electrodes and in most experiments on
encdphale isold preparations, while provoking
an intense and permanent desynchronization in animals with brain-stems sectioned at the postpontine and rostropontine levels. The aim of the present study has been the analysis of the EEG effects of DOPA in cats, depending upon the level of brain-stem section or hemisection. METHODS
The results to be reported are based on studies carried out on thirty-five cats in acute experiments and on six cats and five rabbits with chronically implanted electrodes. Balltipped silver wire epidural electrodes and bipolar concentric steel-silver subcorticai electrodes were used. Acute experiments were performed on gallamine.immobilized male cats (2.5-3.0 kg) with intact neuraxis or with section or hemisection of the brain-stem at various levels. Animals were operated on t~l~d~" ~iethyl ether anaesthesia; the trachea was ¢annulated and a polythene catheter was introduced into the femoral vein. After gallamine injection the application of ether was diseontinaed and artificial respiration was started. Gallamine (4-5 mg/kg, i.e. 0.2 ml/kg of 10~ sol) was further infused every 45-60 min. The wound tissues were carefully infiltrated with a 2 ~ solution of lignocaine in normal saline. In most acute experiments only cortical electrodes were implanted and the head of the animal was not mounted in the stereotaxic unit. When inserting subcortical electrodes stereotaxicaily all pressure points were infiltrated with lignocaine. Care was taken to maintain artificial respiration and body temperature at their proper levels. Electroenceph. c/in. Neurophysiol., 1970, 28:259-265
260
K. KADZIELAWA AND E. WIDY-TYSZKIEWICZ
The compounds studied were injected i.v., i.p. or into the common carotid artery. They were: L-DOPA and DL-DOPA (Koch and Light; Sigma), gallamine triethiodide (Flaxedil-Specia) and lignocaine hydrochloride (Lignocain --Pelf a). DOPA, precisely powdered, was dissolved in warmed saline (50-60°C).
3 Fig. I
The planes of the main sections of the cat brain-stem: I, at Cx or Cg (encdphale isold); 2, postpontine; 3, rostropontine pretrigeminal; 4, rostropontine; 5, precollicular (pretentorial) and intercollicular cerveau isold preparations. The brain-stem was sectioned under ether anaesthesia with a steel spatula at one of the following levels (Fig. l): (1) C1 or C2--the encdphale isold preparation, (2) postpontine section--caudal to the trapezoid body, (3) )ostropontine pretrigeminal, (4) rostropontine, (5) precollicular (pretentorial) and intercollicular --the cerveau isol~ preparation. The hemisection of the brain-stem was performed at the rostropontine level. In about half of the experiments the blood pressure was recorded from the femoral artery by means of a mercury manometer. Ten to 15 rain foUowing section the blood pressure regularly settled at 90-120 mm Hg. The pupil diameter was observed at conswnt intervals during the experiment. Recording was started 2-3 h after the ether had been discontinued. The recruiting response was elicited in the sensori-motor and associative cortex by stimulation of n. centralis medialis thalami at low frequencies: 6-12/see, 2-6 V, 0.5-1.0 msec, for 5-15 sec. A square-wave pulse generator with an isolation unit was used. In a group of six cats and six rabbits electrodes were implanted stereotaxicaUy under Nembutal anaesthesia, as previously described (Kadzielawa 1967). Experiments were performed 1 month later. The positions of electrodes were established accordi,3g to the method of Guzm~in et al. (19581. The recording apparatus consisted of a 16-channelelectroencephalograph (EEG 16-O1 M) with one lead continuously monitored on the screen of an oscilloscope. The electrocardiogram was recorded continuously with the EEG.
RESULTS
The action of DOPA in chronic preparations There was no evident change in the behaviour and EEG pattern in cats with chronically implanted electrodes following i.v. or i.p. injection of 25-50-100-200 mg/kg of DL-DOPA. Only a little increase in frequency and decrease in amplitude lasting for a few hours were noted in two out of six experiments. In two other animals a transient (5-10 min) synchronization of the EEG was noted after 5 rain following i.p. injection of DOPA. However, in rabbits the injection of 25-50 mg/kg of DL-DOPA induced an evident, although not intense, desynchronization persisting for 90-120 rain. The effects of DOPA in acute experiments, depending upon the level of brain.stem section DOPA (DL-DOPA: 25-30 mg/kg, L-DOPA: 5-10 mg/kg) had little apparent influence on the spontaneous EEG pattern of gallamine. immobilized cats. In a few experiments a slight decrease in amplitude and increase in frequency were noted. In encdphale isold preparations this amino acid elicited either no apparent effect or caused desynchronization, although not permane,~ and not so intense as with higher brain-stem sections. Furthermore, this effect seemed to be rather dependent upon the prolonged and large increase in blood pressure observed in this preparation following infusion of DOPA. With postpontine and more anteriorsections this amino acid (L-form: 5-I0 mg/kg, DL-form: 25-30 mg/kg) elicited always an intense desynchronization. When the sections were performed at higher levels, the rostropontine pretrigeminaland cerveau isoI6 preparations, the effect of D O P A was almost the same, although it seemed that in the~e latter cases the desynEtectroenceph. ct.~n.Neuroph.vsiot., 1970, 28:259-265
EEG EFFECTS OF DOPA
261
In gallamine-immobilized cats, in endphale is016 preparations as well as in the preparations with the brain-stem sectioned at the rostropontine, midpontine and postpontine levels, i.v. infusion of DOPA (e-DOPA: 10 mg/kg, LD-DOPA 30 mg/kg) exerted a mild pressor effect accompanied by tachycardia. The blood pressure increased by about 20-40 mm Hg for 10-l 5 min, the increase in heart rate was about 2040%. In the etdphale isole’ preparation there was a large increase in blood pressure, up to 200-230 mm Hg, and the heart rate increased by 5040%. The blood pressure in this case returned to normal values after 20-30 min. In cerveau isok, postpontine and endphale isok preparations in which gallamine was not used, 10-15 min following infusion of DOPA, movements of the animal’s limbs, trunk and head were observed. Therefore most of the
LFig. 2 The influence of DL-DOPA on the cat electrocorticogram depending upon the level of brain-stem section: I, gallamine-immobilized; t, enchphale isolk (Cd; 3, postpontine; 4, rostropontine pretrigeminrl; 5, (‘crueuu is&!, Upper tracings of each record, control; lower tracings, 30 min following i.v. infusion of 25 mgl kg of UL-DOPA. Calibrations: 100 @/. I sec.
chronization was more accentuated. These effects are illustrated in Fig. 2. The desynchronizing effect of DOPA was studied at doses of Xl-30 mg/kg of the DL-form and S-10 mg/kg of L-isomer. In most experiments this effect appeared within 5-10 min following the infusion of DOPA and persisted over 3 h. This action was very intense and appeared in every experiment. It was accompanied by a large increase in the threshold for the recruiting reaction. In some experiments the maxima1 desynchronization appeared within 5 min, in others within 30 min, of DOPA action. Mydriasis was noted at 15-30 min following DO?A and disappeared partially after 60-90 min. The same effects on the EEG were noted when infusing 5-10 mg of L-DOPA into the common carotid artery.
Fig. 3 The influence of DOPA on the cortical and SUbcortical EEG patternsin the cerue~u isok preparationof the cat. 0: control record; b: 30 min following i.v. infusion Of 10 m&kg of L-DOPA. Leads: I-2, SenSori-motorcortex; 3-4, associative cortex; 5, n. amygdalae basalis L-R; 6, hippocampus dorsalis L-R; 7, n. caudatus L-R; 8, thalamus, n. ventr. lat. L-R. Calibrations: 100 CIV,3 Sec. Lkctroenceph. din. Neurophysiol., 1970,28: 259-265
262
K, KADZIELAWA AND E. WIDY-TYSZKIEWICZ
experiments with section of the brain-stem wore performed with gallamine immobflnzation and lignocaine in order to test the effects of DOPA against the same background pharmacological action. The effect of DOPA was always the same and did not depend upon the influence of gallamine or iignocaine. The desynchronizing effect of DOPA was evident in the electrocorticograms and in the leads from subcortical structures. However, i~n the amygdala and hippocampus only a slight decrease in amplitude and no change or slowing of the rhythm were noted following DOPA (Fig. 3, b). The #~fluence of DOPA on electrocorticograms in preparations with hemisection at the rostropontine level Fig. 4 represents a typical response to L-DOPA infused i.v. in a cat with left hemisection at the rostropontine level. In most cases with this section, or with hemisection at the collicular level, the amplitude of the record was greater and the spindles appeared more frequently on the side of the section. During the first 5-10 rain of action in this preparation DOPA caused a typical desynchronization, mainly ipsilateral to the section (Fig. 4, b), whereas equal desyn. chronization on both sides was noted 10-15 rain later (Fig. 4, c). Mydriasis was also much more accentuated and appeared 10-15 rain earlier ipsilateral than contralateral to the section. DISCUSSION
Since the desynchronization following DOPA is dependent upon separation of the lower from the upper brain-stem structures, it seems possible that in normal and curarized cats this effect of DOPA is in some way restrained by the bulbar, and probably high spinal, ascending inhibitory influences. Furthermore, these influences are probably predominantly ipsilateral, as demonstrated in experiments with hemisection at the rostropontine level. Moruzzi and his co]l~borators (see Moruzzi ~q~ for refs.) h~.~e developed a hypothesis suggesting the existence of EEG synchronizing, and probably sleep-inducing, structures located in the lower brain-stem. This hypothesis is
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R Fig. 4 Electrocorticogram of ~he cat encdphale isold preparation with a left hcmisection at the rostropontine Icvvl. a, control; b, tO rain after infusion of 8 mg/kg of L-DOPA i.e.;
c, i 5 rain later. Note the difference in the effect of DOPA on the left (L) and fight (R) ¢lectrocorticograms durin8 the first period of action of the amino acid. Calibrations:
100pV, 1 sec.
supported mainly by the following findings: (I) the activation of the EEG in chronic midpontine pretrigeminal p:eparations, independent of the sensory inflow through cranial nerves; in contrast there is a high voltage slow EEG Electroenceph. clin. NeurophysioL, 1970, 2 8 : 2 5 9 - 2 6 5
263
EEG EFFECTS OF DOPA
rhythm in the rostropontine pretrigeminal preparation; (2) the greater tendency to synchronization in the hemisphere contralateral to the midpontine pretrigeminal hemisection in contrast to the persistent ipsilateral desynchronization; (3) desynchronization of the electrocorticogram following inactivation of the lower brain-stem induced by intravertebral infusion of low doses of thiopental; behavioural and EEG arousal in the encdphale isold cat following the transient inactivation of the buibar centres by localized cooling of the fourth ventricle in front of the obex (Berlucchi et al. 1964). Bloch and Bonvallet (196 I) and Bonvallet and Bloch (1961) have observed the potentiation of arousal, induced by midbrain reticular stimulation, by intrabulbar novocaine or prebulbar section. However, there is a question of the anatomical localization of such a synchronizing centre. Magnes et al. (1961) obtained synchronization in the electrocorticogram of encdphale isold cats by low rate electrical stimulation of n. reticularis ventralis and the nucleus of the solitary tract. However, in studies on cats with chronically implanted electrodes Favale et al. (1961) demonstrated synchronization by low frequency stimulation of many reticular sites. According to Bonvallet and Bloch (1961) there is a feedback mechanism between the bulbar ascending deactivating centres and the reticular activating system. The synchronizing structures of t h e lower brain-stem exert an inhibitory action on the pontine centres (Rosina and Mancia 1966). In the light of these observations it is interesting to note that the studies of Matsumoto and Jouvet (1964) suggest some relationship between the level of cerebral catecholamines and the desynchronized EEG pattern in paradoxical sleep (see Jouvet 1967, 1968 for refs.). It is possible that the desynchronizing action of DOPA is restricted by the ascending influences of the same bulbar structures which are probably responsible for the inhibition of the diencephalic and midbrain desynchronizing influences. The studies of Bueno e t al. (1968) suggest the existence of lower brain-stem EEG ascending synchronizing influences in the rabbit. However, in these animals, at doses ineffective innormal
cats, DOPA is able to induce an evident, although not intense, desynchronization, mainly in neocortex, demonstrated by Monnier and Tissot (! 958), Monnier (1960) and in the present study. 'The hypothesis presented here may also be questioned because in some other instances (see Hornykiewicz 1966 for refs.) DOPA is able to activate behaviour in animals with intact neuraxes and, furthermore, this amino acid is known to have a potent awakening effect in reserpinized animals. Nevertheless it is shown now that in chronic experiments or gailamineimmobilized cats doses of DOPA ten times larger than those effective in animals with prebulbar sections have almost no apparent influence on the EEG and behaviour. According to Everett and Wiegand (1962) there is a cloae cc.rrelation of dopamine levels and the behavioural state and a twofold increase seems to be a threshold level. Thus DOPA may be effective: (1) in normal animals only when applied in large doses (its action may depend also on species differences); (2) when its action is potentiated by monoamine oxidase inhibitors, restricting the deamination of its metal~lites, and (3) when the inhibition of the u p t ~ e of dopamine in reserpinized animals intensifies its action. It was also recently found (Kadzielawa, in preparation) that in rabbits the desynchronization following DOPA, applied either i.v. or into the common carotid artery, is markedly accentuated in preparations with sections at levels above the lower brain-stem. SUMMARY
1. The EEG effects of DOPA were analysed in cats, depending upon the level of brain-stem section. 2. In contrast with cats with chronically implanted electrodes, gallamine.immobilized, and with encdphale isold cats, in which DOPA is unable to induce profound changes in the EEG pattern and behaviour, this amino acid elicits an intense desync.hronization in animals with brain-stems sectioned at the postpontine, rostropontine and collicular levels. The desynchronization is not found in hippocampus and amygdala. Electroenceph. clin. Neurophysiol., 1970, 28:259-265
264
.~. KADZIELAWAAND E. WIDY-TYSZKIEWICZ
3. In the early period of action DOPA induces desynchronization mainly ipsilateral to hemisection at the rostropontine level. 4. The observed effects of DOPA are not very dependent on its autonomic effects. 5. In contrast with the mild presser response in gallamine-immobilized cats and in preparations with the brain-stem sectioned at higher levels there is an intense increase in blood pressure and heart rate in encdphale isold preparations. 6. it is concluded that DOPA-induced desynchronization is probably inhibited in animals with intact neuraxes by the tonic ascending influences of the synchronizing buibar and higher spinal structures. RtSUMI~ ANALYSE I~LECTROENCI~PHALOGRAPH IQUE DE L'ACTION CENTRALE DE LA DIHYDROXYPH~NYLALANINE I. Les effets EEG du DOPA ont ~t~ analys~s chez le chat, en fonction du niveau de section du troac c~r6bral. 2. Par comparaison ~ des chats avec ~lectrodes implant~es chroniquement, des chats immobilis~s par gallamine et des chats enc@hale isol6, chez lesquels le DOPA est incapable d'induire des modifications profondes du pattern EEG et du comportement, cet aminoacide provoque une d~synchronisation intense chez des animaux avec section du trent c~r6bral au niveau r~tropontin et pr6pontin et des tubercules quadrijumeaux. La d6synchronisation n'affecte pas l'hippocampe ni ramygdale. 3. A la p~riode pr6coce de son action, le DOPA provoque une d6synchronisation surtout ipsilat~rale a l'h~misection au niveau pr6pontin. 4. Les effets du DOPA observes sent assez ind6pendants de ses effets v6g~tatifs. 5. Par opposition a la 16g~re r6ponse hypertensive des chats immobilis6s par gaUamine et des preparations avec section haute du tronc c6r~bral, on note une augmentation intense de la pression sanguine et de la vitesse cardiaque dans les pr6parations enc~phale isol~. 6. Les auteurs concluent que la d~synchronisation induite par le DOPA est probablement inhib~e chez les animaux avec n~vraxe
intact par les influences toniques ascendantes des structures synchronisatrices bulbaires et spinales hautes. REFERENCES BERLUCCm,G., MAFt~J,L., MORUZZl,G. and STaATA,P. EEG and behavioral effects elicited by cooling of medulla and pens. Arch. ital. Biol., 1964, 102: 372-392. BERTLER,A., FALCK,B. and R.OSENGREN,E. The direct demonstration of a barrier mech-,nism in the brain capillaries. Acta pharmacol, toxicol., 1964, 20: 317-321. BLOCH,V. et BONVALLET,M. Interaction des formations rc~ticulaires m6senc6phalique et bulbaire. J. Physiol. (Paris), 1961, .53: 280-281. ~3ONVALLET,M. and BLOCtt,V. Bulbar control of cortical arousal. Science, 1961, 133:1133-1134. BUENO,J. R., BesT, K. and HIMWIC'H,H. E. Lower brainstem EFG synchronizing mechanism in the rabbit. Electroenceph. clin. NeurophysioL, 1968, 24: 25-34. COSTA,E., PSCHEtD'r,G. R., VAN MElzR, W. G. and HtMWICH, H. E. Brain concentrations of biogenic amines and EEG patterns of rabbits. J. Pharmacol. exp. Ther., 1960, 130: 81-88. EVEnETT,G. M. and WtEOAND,R. G. Central amines and behavioral states: a critique and new data. Prec. 1st int. pharmacol. Meeting. Pergamon, Oxford, 1962, 8: 85-92. FAVALE,E., LOEB,C., Kosst, G. F. and SAcco, G. EEG synchronization and behavioral signs of sleep following low frequency stimulation of the brain stem reticular formation. Arch. ital. Biol., 1961, 99: 1-22. GLowtNsgl, J. and BALD.~|SAmNt,R. J. Metabolism of norepinephrine in the central nervous system. Pharmacol. Rev., 1966, 1~': 1201-1238. GUZM~,N,C. F., ALCARAZ,M. V. and F~RNANDEZ,A. G. Rapid procedure to localizeelectrodes in experimental neurophysiology. Bol. inst. Estud. todd. biol. ( Mdx.), 1958, 16: 29-31. HORNYKtEWtCZ,O. Dopamine (3-hydroxytyramine) and brain function. PharmacoL Re~,., 1966, 18: 925-964. JOUVET, M. R~gulation neuro-humorale des Stats de sommeil. Aspects blologiques et cliniques du syst~me nerveux central. Symposium de Sandoz SA, Bale, 196/: 103-137. JOUWT, ~L Neuropharmacology of sleep. Ir D. H. EFRON (Ed.), Psychoplmrmacology, A Review of Progress 1957-1967, Public Health Service Publication No. 1836, Washington, 1968: $23-540. KADZt~LAWA,K. Studies on the pharmacology of o~.meth. yl-3,4-dihydroxyphenylalanine (o,-methyl-DOPA) and 0,-methylnorepinephrine. !I. int. ]. Neuropharmacol., 1967, 6: 453-462. L~DEaUa, I. X. et TtssoT, R. Modification de l'activ;t6 ~.lectrique c~r6brale du lapin sous l'effet de microinjections de pr6:urseures de monoamines clans les structures somnog~nes bulbaires et pontiques. Electroenceph. din. NeurophysioL, 1966, 20: 370-381. MAON~, J., Moauzzt, G. and POP,WEtANO,O. SynchroniElectroenceph. clin. Neurophysiol., 1970, 28:259-265
EEG EFFECTS OF DOPA zation of the EEG produced by a low-frequency electrical stimulation of the region of the solitary tract. Arch. ital. Biol., 1961, 99: 33-67. M[ANTEGAZZINI,P. et GL~sr~z, A. Action de la Dt.-3,4dioxyphenylalanine (DOPA) et de la dopamine sur l'activit~ ~lectrique du chat "cerveau isol~". Arch. ital. Biol., 1960, 98: 367-374. MARLEY, E. Behavioral and electrophysiological effects of catecholamines. Pharmacol. Rev., 1966, 18: 753-768. MATSUMOTO, J. et JOUVEZ, M. Effets de r,~'serpine, DOPA et 5.HTP sur les deux ~tats de sommeil. C. R. Soc. Biol. (Paris), 1964, 158: 2137-2140. ]~ONNIER, M. Action ~lectro-physiologiques des stimu!~nts du syst~me nerveux central. I. Syst~mes sdr~nergiques, cholinergiques et neurohumeures s~rotoniques. Arch. int. Pharmacodyn., 1960, 124: 281301.
265
MONNIER:M. et TlssoT, K. Action de la r~serpine et de ses m~diateurs (5-hydroxytryptophan m s~rotonine et Dopa--noradr~naline) sur le cornportement et le cerveau du lapin. Helv. physiol. Acta, 1958, 16: 255267. Moauzzl, G. Synchronizing influences of the brain stem and the inhibitory mechanisms underlying t~.e production of sleep by sensory stimulation. Electroenceph. clin. Neurophysiol., 1960, Suppl. 13: 231-253. OWMAN, C. and ROSaNOREN,E. Dopamine formation in brain capillaries - - an enzymic blood-brain barrier mechanism. J. Neurochem., 196/, 14: 547-550. ROSINA, A. and MANCIA, M. Electrophysiolosical and behaviourai changes following selective and reversible inactivation of lower brain-stem structures in chronic cats. Electroenceph. clin. Neurophysiol., 1966, 21: 157-167.
Reference: KADZIELAWA,K. and WIDY-TYSzKIEWICZ,E. Electroencephalographic analysis of the central action of dihydroxyphenylalanine. Electroenceph. clin. NeurophysioL, 19/0, 28: 259-265.
259
ELECTROENCEPHALOGRAPHIC ANALYSIS OF THE ACTION OF DIHYDROXYPHENYLALANINE
CENTRAL
K. KADZIELAWA AND E. WIDY-TYSZKIEWlCZ
Department of Experimental Pharmacology, Academy of Medicine, Warsaw 64 (Poland) (Ac.c.cpted for publication: July 16, 1969)
The catecholamine precursor /~-(3~4-dihydroxyphenyl)-alanine (DOPA) enters the brain, where it is readily decarboxylated to dopamine (Glowinski and Baldessarini 1966; Hornykiewicz 1966 and Marley 1966 for references). However, it has been recently discovered that DOPA is mainly decarboxylated in the vascular endothelium of brain capillaries, which form an enzymic barrier restricting the penetration of this amino acid into the brain (Bertler et ai. 1964; Owman and Rosengren 1967). In EEG studies it was found that DOPA (20-40 mg/kg) infused i.e. caused desynchronization in rabbit cerveau isold preparations and in locally anaesthetized rabbits (Monnier and Tissot 1958; Monnier 1960). Costa et ai. 0960) found no consistent effects in curarized rabbits with DOPA injected into the common carotid artery. Mantegazzini and Gl~isser 0960) demonstrated an intense desynchronization in cerveau isold cats following intracarotid (8-20 rag) or intravenous (30-50 mg/kg) infusion of DOPA. This effect is not related to the peripheral autonomic actions of the amino acid (Mantegazzini and GlOsser 1960). Ledebur and Tissot (1966) demonstrated d esynchronization in the rabbit EEG following micro-injection of DOPA into the tractus solitarius, n. solitarius and n. pontis ventralis or into n. reticularis pontis caudalis, suggesting the inhibition of the former and activation of the latter centre. In the course of our experiments on the mechanism of action of tricyclic antidepressants and thei:" influence on the central effects of DOPA it was observed that DOPA had almost no influence on the EEG of gallamine-immobilized cats or those with chronically implanted electrodes and in most experiments on
encdphale isold preparations, while provoking
an intense and permanent desynchronization in animals with brain-stems sectioned at the postpontine and rostropontine levels. The aim of the present study has been the analysis of the EEG effects of DOPA in cats, depending upon the level of brain-stem section or hemisection. METHODS
The results to be reported are based on studies carried out on thirty-five cats in acute experiments and on six cats and five rabbits with chronically implanted electrodes. Balltipped silver wire epidural electrodes and bipolar concentric steel-silver subcorticai electrodes were used. Acute experiments were performed on gallamine.immobilized male cats (2.5-3.0 kg) with intact neuraxis or with section or hemisection of the brain-stem at various levels. Animals were operated on t~l~d~" ~iethyl ether anaesthesia; the trachea was ¢annulated and a polythene catheter was introduced into the femoral vein. After gallamine injection the application of ether was diseontinaed and artificial respiration was started. Gallamine (4-5 mg/kg, i.e. 0.2 ml/kg of 10~ sol) was further infused every 45-60 min. The wound tissues were carefully infiltrated with a 2 ~ solution of lignocaine in normal saline. In most acute experiments only cortical electrodes were implanted and the head of the animal was not mounted in the stereotaxic unit. When inserting subcortical electrodes stereotaxicaily all pressure points were infiltrated with lignocaine. Care was taken to maintain artificial respiration and body temperature at their proper levels. Electroenceph. c/in. Neurophysiol., 1970, 28:259-265
260
K. KADZIELAWA AND E. WIDY-TYSZKIEWICZ
The compounds studied were injected i.v., i.p. or into the common carotid artery. They were: L-DOPA and DL-DOPA (Koch and Light; Sigma), gallamine triethiodide (Flaxedil-Specia) and lignocaine hydrochloride (Lignocain --Pelf a). DOPA, precisely powdered, was dissolved in warmed saline (50-60°C).
3 Fig. I
The planes of the main sections of the cat brain-stem: I, at Cx or Cg (encdphale isold); 2, postpontine; 3, rostropontine pretrigeminal; 4, rostropontine; 5, precollicular (pretentorial) and intercollicular cerveau isold preparations. The brain-stem was sectioned under ether anaesthesia with a steel spatula at one of the following levels (Fig. l): (1) C1 or C2--the encdphale isold preparation, (2) postpontine section--caudal to the trapezoid body, (3) )ostropontine pretrigeminal, (4) rostropontine, (5) precollicular (pretentorial) and intercollicular --the cerveau isol~ preparation. The hemisection of the brain-stem was performed at the rostropontine level. In about half of the experiments the blood pressure was recorded from the femoral artery by means of a mercury manometer. Ten to 15 rain foUowing section the blood pressure regularly settled at 90-120 mm Hg. The pupil diameter was observed at conswnt intervals during the experiment. Recording was started 2-3 h after the ether had been discontinued. The recruiting response was elicited in the sensori-motor and associative cortex by stimulation of n. centralis medialis thalami at low frequencies: 6-12/see, 2-6 V, 0.5-1.0 msec, for 5-15 sec. A square-wave pulse generator with an isolation unit was used. In a group of six cats and six rabbits electrodes were implanted stereotaxicaUy under Nembutal anaesthesia, as previously described (Kadzielawa 1967). Experiments were performed 1 month later. The positions of electrodes were established accordi,3g to the method of Guzm~in et al. (19581. The recording apparatus consisted of a 16-channelelectroencephalograph (EEG 16-O1 M) with one lead continuously monitored on the screen of an oscilloscope. The electrocardiogram was recorded continuously with the EEG.
RESULTS
The action of DOPA in chronic preparations There was no evident change in the behaviour and EEG pattern in cats with chronically implanted electrodes following i.v. or i.p. injection of 25-50-100-200 mg/kg of DL-DOPA. Only a little increase in frequency and decrease in amplitude lasting for a few hours were noted in two out of six experiments. In two other animals a transient (5-10 min) synchronization of the EEG was noted after 5 rain following i.p. injection of DOPA. However, in rabbits the injection of 25-50 mg/kg of DL-DOPA induced an evident, although not intense, desynchronization persisting for 90-120 rain. The effects of DOPA in acute experiments, depending upon the level of brain.stem section DOPA (DL-DOPA: 25-30 mg/kg, L-DOPA: 5-10 mg/kg) had little apparent influence on the spontaneous EEG pattern of gallamine. immobilized cats. In a few experiments a slight decrease in amplitude and increase in frequency were noted. In encdphale isold preparations this amino acid elicited either no apparent effect or caused desynchronization, although not permane,~ and not so intense as with higher brain-stem sections. Furthermore, this effect seemed to be rather dependent upon the prolonged and large increase in blood pressure observed in this preparation following infusion of DOPA. With postpontine and more anteriorsections this amino acid (L-form: 5-I0 mg/kg, DL-form: 25-30 mg/kg) elicited always an intense desynchronization. When the sections were performed at higher levels, the rostropontine pretrigeminaland cerveau isoI6 preparations, the effect of D O P A was almost the same, although it seemed that in the~e latter cases the desynEtectroenceph. ct.~n.Neuroph.vsiot., 1970, 28:259-265
EEG EFFECTS OF DOPA
261
In gallamine-immobilized cats, in endphale is016 preparations as well as in the preparations with the brain-stem sectioned at the rostropontine, midpontine and postpontine levels, i.v. infusion of DOPA (e-DOPA: 10 mg/kg, LD-DOPA 30 mg/kg) exerted a mild pressor effect accompanied by tachycardia. The blood pressure increased by about 20-40 mm Hg for 10-l 5 min, the increase in heart rate was about 2040%. In the etdphale isole’ preparation there was a large increase in blood pressure, up to 200-230 mm Hg, and the heart rate increased by 5040%. The blood pressure in this case returned to normal values after 20-30 min. In cerveau isok, postpontine and endphale isok preparations in which gallamine was not used, 10-15 min following infusion of DOPA, movements of the animal’s limbs, trunk and head were observed. Therefore most of the
LFig. 2 The influence of DL-DOPA on the cat electrocorticogram depending upon the level of brain-stem section: I, gallamine-immobilized; t, enchphale isolk (Cd; 3, postpontine; 4, rostropontine pretrigeminrl; 5, (‘crueuu is&!, Upper tracings of each record, control; lower tracings, 30 min following i.v. infusion of 25 mgl kg of UL-DOPA. Calibrations: 100 @/. I sec.
chronization was more accentuated. These effects are illustrated in Fig. 2. The desynchronizing effect of DOPA was studied at doses of Xl-30 mg/kg of the DL-form and S-10 mg/kg of L-isomer. In most experiments this effect appeared within 5-10 min following the infusion of DOPA and persisted over 3 h. This action was very intense and appeared in every experiment. It was accompanied by a large increase in the threshold for the recruiting reaction. In some experiments the maxima1 desynchronization appeared within 5 min, in others within 30 min, of DOPA action. Mydriasis was noted at 15-30 min following DO?A and disappeared partially after 60-90 min. The same effects on the EEG were noted when infusing 5-10 mg of L-DOPA into the common carotid artery.
Fig. 3 The influence of DOPA on the cortical and SUbcortical EEG patternsin the cerue~u isok preparationof the cat. 0: control record; b: 30 min following i.v. infusion Of 10 m&kg of L-DOPA. Leads: I-2, SenSori-motorcortex; 3-4, associative cortex; 5, n. amygdalae basalis L-R; 6, hippocampus dorsalis L-R; 7, n. caudatus L-R; 8, thalamus, n. ventr. lat. L-R. Calibrations: 100 CIV,3 Sec. Lkctroenceph. din. Neurophysiol., 1970,28: 259-265
262
K, KADZIELAWA AND E. WIDY-TYSZKIEWICZ
experiments with section of the brain-stem wore performed with gallamine immobflnzation and lignocaine in order to test the effects of DOPA against the same background pharmacological action. The effect of DOPA was always the same and did not depend upon the influence of gallamine or iignocaine. The desynchronizing effect of DOPA was evident in the electrocorticograms and in the leads from subcortical structures. However, i~n the amygdala and hippocampus only a slight decrease in amplitude and no change or slowing of the rhythm were noted following DOPA (Fig. 3, b). The #~fluence of DOPA on electrocorticograms in preparations with hemisection at the rostropontine level Fig. 4 represents a typical response to L-DOPA infused i.v. in a cat with left hemisection at the rostropontine level. In most cases with this section, or with hemisection at the collicular level, the amplitude of the record was greater and the spindles appeared more frequently on the side of the section. During the first 5-10 rain of action in this preparation DOPA caused a typical desynchronization, mainly ipsilateral to the section (Fig. 4, b), whereas equal desyn. chronization on both sides was noted 10-15 rain later (Fig. 4, c). Mydriasis was also much more accentuated and appeared 10-15 rain earlier ipsilateral than contralateral to the section. DISCUSSION
Since the desynchronization following DOPA is dependent upon separation of the lower from the upper brain-stem structures, it seems possible that in normal and curarized cats this effect of DOPA is in some way restrained by the bulbar, and probably high spinal, ascending inhibitory influences. Furthermore, these influences are probably predominantly ipsilateral, as demonstrated in experiments with hemisection at the rostropontine level. Moruzzi and his co]l~borators (see Moruzzi ~q~ for refs.) h~.~e developed a hypothesis suggesting the existence of EEG synchronizing, and probably sleep-inducing, structures located in the lower brain-stem. This hypothesis is
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R Fig. 4 Electrocorticogram of ~he cat encdphale isold preparation with a left hcmisection at the rostropontine Icvvl. a, control; b, tO rain after infusion of 8 mg/kg of L-DOPA i.e.;
c, i 5 rain later. Note the difference in the effect of DOPA on the left (L) and fight (R) ¢lectrocorticograms durin8 the first period of action of the amino acid. Calibrations:
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supported mainly by the following findings: (I) the activation of the EEG in chronic midpontine pretrigeminal p:eparations, independent of the sensory inflow through cranial nerves; in contrast there is a high voltage slow EEG Electroenceph. clin. NeurophysioL, 1970, 2 8 : 2 5 9 - 2 6 5
263
EEG EFFECTS OF DOPA
rhythm in the rostropontine pretrigeminal preparation; (2) the greater tendency to synchronization in the hemisphere contralateral to the midpontine pretrigeminal hemisection in contrast to the persistent ipsilateral desynchronization; (3) desynchronization of the electrocorticogram following inactivation of the lower brain-stem induced by intravertebral infusion of low doses of thiopental; behavioural and EEG arousal in the encdphale isold cat following the transient inactivation of the buibar centres by localized cooling of the fourth ventricle in front of the obex (Berlucchi et al. 1964). Bloch and Bonvallet (196 I) and Bonvallet and Bloch (1961) have observed the potentiation of arousal, induced by midbrain reticular stimulation, by intrabulbar novocaine or prebulbar section. However, there is a question of the anatomical localization of such a synchronizing centre. Magnes et al. (1961) obtained synchronization in the electrocorticogram of encdphale isold cats by low rate electrical stimulation of n. reticularis ventralis and the nucleus of the solitary tract. However, in studies on cats with chronically implanted electrodes Favale et al. (1961) demonstrated synchronization by low frequency stimulation of many reticular sites. According to Bonvallet and Bloch (1961) there is a feedback mechanism between the bulbar ascending deactivating centres and the reticular activating system. The synchronizing structures of t h e lower brain-stem exert an inhibitory action on the pontine centres (Rosina and Mancia 1966). In the light of these observations it is interesting to note that the studies of Matsumoto and Jouvet (1964) suggest some relationship between the level of cerebral catecholamines and the desynchronized EEG pattern in paradoxical sleep (see Jouvet 1967, 1968 for refs.). It is possible that the desynchronizing action of DOPA is restricted by the ascending influences of the same bulbar structures which are probably responsible for the inhibition of the diencephalic and midbrain desynchronizing influences. The studies of Bueno e t al. (1968) suggest the existence of lower brain-stem EEG ascending synchronizing influences in the rabbit. However, in these animals, at doses ineffective innormal
cats, DOPA is able to induce an evident, although not intense, desynchronization, mainly in neocortex, demonstrated by Monnier and Tissot (! 958), Monnier (1960) and in the present study. 'The hypothesis presented here may also be questioned because in some other instances (see Hornykiewicz 1966 for refs.) DOPA is able to activate behaviour in animals with intact neuraxes and, furthermore, this amino acid is known to have a potent awakening effect in reserpinized animals. Nevertheless it is shown now that in chronic experiments or gailamineimmobilized cats doses of DOPA ten times larger than those effective in animals with prebulbar sections have almost no apparent influence on the EEG and behaviour. According to Everett and Wiegand (1962) there is a cloae cc.rrelation of dopamine levels and the behavioural state and a twofold increase seems to be a threshold level. Thus DOPA may be effective: (1) in normal animals only when applied in large doses (its action may depend also on species differences); (2) when its action is potentiated by monoamine oxidase inhibitors, restricting the deamination of its metal~lites, and (3) when the inhibition of the u p t ~ e of dopamine in reserpinized animals intensifies its action. It was also recently found (Kadzielawa, in preparation) that in rabbits the desynchronization following DOPA, applied either i.v. or into the common carotid artery, is markedly accentuated in preparations with sections at levels above the lower brain-stem. SUMMARY
1. The EEG effects of DOPA were analysed in cats, depending upon the level of brain-stem section. 2. In contrast with cats with chronically implanted electrodes, gallamine.immobilized, and with encdphale isold cats, in which DOPA is unable to induce profound changes in the EEG pattern and behaviour, this amino acid elicits an intense desync.hronization in animals with brain-stems sectioned at the postpontine, rostropontine and collicular levels. The desynchronization is not found in hippocampus and amygdala. Electroenceph. clin. Neurophysiol., 1970, 28:259-265
264
.~. KADZIELAWAAND E. WIDY-TYSZKIEWICZ
3. In the early period of action DOPA induces desynchronization mainly ipsilateral to hemisection at the rostropontine level. 4. The observed effects of DOPA are not very dependent on its autonomic effects. 5. In contrast with the mild presser response in gallamine-immobilized cats and in preparations with the brain-stem sectioned at higher levels there is an intense increase in blood pressure and heart rate in encdphale isold preparations. 6. it is concluded that DOPA-induced desynchronization is probably inhibited in animals with intact neuraxes by the tonic ascending influences of the synchronizing buibar and higher spinal structures. RtSUMI~ ANALYSE I~LECTROENCI~PHALOGRAPH IQUE DE L'ACTION CENTRALE DE LA DIHYDROXYPH~NYLALANINE I. Les effets EEG du DOPA ont ~t~ analys~s chez le chat, en fonction du niveau de section du troac c~r6bral. 2. Par comparaison ~ des chats avec ~lectrodes implant~es chroniquement, des chats immobilis~s par gallamine et des chats enc@hale isol6, chez lesquels le DOPA est incapable d'induire des modifications profondes du pattern EEG et du comportement, cet aminoacide provoque une d~synchronisation intense chez des animaux avec section du trent c~r6bral au niveau r~tropontin et pr6pontin et des tubercules quadrijumeaux. La d6synchronisation n'affecte pas l'hippocampe ni ramygdale. 3. A la p~riode pr6coce de son action, le DOPA provoque une d6synchronisation surtout ipsilat~rale a l'h~misection au niveau pr6pontin. 4. Les effets du DOPA observes sent assez ind6pendants de ses effets v6g~tatifs. 5. Par opposition a la 16g~re r6ponse hypertensive des chats immobilis6s par gaUamine et des preparations avec section haute du tronc c6r~bral, on note une augmentation intense de la pression sanguine et de la vitesse cardiaque dans les pr6parations enc~phale isol~. 6. Les auteurs concluent que la d~synchronisation induite par le DOPA est probablement inhib~e chez les animaux avec n~vraxe
intact par les influences toniques ascendantes des structures synchronisatrices bulbaires et spinales hautes. REFERENCES BERLUCCm,G., MAFt~J,L., MORUZZl,G. and STaATA,P. EEG and behavioral effects elicited by cooling of medulla and pens. Arch. ital. Biol., 1964, 102: 372-392. BERTLER,A., FALCK,B. and R.OSENGREN,E. The direct demonstration of a barrier mech-,nism in the brain capillaries. Acta pharmacol, toxicol., 1964, 20: 317-321. BLOCH,V. et BONVALLET,M. Interaction des formations rc~ticulaires m6senc6phalique et bulbaire. J. Physiol. (Paris), 1961, .53: 280-281. ~3ONVALLET,M. and BLOCtt,V. Bulbar control of cortical arousal. Science, 1961, 133:1133-1134. BUENO,J. R., BesT, K. and HIMWIC'H,H. E. Lower brainstem EFG synchronizing mechanism in the rabbit. Electroenceph. clin. NeurophysioL, 1968, 24: 25-34. COSTA,E., PSCHEtD'r,G. R., VAN MElzR, W. G. and HtMWICH, H. E. Brain concentrations of biogenic amines and EEG patterns of rabbits. J. Pharmacol. exp. Ther., 1960, 130: 81-88. EVEnETT,G. M. and WtEOAND,R. G. Central amines and behavioral states: a critique and new data. Prec. 1st int. pharmacol. Meeting. Pergamon, Oxford, 1962, 8: 85-92. FAVALE,E., LOEB,C., Kosst, G. F. and SAcco, G. EEG synchronization and behavioral signs of sleep following low frequency stimulation of the brain stem reticular formation. Arch. ital. Biol., 1961, 99: 1-22. GLowtNsgl, J. and BALD.~|SAmNt,R. J. Metabolism of norepinephrine in the central nervous system. Pharmacol. Rev., 1966, 1~': 1201-1238. GUZM~,N,C. F., ALCARAZ,M. V. and F~RNANDEZ,A. G. Rapid procedure to localizeelectrodes in experimental neurophysiology. Bol. inst. Estud. todd. biol. ( Mdx.), 1958, 16: 29-31. HORNYKtEWtCZ,O. Dopamine (3-hydroxytyramine) and brain function. PharmacoL Re~,., 1966, 18: 925-964. JOUVET, M. R~gulation neuro-humorale des Stats de sommeil. Aspects blologiques et cliniques du syst~me nerveux central. Symposium de Sandoz SA, Bale, 196/: 103-137. JOUWT, ~L Neuropharmacology of sleep. Ir D. H. EFRON (Ed.), Psychoplmrmacology, A Review of Progress 1957-1967, Public Health Service Publication No. 1836, Washington, 1968: $23-540. KADZt~LAWA,K. Studies on the pharmacology of o~.meth. yl-3,4-dihydroxyphenylalanine (o,-methyl-DOPA) and 0,-methylnorepinephrine. !I. int. ]. Neuropharmacol., 1967, 6: 453-462. L~DEaUa, I. X. et TtssoT, R. Modification de l'activ;t6 ~.lectrique c~r6brale du lapin sous l'effet de microinjections de pr6:urseures de monoamines clans les structures somnog~nes bulbaires et pontiques. Electroenceph. din. NeurophysioL, 1966, 20: 370-381. MAON~, J., Moauzzt, G. and POP,WEtANO,O. SynchroniElectroenceph. clin. Neurophysiol., 1970, 28:259-265
EEG EFFECTS OF DOPA zation of the EEG produced by a low-frequency electrical stimulation of the region of the solitary tract. Arch. ital. Biol., 1961, 99: 33-67. M[ANTEGAZZINI,P. et GL~sr~z, A. Action de la Dt.-3,4dioxyphenylalanine (DOPA) et de la dopamine sur l'activit~ ~lectrique du chat "cerveau isol~". Arch. ital. Biol., 1960, 98: 367-374. MARLEY, E. Behavioral and electrophysiological effects of catecholamines. Pharmacol. Rev., 1966, 18: 753-768. MATSUMOTO, J. et JOUVEZ, M. Effets de r,~'serpine, DOPA et 5.HTP sur les deux ~tats de sommeil. C. R. Soc. Biol. (Paris), 1964, 158: 2137-2140. ]~ONNIER, M. Action ~lectro-physiologiques des stimu!~nts du syst~me nerveux central. I. Syst~mes sdr~nergiques, cholinergiques et neurohumeures s~rotoniques. Arch. int. Pharmacodyn., 1960, 124: 281301.
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Reference: KADZIELAWA,K. and WIDY-TYSzKIEWICZ,E. Electroencephalographic analysis of the central action of dihydroxyphenylalanine. Electroenceph. clin. NeurophysioL, 19/0, 28: 259-265.