Progress in Brain Research Volume 28 Anticholinergic Drugs

PROGRESS I N B R A I N RESEARCH V O L U M E 28 ANTICHOLINERGIC DRUGS AND BRAIN FUNCTIONS I N ANIMALS AND MAN

PROGRESS I N BRAIN RESEARCH

ADVISORY BOARD W. Bargmann H. T. Chang

E. De Robertis

J. C. Eccles J. D. French H. Hydtn J. Ariens Kappers S. A. Sarkisov

J. P. SchadC F. 0. Schmitt

Kiel

Shail ghai Buenos Aires Canberra Los Angeles Goteborg Amsterdam Moscow Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 28

ANTICHOLINERGIC DRUGS AND BRAIN FUNCTIONS I N ANIMALS AND MAN EDITED B Y

P. B. B R A D L E Y Department of Experimental Neuropharmacology, The Medical School, Birmingham (England)

AND

M. F I N K Department of Psychiatry, New York Medical College, New York (U.S.A.)

ELSEVIER P U B L I S H I N G C O M P A N Y A M S T E R D A M / LONDON / N E W Y O R K 1968

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This volurrie contains the Proceedings of the Vltli Symposiurn on Anticholinergic Drugs and Brain Firnciions in Aiiinials a d Man, held in Conneziion with the Vtli International Congress Collegiuni Intenlationale Neuro-psychopharmacologicuni, at Washi!igton D.C., March 20-31, 1966 (chairmen: P. B. Bradley and M . Fink)

L I B R A R Y O F C O N G R E S S C A T A L O G C A R D N U M B E R 67-25155

W I T H 102 I L L U S T R A T I O N S A N D 13 T A B L E S

COPYRIGHT 0 1968 BY ELSEVIER PUBLISHING COMPANY, AMSTERDAM

ALL RIGHTS RESERVED TI3IS BOOK O R A N Y P A R T T H E R E O F M U S T N O T BE R E P R O D U C E D I N A N Y F O R M

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List of Contributors

0. BENESOVA, Department of Pharmacology, Charles University, Prague (Czecholovakia). Z. BOHDANECK?,Department of Pharmacology, Charles University, Prague (Czechoslovakia). BOST, Thudichum Psychiatric Research Laboratory, Galesburg Research KATHRYN Hospital, Galesburg, Ill. (U.S.A.). P. B. BRADLEY, The Medical School, Birmingham (U.K.). J. BURES,Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia). P. L. CARLTON, Rutgers, The State University, New Brunswick, New Jersey (U.S.A.). Z. CUCULIC, Douglas Hospital, Verdun, Quebec (Canada). E. F. DOMINO,Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U.S.A.). A. T. DREN,Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U. S.A.). M. FINK,Department of Psychiatry, New York Medical College, New York (U.S.A.). S. GROF,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). 0. GROFOVA, Department of Pharmacology, Medical Faculty of Hygiene, Charles University, Prague (Czechoslovakia). A. HERZ,Deutsche Forschungsanstalt fur Psychiatrie, Max-Planck Institute, Munich (Germany). H. E. HrMwrc~,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, 111. (U.S.A.). R. Yu. ILYUTCHENOK, Institute of Cytology and Genetics, Pharmacological Laboratory, Siberian Branch, Academy of Sciences of the USSR, Novosibirsk (USSR). T. ITIL,Department of Psychiatry of the Missouri Institute of Psychiatry, University of Missouri School of Medicine, St. Louis, Missouri (U.S.A.). E. JACOBSON, Department of Pharmacology, Royal Danish School of Pharmacy, Copenhagen (Denmark). D. KRUS,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). K. KUNZ,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). V. G. LONCO,Istituto Superiore di Sanita, Rome (Italy). A. S. RUDOLPH,Department of Pharmacology and Brain Research Institute, University of Tennessee Medical Units, Memphis, Tennessee (U.S.A.).

VI

LIST OF CONTRIBUTORS

K. RYSANEK,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). A. SCOTTIDE CAROLIS, Istituto Superiore di Saniti, Rome (Italy). J. SKALA,Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). V. V~TEK, Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). M. VOJTECHOVSK?, Laboratory of Experimental Psychopathology, Institute of Pharmacology and School of Public Health, Caroline University, Prague (Czechoslovakia). Z. VOTAVA,Department of Pharmacology, Medical Faculty of Hygiene, Charles University, Prague (Czechoslovakia). R. P. WHITE,Department of Pharmacology and Brain Research Institute, University of Tennessee Medical Units, Memphis, Tennessee (U.S.A.). A. WIKLER,Lexington, Kentucky (U.S.A.). K. YAMAMOTO, Department of Pharmacology, University of Michigan, Ann Arbor, Michigan (U.S.A.). a

Other volumes in this series:

Volume 1: Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener? and J. P. Schade Volume 3 : The Rhinencephalon and Related Structures Edited by W. Bargmann and J. P. Schade Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schade Volume 5 : Lectures on the Diencephalon Edited by W. Bargmann and J. P. Schade Volume 6: Topics in Basic Neurology Edited by W. Bargmann and J. P. Schade Volume 7: Slow Electrical Processes in the Brain by N. A. Aladjalova Volume 8: Biogenic Ainines Edited by Harold E. Himwich and Williamina A. Himwich Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich Volume 10: The Structure and Function of rhe Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. Schadd Volume 11: Organization of the Spinal Cord Edited by J . C. Eccles and J. P. Schade Volume 12: Physiology of Spinal Neurons Edited by J . C. Eccles and J. P. Schade Volume 13 : Mechanisms of Neural Regeneration Edited by M. Singer and J. P. Schadt Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P, Schade

VIlI

Volume 15 : Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrea Volume 16: Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schadk Volunie 17 : Cybernetics of the Nervous System Edited hy Norbert Wiener1 and J. P. Schadt Volume 18 : Sleep Mechanisms Edited by I62.Moreover, the literature clearly indicates that such compounds exert both actions clinically53.Parsidol (ethopropazine), for example, is a phenothiazine used in the treatment of Parkinson’s disease which, in contrast to the ataractic phenothiazines, will block the EEG activation induced by physostigmine62, will arrest the pseudoparkinsonism produced by chlorpromazine29, and in adequate dosage will induce hallucinations39. Similarly, benactyzine readily blocks the EEG effects of physostigmine54; may be used to treat parkinsonism24; and is capable of producing psychotic episodes51. Lastly, the psychotomimetic JB-3 18 has an antitremor action in patients’. The present study affords new evidence that anticholinergic compounds can antagonize an effect of a cholinergic drug at the midbrain level. Although the changes induced by physostigmine in the midbrain reticulum may be indirect, e.g., secondary to excitation of other brainstem structures, the results clearly show that these changes take place in the absence of the prosencephalon and indicate they are central in origin. Therefore, the antagonism of atropine, scopolamine, JB-329 (Ditran) and JB-3 18 to this effect ofphysostigmine can occur centrally at sites below the diencephalon. This is not true of JB-340, which exerts its strong anticholinergic actions only peripherally. These results, coupled with the finding that JB-318 is less active on the iris than JB-340, but more active centrally in antagonizing physostigmine, emphasize that central and peripheral cholinolytic properties of a drug may be of a different order of magnitude and that inferences concerning the central actions of these drugs should not be based upon results obtained from peripheral tissue53. In high doses, for example, Darstine (mepiperphenidol) mimics many of the peripheral effects of atropine in humans but is far less active centrally60; whereas, the newer anticholinergic psychotomimetics (piperidyl benzilates) are evidently superior tools for neuropsychiatric research because they produce more gradual EEG and psychological changes as the dose is increased, induce richer hallucinogenic episodes, and have less autonomic side effects than the belladonna alkaloidss3. There is a growing body of pharmacological evidence indicating that a family of related cholinergic receptors are involved in synaptic transmission. There is, for example, pharmacological evidence indicating both muscarinic and nicotinic receptors are capable of independently causing EEG activationz5150but that nicotinic receptors may not be present in the cerebral cortex37. Atropine readily blocks the EEG arousal caused by acetylcholine42 but does not block the effects of this substance on the Renshaw ce1112. Conversely, dihydro-/3-erythroidine inhibits the Renshaw neuron

SUBCORTICAL ACTIONS OF ANTICHOLINERGICS

21

but fails to block EEG activation. At least two distinct cholinergic receptors are involved in sympathetic ganglionic transmissionl3. Also, atropine and scopolamine inhibit spinal flexor reflexes only at subthalamic sites; whereas, caramiphen produces a similar inhibition at areas below this level of the neuraxislo. The intracerebral injection of cholinergic substances will produce a wide variety of behavioral effects (rage, sleep, arousal, catatonia, etc.) depending on the area injected 21.It is not surprising, therefore, that differences have been reported among the “anticholinergic psychotomimetics” including the belladonna alkaloids. A survey of the literature, however, indicates these differences are quantitative in nature rather than qualitative53. These compounds apparently also have a dual action in impairing central cholinergic mechanisms: they block the usual EEG effects of cholinergic agents55 and they decrease brain acetylcholine content20. These two actions appear to be related; the most potent blockers of cholinergic drugs seem to be most active in decreasing brain acetylcholine. Moreover, the effects on the EEG53,55*59, behavior55959 and on brain acetylcholine levels20 caused by the anticholinergic psychotomimetics reach a maximum with comparatively low doses. Since scopolamine36.59 and atropine59 d o not produce notable sedation in intact rabbits even in enormous doses59 but do produce a “Iissive” effect in decorticate rabbits36959, it is apparent that the subcortical and cortical actions of the drugs differ in this species. The subcortical effects of these drugs may be related to their antiparkinson actions in humans. At least, our findings lend support to the hypothesis that antiparkinson agents counteract a subcortical hyperactive cholinergic mechanism24,28,49,62. The early work of Veit and Vogt48 may help explain why low doses of these belladonna alkaloids produce sedation in dogs and in higher doses disorientation or deliriumlike behavior59. They found the concentration of scopolamine in the cerebral cortex and midbrain to be comparable after low doses of scopolamine (2 mg/kg), but after high doses (10 mg/kg) the concentration in the cortex was about 2.5 times greater than in the midbrain. Therefore, in low doses normal function may be inhibited throughout the neuraxis producing effects on the EEG and midbrain reticular evoked responses similar to those produced by other sedatives in animals53-61. In low doses both are also sedatives in man32138~60.In higher doses, only the functions of the cerebrum may be further impaired significantly, producing in dogs many of the characteristics of decortication5.59 without producing classical signs of anesthesia, either behaviorally, electroencephalographically, or on midbrain evoked responses34.53. Indeed, the behavioral changes induced in dogs (blindness, slow compulsive gait, etc.) resemble those obtained with LSD256. Moreover, some motor effects produced by amphetamine are enhanced8359 suggesting that adrenergic or non-cholinergic mechanisms may be “released” during atropine or scopolamine toxicity. However, the so-called stimulation produced by these drugs does not mimic that induced by amphetamine in dogs59 and has no reliable analeptic value clinically. Indeed the “stimulation” seen with these alkaloids is interrupted by periods of sleep in dogs, monkeys and man53. Moreover, the EEG activation caused by adrenergic drugs is blocked by anticholinergic agents56 suggesting some central “adrenergic” systems depend ultimately on cholinergic References p . 24-26

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A. S. R U D O L P H

processes. Also, the belladonna alkaloids greatly potentiate the depressant actions of ether (personal observations) and pentobarbital69 in dogs. They also potentiate the action of barbiturates in rats19 and monkeys5Q.Such synergism is a common characteristic of CNS depressants. Hence the “excitation” or disorientation induced by toxic doses of the belladonna alkaloids may be considered “pseudostimulation” caused by an inhibition of cholinergic mechanisms and an imperfect “release” of non-cholinergic mechanisms. Most reports dealing with the mechanism by which anticholinergics produce central actions are consonant with the hypothesis that they inhibit cholinergic systems and that their behavioral effects may result from a functional “imbalance”4,53. The tricyclic antidepressants (e.g., desipramine) evidently possess mild anticholinergic actions centrally, reducing the effects of cholinergic agents3.40, while simultaneously may enhancing the actions of adrenergic agents40943, so that a dual a~tion4~~43~46~53 account for their clinical efficacy. In this regard, it would be of interest to administer to endogenously depressed patients small doses of both atropine and amphetamine to ascertain whether this combination is also beneficial to such patients. In humans, of atropine toxicity the EEG synchrony64 and hallucinogenic manife~tationsl~9~~964 revert to normal after the intramuscular administration of 4 mg of physostigmine, presumably by restoring cholinergic functions centrally. Similarly, the belladonna alkaloids and physostigmine are antagonists in their effects on conditioned behavior of laboratory animals22135. Another cholinergic drug, tetrahydroaminoacrin (THA), in 60 mg doses i.v. will antagonize the hallucinations, stupor and other effects of 10 mg of Ditran (JB-329) in humanslB.Moreover, the psychotomimetic effects of Ditran are changed to a coma-like condition with small doses of chlorpr0mazine2~, and perhaps because the sedative properties of chlorpromazine are intensified or “released”, adrenergic phenomena are antagonized. The independent nature of the EEG and the evoked responses seen in this study indicate that different processes or neurons are involved in each phenomenon. The evoked phenomenon is also more specific, being obtained only in certain leads; whereas, an EEG was obtained from all locations of the midbrain. Independent variations between the EEG and evoked responses are also evident in intact a n i m a W . It is possible that the neuroglia contribute significantly to the EEG pattern16, and evoked potentials are specific signals so that, at least under the influence of drugs, they may vary independently. Since the anticholinergic agents failed to alter the EEG pattern from these “midbrain animals”, but do change this pattern in intact rabbits61, it is possible that their main site of action is on cholinergic links above the midbrain23v40.52.On the other hand, such drugs may not be able to affect these abnormal waves because impulses responsible for normal EEG patterns were destroyed, thereby preventing any cholinolytic action at the midbrain level. In this regard, atropine fails to change the electrocorticogram obtained from the acute “isolated hemisphere” preparation42 so that the drug apparently does not affect the electroencephalogram in such abnormal preparations. From more physiological experiments, however, Rinaldi41 concluded that atropine must have actions both at cortical and midbrain sites,

SUBCORTICAL ACTIONS OF ANTJCHOLINERGICS

23

Although it is questionable whether the results described here are specifically related to the many diverse behavioral effects produced by anticholinergic compounds, they do provide evidence that the midbrain reticulum is affected - probably directly by cholinergic and anticholinergic agents. They further show that pharmacological changes may be induced in the midbrain reticular formation that are not dependent on higher centers and demonstrate the importance of testing anticholinergic compounds upon a background of cholinergic stimulation. Alone, none of the anticholinergic drugs given systemically depress midbrain evoked responses, but neither do ataractics or sedatives61. However, the latter two groups of drugs fail to block the effect ofphysostigmine on single shock responses recorded from the midbrain of intact rabbits; whereas, the piperidyl benzilates and belladonna alkaloids are antagonistic to physostigmine53~61.Similarly, atropine alone will not inhibit synaptic activity of the cerebral cortex, but will block the effects of acetylcholine given by close arterial injection44. Lastly, our positive findings question the implication of Giarman and Pepeu20 that scopolamine has no important action on the “rostral” midbrain because in rats it failed to reduce significantly acetylcholine levels in this area. The fact that these investigators found a reduction of 14% in “rostral” midbrain agrees with the report of Veit and Vogt48 that scopolamine does enter the midbrain. Also, with higher doses Veit and Vogt found about 2.5 times more scopolamine in the cortex than in the midbrain. Giarman and Pepeu showed, similarly, that scopolamine reduced acetylcholine levels of the cerebrum 2.5 times greater than the 14% in the midbrain. Our findings indicate that at least some anticholinergic agents enter regions of the brainstem to antagonize the actions of cholinergic agents and support the inferences of 0 t h e r s 7 J 5 ~ ~that 8 ~ ~chclinoceptive ~ neurons are present in the brain below the diencephalon which may be pharmacologically altered by anticholinergic compounds. SUMMARY

Electrographic recordings were obtained from the midbrain reticular formation of rabbits in which the prosencephalon (cerebrum and diencephalon) was extirpated. These recordings consisted of “spontaneous” brain waves (EEG) and evoked potentials produced by applying single shock stimuli to one sciatic nerve or to the pontine region of the reticular formation. Physostigmine (0.2 mg/kg) significantly reduced or abolished the single shock responses. In contrast, atropine, scopolamine, and four piperidyl benzilates (JB-329, JB-3 18, JB-340, JB-305) did not reduce the amplitude of the evoked potentials. However, atropine, scopolamine, JB-3 18 and JB-329 completely blocked the effect of physostigmine on the single shock responses; whereas, JB-340 and JB-305 had no such effect. Possible relationships between the ability of anticholinergic compounds to produce psychotic episodes or ameliorate Parkinson’s disease and their ability to block the electrographic effects of physostigmine in experimental animals were discussed. It was concluded that the reduction of the single shock response caused by physostigmine was central in origin because (1) this same effect was obtained by high freReferences p . 24-26

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quency stimulation of the pontine reticulum, (2) it was not produced by neostigmine, and (3) JB-340, a compound which exerts strong anticholinergic actions peripherally, was unable to block this action of physostigmine. The “spontaneous” electrical activity recorded from the midbrain reticulum in these experiments was low in amplitude and was not significantly changed by the above drugs. Pentobarbital also failed to change these patterns but a Metrazol seizure pattern could be induced. Important differences between recording antagonistic actions of drugs on single shock responses and on “spontaneous” electrical activity was therefore emphasized. It was also stressed that the use of a suitable agonist (e.g., physostigmine) may be necessary to reveal significant effects of, and differences among, many centrally acting drugs. REFERENCES 1 AeooD, L. G. (1957) Some relations between chemicalstructure andphysiologicalaction of mescaline and related compounds, in Neuropharmacology. Josiah Macy, Jr. Foundation, New York, pp. 229-234. 2 ABOOD,L. G., OSTFELD, A. AND BIEL,J. H. (1959) Structure-activity relationship of 3-piperidyl benzilates with psychotogenic properties. Arch. int. Pharmacodyn., 120, 186-200. 3 BENESOVA, O., BOHDANECK~, Z. AND GROFOVA, I. (1964) Electrophysiological analysis of the neuroleptic and antidepressant actions of psychotropic drugs in rabbits. Znt. J. Neuropharmacol., 3,479-488. 4 BIEL,J. H., NUHFER, P. A., HOYA,W. K., LEISTER, H. A. AND ABOOD,L. G. (1962) Cholinergic blockade as an approach to the development of new psychotropic agents. Ann. N. Y.Acad. Sci., 96, 251-262. 5 BIJLSMA, U. G. AND BROUWER, J. E. (1928) Die Wirkung des Skopolamins in Kombination mit Cyanid, Kohlenoxyd und Luftverdunnung, Arch. exp. Path. Pharmakol., 138, 190-207. 6 BOGDANSKI, D. F., WEISSBACH, H. AND UDENFRIEND, S. (1958) Pharmacological studies with the serotonin precursor, 5-hydroxytryptophan. J. Pharrnacol., 122, 182-194. 7 BRADLEY, P. B. (1957) Microelectrode approach to the neurnpharmacology of the reticularformatiorr. Psychotropic Drugs. Eds. S. Garattini, V. Ghetti. Elsevier, Amsterdam, 207-216. 8 CARLTON, P. L. AND DIDAMO, P. (1961) Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmacol., 132, 91-96. 9 CHATFIELD, P. 0. AND PURPURA, D. P. (1954) Augmentation of evoked cortical potentials by topical application of prostigmine and acetylcholine after atropinization of cortex. EEG Clin. Neurophysiol., 6, 287-298. 10 DEMAAR, E. W. J. (1956) Site and mode of action in the central nervous system of some drugs used in the treatment of Parkinsonism. Arch. int. Pharmacodyn., 105, 349-365. 11 DESMEDT, J. E. AND SCHLAG, J. (1957) Mise en evidence d’elements cholinergiques dans la formation reticulee mesendphalique. J . Physic/. (Paris), 49, 136-1 38. 12 ECCLES, J. C., ECCLES, R. M. AND FATT,P. (1956) Pharmacological investigations on a central synapse operated by acetylcholine. J. Physiol. (Lond.), 131, 154-169. 13 ECCLES, R. M. AND LIBET,B. (1961) Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol., 157, 484-503. E. AND MARSALA, J. (1962) Sereotaxic atlases for the cat, rabbit and rat. In J. BureS 14 FIFKOVA, et al., Electrophysiological Methods in Biological Research. Academic Press, New York, Appendix I: 426-467. 15 FORRER, G. R. (1958) Atropine coma therapy: Report of a death. J. Michigan State Med. Soc., 57, 996-998. 16 GALAMBOS, R. (1961) A glia-neural theory of brain function. Proc. Nut. Acad. Sci.,47, 129-136. 17 GERSHON, S. AND BELL,C. (1963) A study of the antagonism of some indole alkaloids to the behavioural effects of “Ditran”. M e d exp., 8, 15-27. 18 GERSHON, S. AND OLARIU,J. (1960) JB-329 - A new psychotomimetic. Its antagonism by tetrahydroaminacrin and its comparison with LSD, mescaline and sernyl. J. Neuropsychiat., 1,283-292.

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19 GIARMAN, N. J. AND PEPEU,G. (1962) Drug-induced changes in brain acetylcholine. Brit. J. Pharmacol., 19, 226-234. 20 GIARMAN, N. J. AND PEPEU,G. (1964) The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Brit. J. Pharmacol., 23, 123-1 30. 21 HERNANDEZ-PEON, R., CHAVEZ-IBARRA, G., MORGANE, P. J. AND TIMO-IARIA, C. (1963) Limbic cholinergic pathways involved in sleep and emotional behavior. Exper. Neurul., 8, 93-1 11. 22 HERZ,A. (1967) Some actions of cholinergic and anticholinergic drugs on behaviour. This volume. 23 HIMWICH, H. E. AND CUCULIC, Z. (1967) An examination of a possible cholinergic link in the EEG arousal reaction. This volume. 24 HIMWICH,H. E. AND RINALDI,F. (1957) The antiparkinson activity of benactyzine. Arch. int. Pharmacodyn., 110, 119-127. 25 ILYUTCHENOK, R. J. (1963) Problems of chemical perceptibility of the bruin stem reticular formation. Psychopharmacological Methods, Eds: Z. Votava, M. Horvath, 0. Vinaf. Pergamon Press, Oxford, England, pp. 115-122. 26 ISBELL, H., ROSENBERG, D. E., MINER,E. J. AND LOGAN, C. R. (1964) Tolerance and cross tolerance to scopolamine, n-ethyl-3-piperidyl benzylate (JB-318) and LSD-25. Neuropsychopharmacology, 3,440-446. 27 ITIL,T. M. (1966) Quantitative EEG changes induced by anticholinergic drugs and their behavioral cxrelates in man. Rec. Adv. B i d . Psychiat., 8, 151-173. 28 JENKNER, F. L. AND WARD,JR., A. (1953) Bulbar reticular formation and tremor. Arch. Neirrol. Psychiat. (Chicago), 70,489-502. 29 KRUSE,W. (1960) Treatment of drug-induced extrapyramidal symptoms. Dis. New. Syst., 21, 79-8 1, 30 LOEB,C., MAGNI,F. AND ROW,G. F. (1960) Electrophysiological analysis of the action of atropine on the central nervous system. Arch. ital. Biol., 98, 293-307. 31 LONGO,V. G. (1956) Effects of scopolamine and atropine on electroencephalographic and behavioral reactions due to hypothalamic stimulation. J. Pharmacol., 116, 198-208. 32 LONGO, V. G. (1966) Mechanisms of the behavioral and electroencephalographic effects of atropine and related compounds. Pharmacol. Rev., 18, 965-996. 33 LONGO,V. G. AND SILVESTRIM, B. (1957) Effects of adrenerkic and cholinergic drugs injected by the intra-carotid route on electrical activity of brain. Proc. Soc. exp. Biol., 95, 43-47. 34 LONGO,V. G. AND SILVESTRINI, B. (1958) Contribution a l’etude des rapports entre le potentiel reticulaire Bvoque, l’etat d’anesthtsie et l’activite electrique cerebrale. EEG Clin. Neurophysiol., 10,111-120. 35 MCGAUGH, J. L., DEBARAN, L. AND LONGO,V. G. (1963) Electroencephalographic and behavioral analysis of drug effects on an instrumental reward discrimination in rabbits. Psychophurmacofogia, 4, 126-1 38. 36 MEHES,J. (1929) Studien iiber den Skopolaminschlaf und seine Verstarkung durch Morphium. Arch. exp. Path. Pharmakol., 142, 309-322. 37 NICKANDER, R. C. AND YIM,G. K. W. (1964) Effects of tremorine and cholinergic drugs on the isolated cerebral cortex. Int. J. Neuropharmacol., 3, 571-578. 38 OSTFELD, A. M. AND ARUGUETE, A. (1962) Central nervous system effects of hyoscine in man. J. Pharmacol., 137, 133-139. 39 PFEIFFER, C. C., (1959) Parasymphathetic neurohumors; possible precursors and effect on behavior. Int. Rev. Neurobiol., 1, 195-244. 40 RATHBUN, R. C. AND SLATER, I. H. (1963) Amitriptyline and nortriptyline as antagonists of central and peripheral cholinergic action. Psychopharmacologiu, 4, 114-125. 41 RINALDI,F. (1956) Direct action of atropine on the cerebral cortex of the rabbit. Progr. Brain Res., 16, 229-244. 42 RINALDI,F. AND HIMWICH, H. E. (1955) Cholinergic mechanisms involved in function of mesodiencephalic activating system. Arch. Nrurol. Psychiat., 73, 396-402. 43 SIGG,E. 3.(1962) The pharmacodynamics of imipramine. The first Hahnemann Symposium on Psychosomatic Medicine. Lea and Febiger, Pub., 671-678. 44 SIGG,E. B., DRAKONTIDES, A. B. A N D DAY,C. (1965) Muscarinic inhibition of dendritic postsynaptic potentials in cat cortex. Int. J. Neurupharmucol., 4,281-289. 45 STEINER, W. G. AND HIMWICH, H. E. (1962) Central cholinolytic action of chlorpromazine. Science, 136, 873-874. 46 SULSER, F., BICKEL, M. H. AND BRODIE, B. B. (1964)The action of desmethylimipramine in counteracting sedation and cholinergic effects of reserpine-like drugs. J. Pharmacol., 141, 321-330.

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47 TEUCHMANN, J. (1949) The action of diallilbarbituric acid and of scopolamine on the spinal reflexes of the decapitated, the decerebrated and the decorticated cat. Arch. in/. Pharmacodyn., 79, 257-262. 48 VEIT,F. AND VOGT,M. (1935) Verteilung von Arzneistoffen auf verschiedene Regionen des Zentralnervensystems, zugleich ein Beitrag zu ihrer quantitativen Mikrobestimmung im Gewebe. Arch. f: exper. Path. u. Pharmakol., 178, 534-559. 49 VERNIER, V. G. AND UNNA,K. R. (1956) Theexperimental evaluationofantiparkinsoncompounds. Ann. N . Y.Acad. Sci., 64,690-704. 50 VILLARREAL, J. E. AND DOMINO,E. F. (1964) Evidence for two types of cholinergic receptors involved in EEG desynchronization. The Pharmacologist, 6, 192. 51 VOJTECHOVSKY, M. (1967) Experimental psychosis induced by benactyzine. This volume. 52 WHITE,R. P. (1963) Relationship between cholinergic drugs and EEG activation. Arch. in/. Pharmacodyn., 145, 1-17. 53 WHITE,R. P. (1966) Electrographic and behavioral signs of anticholinergic activity. Rec. Adv. Biol. Psychiat., 8, 127-139. 54 WHITE,R. P. AND BOYAJY, J. D. (1960) Neuropharmacological comparison of atropine, scopolamine, benactyzine, diphenhydramine and hydroxyzine, Arch. in/. Pharmacodyn., 127, 260-273. 55 WHITE,R. P. AND CARLTON, R. A. (1963) Evidence indicating central atropine-like actions of psychotogenic piperidyl benzilates. Psychopharmacologia, 4, 459-47 I . 56 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effects of adrenergic drugs. J. Pharmacol., 125, 339-346. 57 WHITE,R. P. AND HIMWICH,H. E. (1957) Analysis of forced circling induced by DFP and ablation of cerebral structures. Am. J. Physiol., 189, 513-516. 58 WHITE,R. P. AND HIMWICH, H. E. (1957) Circus movements and excitation of striatal and mesodiencephalic centers in rabbits. J . Neurophysiol., 20, 81-90. 59 WHITE,R. P., NASH,c. B., WESTERBEKE, E. J. AND POSSANZA, G . 3. (1961) Phylogenetic comparison of central actions produced by different doses of atropine and hyoscine. Arch. int. Pharmacodyn., 132, 349-363, 60 WHITE,R. P., RINALDI, F. AND HIMWICH, H. E. (1956) Central and peripheral nervous effects of atropine sulfate and mepiperphenidol bromide (Darstine) on human subjects. J. Appl. Physiol., 8,635-642. 61 WHITE,R. P., SEWELL, H. H., JR. AND RUDOLPH, A. S. (1965) Drug-induced dissociation between evoked reticular potentials and the EEG. EEG Clin. Neurophysiol., 19, 16-24. 62 WHITE,R. P. AND WESTERBEKE, E. J. (1961) Differences in central anticholinergic actions of phenothiazine derivatives. Exp. Neurol., 4, 317-329. 63 WIKLER, A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: Morphine, N-allylnormorphine and atropine. Proc. Soc. exp. Biol., N . Y., 79, 261-264. 64 WILSCN, W. P. (1961) Observations on the effect of toxic doses of atropine on the electroencephalogram of man. J. Neuropsychiat., 2, 186-190.

27

An Examination of a Possible Cortical Cholinergic Link in the EEG Arousal Reaction Z. CUCULIC*, K A T H R Y N BOST

AND

H. E. H I M W I C H

Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, lllii7ois 61401 (U.S.A.)

Knowledge concerning the function of the reticular formation received a great impetus with the discoveries of Moruzzi and Magoun27 and their schools. The part played by the thalamic diffuse projection systems has also been intensively studied, especially by Jasperl5. Because these two systems may function together in the production of EEG arousal, Rinaldi and H i m ~ i c hsuggested ~ ~ ? ~ ~the term mesodiencephalic activating system (MDAS) to denote their combined action. They also suggested that the EEG arousal reaction is cholinergic i n nature for not only was the response evoked by cholinergic drugs but it was blocked by atropine. Other cholinolytics, like benztropine methane sulfonate (Cogentin) and benzilic acid diethylaminoethylester (benactyzine)l3J4 also prevent EEG arousal. In seeking a rostral link in the MDAS, it was found that EEG arousal could still be obtained after post-collicular post-pontine secti0n3~.With a more rostral transection, at the pre-collicular pre-pontine level, thus excluding the midbrain, Steiner and Himwich38 observed that the administration of acetylcholine was followed by EEG arousal which was blocked by atropine. In this regard it is pertinent that Smirnov and Ilyutchenok36 found that atropine-like drugs applied topically to the cortex blocked EEG arousal, thus indicating the possibility of cholinergic structures at or close beneath the cortical surface, a suggestion of importance in establishing a cortical cholinergic link in EEG arousal. In agreement with these workers are the observations of Rinaldi30, who administered atropine not only topically but also by injection into a cortical artery. The present report is concerned with further studies of the above suggestion. METHOD A N D MATERIAL

Our experiments were performed on 90 adult albino rabbits weighing between 2.5 to 3.0 kg. The rabbits were prepared under ether and local 0.2 % pontocaine anesthesia and were studied under artificial respiration, using small doses of curarz sufficient to establish physical immobilization but not to affect the electrical phenomenon. The electrocardiogram was recorded throughout the experiment and blood pressure

*

Present address: Douglas Hospital, Verdun, Quebec (Canada).

References p. 38-39

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readings were obtained from the femoral artery by means of a mercury manometer. A polyethylene cannula was inserted in the femoral vein for the intravenous administration of the drugs. The cortical electrodes were of the silver ball type with flexible insulated silver wires and placed in Plexiglas holders. In some experiments, deep coaxial electrodes were implanted according to the maps of Sawyer, Everett and Green33a for the rabbit. The recordings were registered from the following areas: anterior cortex (motor), posterior cortex (limbic), caudate nucleus, hippocampus and thalamus (see Fig. 1). All drugs were dissolved in distilled water and for

SAGITTAL SUTURE

SAGITTAL SUTURE

Fig. 1. Schematic representation of the electrode sites used in present experiments. a, b, c, d are cortical leads. In these experiments monopolar coaxial electrodes were used. The deep electrodes include nucleus caudatus (NC), thalamus (TH), reticular formation (RF) and hippocampus (H). Four different arrangements of electrode sites are presented: A, B, C, D.

topical application were placed on the exposed cortex in gel-foam pledgets (6 x 8 mm) saturated with the solution of the drug used. The pH was adjusted to approximately 5. The drugs used and the concentrations employed were : benactyzine 0.3 %, scopolamine 0.3 %, atropine 0.34.5 %, eserine 0.1 %, pilocarpine 0.1 %, Metrazol 0.5 %, pontocaine 2 % and 1 %, carbocaine 1 %, and strychnine 0.05%. The intravenous injections included cholinolytic drugs given in the following ranges : scopolamine 0.9-1 .O mg/kg, benactyzine 1.5-2 mg/kg, atropine 1-3 mg/kg. The dosage ranges of the cholinergic agents included i.v. eserine at 0.1-0.2 mg/kg and i.v. pilocarpine 14 mg/kg. One convulsant was given i.v., strychnine 0.1-0.3 mg/kg. The ranges of the other two drugs used i.v. were d-amphetamine 4.5-6 mg/kg and 5-hydroxytryptophan with a toral dose of 33 mg. In each experiment the dose employed was sufficient to produce the required results, whether blocking or activation. To stimulate the midbrain reticular substance, direct current pulses of a frequency of 300/second and a duration of 2 msec were employed. The voltage varied from 1 to 10 with total durations from

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3 to 10 seconds. The topical effects of the local anesthetics, pontocaine and carbocaine, were examined against the EEG alerting produced by 0.1 mg/kg eserine i.v. RESULTS

A . Results o j topicul application of the cholinolytic drugs (benactyzine, scopolamine, atropine) upon cortical alert patterns previously established with i.v. eserine or pilocarpine In 24 animals, soon after the topical application of gel-foam pledgets saturated with the anticholinergic agents, the alert pattern evoked by eserine (16 experiments) and pilocarpine (8 experiments) changed to high slow waves but there were differences between the three cholinolytic drugs. Following the application of atropine, a period of from 5 to 6 minutes elapsed before the synchronization was observed. Scopolamine required about 3 minutes and benactyzine synchronized the brain waves during the first 2 minutes of application. With simultaneous cortical and subcortical recordings (12 experiments) the synchronization was seen only in the area of the cerebral cortex EFFECT OF TOPICAL APPLICATION OF BENACTYZJNE O N THF AROUSAL REACTION EVOKED BY ESERINE I.V. a

b C

EKG 1 0 0 p v ~ I-SCC.

R.M

\

C

N

.

R s=zc-s

Fig. 2. Blocking effect of topical application of benactyzine (0.3 % solution) to the left cortical sites (a, c) on the arousal reaction evoked by i.v. eserine (0.2 mg/kg). For the designation of electrode sites, refer to Fig. 1, C.

where the cholinolytic drug was applied (Fig. 1, B,C,D) Other cortical as well as subcortical areas (nucleus caudatus, hippocampus, thalamus) continued unchanged (Fig. 2). Moreover, of the three drugs, benactyzine proved to be the most effective in the complete elimination of alerting and was especially superior to atropine which intermittently permitted alerting patterns to break through the inhibition. The blocking action of benactyzine was most pronounced in the immediate vicinity of the coronal suture. Single topical applications of benactyzine or scopolamine were followed by periods of EEG synchronization which endured for 30-45 minutes. But after atropine References p. 38-39

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the synchronization lasted only 15-20 minutes. After these periods, however, alerting procedures evoked the EEG arousal in all leads although the arousal was still somewhat less pronounced in the area of cortical application of the anticholinergic agents. The differences between the inhibitory influences exerted by benactyzine on eserine or pilocarpine alerting were slight.

B. Eflect of the topical application of the cholinolytic drugs upon the cortical EEG resting pattern In 6 animals the areas of application of benactyzine revealed waves of comparatively higher amplitude and lower frequency than the other cortical regions; these differences endured for approximately 60 minutes. In 6 other rabbits, however, the resting patterns did not disclose differences between the sites of application and the contralateral control area until after the usual alerting procedures when the treated portions of the cerebral cortex failed to show alerting either to sound or pain or the intravenous administration of eserine, similar to the patterns observed in Fig. 2. C . Results o j the topical application of cholinolytic drugs on the EEG arousal evoked by the electrical stimulation of the midbrain Approximately 2 minutes after the topical application of benactyzine (8 animals) the strength of the current stimulating the midbrain reticular formation had to be increased from 3-4 volts with a duration of 5 seconds to a current of from 6-8 volts with the same duration before the arousal reaction could be evoked. But 10 minutes after the application of benactyzine even a current of 10 V for 10 seconds could no longer induce the alert pattern though it was seen clearly in non-treated areas (Fig. 3).

EFFECT OF TOPICAL APPLICATION OF BENACTYZINE ON THE AROUSAL REACTION EVOKED BY ELECTRICAL STIMULATION OF THE MIDBRAIN RETICULAR FORMATION

d

EKG

Fig. 3. Contrasting effects of previous application of benactyzine (0.3 % solution) to left cortical areas (a, c) on the arousal reaction subsequently evoked by electrical stimulation of the midbrain reticular formation (direct current pulses with a frequency of 300/second and a duration of 2 msec; 4 volts with a total duration of 5 seconds). For the designation of electrode sites, refer to Fig. 1, B.

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D . Effects of topical application of’ eserine followed by the intravenous administration of either benactyzine or scopolamine In 5 animals after the topical application of eserine, spiking appeared in the area of application in approximately 5 minutes. Subsequently, in most instances the intensity increased to seizure-like outbursts. These seizure patterns were triggered by the intravenous administration of benactyzine or of scopolamine as well as by various alerting procedures. E. Eflects of topical application with pentamethylenetetrazol (Metrazol) followed by i.v. eserine on EEG alerting In 5 rabbits the topical application of pentamethylenetetrazol (0.5 solution) was followed by the appearance of rounded waves of high amplitude and slower frequency though sometimes spikes were also observed as well as seizure-like patterns. But the usual EEG arousal patterns evoked by 0.2 mg/kg of eserine continued interspersed between the abnormal waves similar in general design to that shown in Fig. 4 where the abnormal waves are caused by strychnine. F. Effects of topical applications of strychnine against the intravenous administration of benactyzine, eserine and pilocarpine In 7 experiments after the application of strychnine, sporadic spikes, later becoming s-izurz-like outbursts were observed. The outbursts were readily triggered by peripheral stimulation (sound or pain) or by the intravenous administration of eserine. The intravenous administration of benactyzine intensified the local effects of strychnine but in untreated cortical areas, waves of high amplitude and low frequency indicating synchronization were observed. In 6 of these 7 experiments strychnine spiking spread t o all leads but was most pronounced in the areas of topical application where the frequency was greater and seizure-like outbursts were more numerous. In 6 additional experiments the intravenous administration of eserine (0.2 mg/kg) or pilocarpine (14 mg/kg) induced alert patterns in all leads though in the cortical area covered with the gel-foam pledgets saturated with strychnine, the alerting patterns were interspersed between areas of multiple spiking even more pronounced than with strychnine alone (Fig. 4). G. Interactions resulting from the topical application of benactyzine and the intra-

venous administration of strychnine Benactyzine was applied topically prior to the intravenous administration of strychnine in 6 animals. Following strychnine, blood pressure increased from 80 mm Hg to 160 mm Hg, two minutes after the injection. Simultaneously EEG alert patterns were observed in the areas other than those of topical application. This alert pattern lasted for 3 minutes after the first injection (0.1 mg/kg) but after two or three injections the arousal persisted for 10-1 5 minutes. The arousal reaction was followed by spiking first in the areas of the application of the cholinolytics but later spreading to all areas recorded. References p . 38-39

32

2. C U C U L I C et al. INTER-REACTION OF TOPICAL APPLICATION O f STRYCHNINE AND THE AROUSAL REACTION EVOKED BY ESERINE I.V.

a

c d 100 p

v

L

I-SEC.

EKG OCZC-3

Fig. 4. The arousal reaction evoked by the intravenous administration of eserine (0.2 rng/kg) following previous topical application of strychnine (0.05% solution) on the left hemicortex (areas a, c). For the designation of electrode sites, refer to Fig. I A .

H . Efects of the topical application of benactyzine on the alert pattern following the intravenous administration of amphetanzine In 4 experiments benactyzine was applied topically and in 2 others atropine was used after the amphetamine-induced alert pattern had been established. Approximately two minutes after benactyzine the alert pattern was changed to high slow waves in the treated areas (Fig. 5 ) . In one experiment with successive applications of benactyzine EFFECT OF TOPJCAL APPLICATION OF BENACTYZINE ON THE AROUSAL REACTION EVOKED BY 0-AMPHETAMINE I.V. AMPHETAMINE AROUSAL

AFTER BENACTYZINE

I-SEC.

DLZC-6

Fig. 5. l h e blocking effect of topical application of benactyzine (0.3%solution) to the left cortical areas (a, c) on the arousal reaction evoked by the intravenous administration of d-amphetamine (total dose 9 mg). For the designation of electrode sites, refer to Fig. l,A.

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to all cortical areas, amphetamine arousal was gradually extinguished until all exposed cortical sites exhibited blocked patterns as previously described in B. With atropine the synchronization was not so prominent and required about 10 minutes before blocking occurred.

EKG

5-HTP ACTIVATION a-

b C

EKG EFFECT OF TOPICAL APPLICATION OF BENACTYZINE ON THE AROUSAL REACTION EVOKED BY 5-HTP I.V.

a

>

b C-

&c-2 'OQ+z

EKG

Fig. 6 . Interactions of 5-hydroxytryptophan induced alerting and topical application of benactyzine. Top series of tracings, controls with alerting response to sound of hand clapping. Middle series of tracings, alerting induced by 3 intracarotid injections of 5-hydroxytryptophan (5-HT), 11 mg each. Lowest series of tracings portray effects of the topical application of benactyzine (left cortical areis a, c) on the arousal reaction evoked by 5-HTP. For the designation of electrode sites, refer to Fig. 1, A. References p . 38-39

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I. Effects of topical application of benactyzine on the EEG alert pattern obtained by the administration of 5-hydroxytryptop~an(5-HTP) According to the technique of Schweigerdt and Himwich34, EEG arousal was obtained by intracarotid injections of 11 mg of 5-HTP given three times. Usually the alerting evoked by 5-HTP was not as marked as that produced by eserine nor was the inhibition exerted by benactyzine always as clearly demarcated, but in 3 of 4 experiments a change from the resting pattern followed the intravenous administration of the serotonin precursor and in each benactyzine synchronized the pattern of the cortical surfaces to which it had been applied (Fig. 6). J . Effects of pontocaine and carbocaine on EEG arousal patterns In 4 experiments gel-foam pledgets saturated with 2 % pontocaine were applied to the left side of the exposed cortex causing some diminution of amplitude on that side. Nevertheless arousal patterns continued, occurring apparently spontaneously and after auditory and pain stimuli as well as after i.v. eserine, 0.1 mg/kg. The arousal responses evoked by i.v. eserine continued for 20-30 minutes (Fig. 7) despite the topical application of pontocaine. Four other experiments with 1 % pontocaine INTER - REACTION OF TOPICAL APPLICATION OF 2 % PONTOCAINE A N D THE AROUSAL REACTION EVOKED BY ESERlNE Apparently. spontaneous Control

a r o u d 16 min. after 2 I Pontocaine on a & c

Arousal 3 min. after h e r i n e 0.1 m g / k g I.V.

b-

v

d-

K E G -

100 p v

r , I-See.

9c-zc--7

Fig. 7. Interreaction of topical application of 20/, pontocaine and the arousal reaction evoked by eserine.

yielded similar results except that in addition, biphasic spikes of slow frequencies occurred in the medicated side. In these animals the alert patterns were continuous on the right side of the cortex but on the left side were observed only between the slow spikes, a mixture of frequencies similar to the alerting patterns which occurred between strychnine spikes as illustrated in Figure 4. After 0.1 mg/kg eserine i.v. in one of these four observations, the slow spikes gradually disappeared and the alerting patterns were maintained throughout all observed areas. In four additional experiments 1 % carbocaine saturated gel-foam pledgets were applied to the left side of the exposed cortex with a resulting slight increase in amplitude and slowing of the brain waves on that

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side. Arousal reponses again occurred spontaneously, after auditory and pain stimuli and following the i.v. injection of 0.1 mg/kg eserine. The eserine-induced arousal responses ranged in duration from 16-30 minutes. These 12 experiments illustrate EEG arousal after the application of local anesthetics. DISCUSSION

The purpose of the present experiments was to apply various pharmacologically active drugs on the cerebral cortex and to compare their influences on the EEG arousal reaction. The results have revealed that drugs which inhibit structures innervated by post-ganglionic cholinergic nerves in the peripheral system also block the EEG arousal reaction. In this regard it is well to point out that Dale and Sherrington6, in discussing possibilities for mechanisms of central transmission, pointed to the phenomena of peripheral transmission as a heuristic guide. Many diverse methods have produced evidence in favor of a cortical cholinergic mechanism and our results are discussed in relation to these findings. The present experiments have revealed that topical application of all three cholinolytics used have been effective in blocking EEG arousal; but atropine sulfate was less so than either benactyzine or scopolamine. Here we may refer to Giarman and PepeuQ who found that atropine sulfate was also less effective than scopolamine in reducing the acetylcholine levels of cerebral hemispheres. It is also worthy of comment that such reductions in acetylcholine concentrations did not occur in the subcortical areas of the rostra1 mesencephalon nor of the caudal mesencephalon and myelencephalon. long^^^, studying th.: EEG blocking effects of intravenously administered scopolamine and atropine on the arousal reaction evoked by eserine, found similar correlations, scopolamine being 10 to 15 times as active as atropine. It is important to notice that every drug applied topically in our experiments caused spiking in higher concentrations but only cholinolytic drugs blocked the EEG arousal reaction. The topical application of acetylcholine usually produced a localized depression, often succeeded by enhanced electrical activity8 and in some instances this activity was organized into distinct groups of paroxysmal spikes. There is no doubt that eserine, causing the accumulation of acetylcholine, can evoke alerting but the intravenous or intracarotid administration of anticholinesterase can also produce EEG spikingll. The use of anticholinesterases is associated with accumulations of acetylcholine in the brainl. This is not to say that excessive EEG activity must have an exclusive basis only in large amounts of acetylcholine. Our observations with topical applications of strychnine, pentamethylenetetrazol and 1 % pontocaine revealed abnormal electroencephalographic activity evoked by mechanisms other than those involving acetylcholine. In fact, the EEG changes brought on by these three drugs became apparent only when their influence was prepotent over that of acetylcholine and replaced the EEG alerting pattern evoked by eserine (Fig. 4). These EEG results indicate that eserine induces arousal independently of the abnormalities associated with the topical application of strychnine, thus indicating that they are two unrelated phenomena. References p. 38-39

36

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al.

In evoking EEG arousal we used sensory modalities (audition or pain) and electrical stimulation of the midbrain reticular formation as well as pharmacologically active substances. It is significant that chemically induced EEG arousal is mediated by different classes of agents : (a) the anticholinesterase, physostigmine, (b) the directly reactive cholinergic drug, pilocarpine, (c) the adrenergic compound, d-amphetamine and (d) the precursor of serotonin, 5-HTP. The latter is known to provoke arousal associated with a large and rapid rise of serotonin in the midbrain and without any marked changes in norepinephrine levels4. In this connection it will be well to remember the conclusion of Rinaldi and Himwich31, who regarded atropine as a nniversal inhibitor of alerting reactions, when referring to the effects of atropine-induced blocking of EEG activation, whether the source of stimulation was peripheral or central, including the administration of cholinergic substancesl3J4. White and D a i g n e a ~ l thave ~ ~ carried this idea further and showed that atropine blocked the alerting produced by adrenergic agents. In the present experiments topical applications of benactyzine blocked EEG arousal induced by cholinergic and adrenergic agents as well as by 5-HTP, a precursor of serotonin. Though this communication is not directly concerned with the midbrain and medullary portion of the MDAS, we may point out that the reticular formation is not homogeneous pharrnacologically3~4onor in regard to possible neurotransmitters5~28. Irrespective of thc mechanisms involved in EEG arousal it can be blocked by the topical applications of cholinolytics. We must therefore conclude that this type of inhibition of arousal must act upon structures, whether axons, synapses or dendrites, with cholinergic function situated near the cortical surface. These cortical structures present sites where drugs with cholinolytic activities, including chlorpromazine administered intravenously38 prevent the EEG arousal reaction. Histochemical studies have revealed fibers containing acetylcholinesterase and travelling subcortically extending through layers V and IV, 0.8 to 1.3 mm below the cortical surface. KrnjeviE and Silver22 suggest that these fibers provide a cholinergic innervation of some deep pyramidal cells and that the cholinergic fibers represent the final corticopetal link in the MDAS. KrnjeviC and Phillis21 found that acetylcholine-sensitive cells are present in most regions of the cortex but occur in greatest concentration in the primary sensory areas, an observation agreeing with our finding that the topical effects of the cholinolytic drugs were most marked in the region of the coronal suture. Direct evidence for the presence of acetylcholine in cholinergic neurons has been demonstrated for the cerebral cortex and nutria of guinea pig42. Of the three cliolinolytic substances we applied topically, atropine and benactyzine are known to possess local anesthetic properties. Atropine has weak anesthetic properties, but benactyzine is more effective with double the activity of cocainelo. The potency of carbocaine is of the same order as cocaine but pontocaine is much stronger, in fact 15 times as effective as cocainel7. The fact that pontocaine failed to block EEG arousal suggests that the much less effective atropine and benactyzine do not block cortical arousal because of a local anesthetic property but because of a specific cholinolytic activity. Other observations can also be explained by the presence of such cholinergic

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structures just beneath the cortical surfaces. MacIntosh and Oboris25 as well as Mitchell26 have found that excitation of the cortex through sensory nerves or by direct electrical stimulation increased the acetylcholine output in the fluid collected on the surface of the cortex, thus indicating that a cholinergic agent had been liberated from nervous structures within the cerebral cortex. Beleslin et a1.2 in cats under amytal anesthesia, perfused the cerebral subarachnoid space with neostigmine and the acetylcholine appeared in the effluent. It is noteworthy that output of the acetylcholine was most pronounced on the cerebral cortex bounding the ventral parts of the fissures of Sylvius. In contrast the acetylcholine content of the perfusion fluid obtained from interpeduncular fossa and cisterna magna was minimal. KrnjeviC and Phillis19920 and Spehlmann37,using microiontophoretic methods, found that neurons concentrated in the deeper layers of the cortex can be excited with acetylcholine while on the other hand anti-muscarinic substances like atropine and scopolamine are more potent antagonists of acetylcholine than the antinicotinic substances. For this reason KrnjeviElB suggested that the cortex receives a cholinergic innervation with muscarinic properties. Riehl et ~ 1 . ~ 9in, order to obtain more information on cholinergic receptors in the central nervous system, injected muscarine and arecoline in the enckphale isole' preparations of cats and found that belladonna alkaloids blocked peripheral and central effects of muscarine almost equally while with arecoline only peripheral inhibition was obtained. Histochemical studies yield additional evidence for fibers containing acetylcholinesterase activity. Shute and Lewis35 found that such fibers project from subcortical centers to the cortex. KrnjeviC and Silver22 reported that fibers originating from the striatum invade the fetal neocortex and presumably give rise to the cortical horizontal connections which in the adult are found in layers V and IV and are under control of striated and septa1 cells. Further evidence in favor of tangential cortical fibers comes from Hebbl2 who reported that cortical acetylcholinesterase activity is decreased after cortical undercutting. These observations have been confirmed by Rosenberg and EchIin33 in the chronic partially isolated cerebral cortex of the monkey with pial circulation intact. SUMMARY

A group of 90 adult rabbits weighing between 2.5 to 3.0 kg were subjects of an EEG analysis on the effects of various drugs topically applied to cerebral cortex or administered systemically. The topical application of the cholinolytic drugs benactyzine, scopolamine and atropine blocked previously established EEG alerting evoked by eserine or pilocarpine administered intravenously. The cholinolytic drugs also evoked synchronization when alerting was induced by peripheral stimulation as well as after the electrical stimulation of the midbrain. In addition, the cholinolytic drugs were effective in blocking alerting induced by i.v. amphetamine and 5-HTP. Carbocaine as well as pontocaine, and the latter is a far more potent anesthetic than either atropine or benactyzine, failed to interfere with arousal patterns. It is therefore suggested that the ability of the cholinolytic drugs we used to interfere with the EEG alerting References p . 38-39

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et al.

reaction is a specific effect and not due to local anesthesia. Thus our experiments bring additional evidence for the concept of structures cholinergic in function and situated close to the cortical surface being the final corticopetal link in EEC alerting.

R E F E R E NCES 1 APRISON,M. H. AND NATHAN,P. (1956) Brain acetylcholine and behavior. Fed. Proc., 15, 5. 2 BELESLIN, D., POLAK,R. L. AND SPROULL, D. H. (1965) The release of acetylcholine into the cerebral subarachnoid space of anesthetized cats. J. Physiol., 177,420-428. 3 BRADLEY, P. B. (1957) Microelectrode approach to the neuropharmacology of the reticular formation. Psychotropic Drugs, S. Garattini and V. Ghetti, Eds., Elsevier, Amsterdam, pp. 207-216. 4 COSTA, E., PSCHEIDT, G. R., VANMETER,W. G. AND HIMWICH, H. E. (1960) Brain concentrations of biogenic amines and EEG patterns of rabbits. J. Pharmacol. Exper. Therap., 130,81-88. 5 DAHLSTROM, A. AND FUXE,K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. Acta physiol. s c a d , 62, Suppl. 232, 1-52. 6 DALE,SIR HENRY(1934) Chemical transmission of the effects of nerve impulses. Brit. Med. J., 1, 835-841. 7 DEROBERTIS, E. (1964) Electron microscope and chemical study of binding sites of brain biogenic amines. Biogenic Amines, Vol. 8, Progress in Brain Research, H. E. Himwich and W. A. Himwich, Eds., Elsevier, Amsterdam, pp. 118-136. 8 ESSIG,C. F., ADKINS, F. J. AND BARNARD, G. L. (1953) Observations on electrocorticographic effects of acetylcholine in monkeys and cats. Proc. SOC.Exper. Biol. Med., 82, 551-553. 9 GIARMAN, N. J. AND PEPEU,G. (1964) The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Brit. J. Pharmacol. Chemotherap., 23, 123-1 30. 10 GOODMAN, L. AND GILMAN,A. (1965) Pharmacological Basis of Therapeutics, Third edition, Macmillan Company, New York. 11 HAMPSON, J. L., ESSIG,c. F., MCAULEY, A. AND HIMWICH, H. E. (1950) Effects of di-isopropyl fluorophosphate (DFP) on electroencephalogram and cholinesterase activity. EEG Clin. Neurophysiol., 2, 41. 12 HEBEI,C. O., KRNJEVIC,K. AND SILVER,A. (1963) Effect of undercutting on the acetylcholinesterase and choline acetyltransferase activity in the cat’s cerebral cortex. Nature, 198, 692. 13 HIMWICH, H. E. AND RINALDI,F. (1957a) Analysis of the action of benztropine methanesulfonate against parkinsonism. Tranquilizing Drugs, H. E. Himwich, Ed., American Association for Advancement of Science, pp. 41-57. 14 HIMWICH, H. E. AND RINALDI, F. (1957b) The antiparkinson activity of benactvzine. Arch. Intern. Pharmacodynamie, 110, 119-127. 15 JASPER,H. H. (1949) Diffuse projection systems: the integrative action of the thalamic reticular system. EEG Clin. Neurophysiol., 1, 405-420. 17 JORDAN, E. P. (1958) Modern Drug Encyclopedia and Therapeutic Index, Drug Publications, Inc., New York. 18 KRNJEVIC, K. (1965) Actions of drugs on single neurones in cerebral cortex. Brit. Men. Bull., 21, 10-14. 19 K R N JEVIK. ~ , AND PHILLIS,J. W. (1963a) Iontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol., 165, 274. 20 KRNJEVIC, K. A N D PHILLIS, J. W. (1963b) Actions of certain amines on cerebral cortical neurones. Brit. J. Pharmacol., 20, 471-490. 21 KRNJEVIC, K. AND PHILLIS, J. W. (1963~)Acetylcholine-sensitive cells in the cerebral cortex. J. Physiol., 166, 296-327. 22 KRNJEVIC, K. AND SILVER, A. (1963) The distribution of ‘cholinergic’ fibres in the cerebral cortex. J. Physiol., 168, 39P. 23 KRNJEVIC, K. AND SILVER, A. (1965) A histochemical study of cholinergic fibres in the cerebral cortex, J. Anat., 99, 711-759. 24 LONGO,V. G . (1956) Effects of scopolamine and atropine on electroencephalographic and behavioral reqctioos due to hypothalamic stimulation, J, Pharmacol. Exper. Therap., 116, 198.

EEG

AROUSAL A N D CORTICAL CHOLINERGIC LINK

39

25 MACINTOSH, F. C. AND OBORIS, P. E. (1953) In: XIXInternationalPhysiologicalCongress, Montreal, August 31-September 4 ; Abstracts of Communications, p. 580. 26 MITCHELL, J. P. (1963) The spontaneous and evoked release of acetylcholine from the cerebral cortex. J . Physiol., 165, 98. 27 MORUZZI, G. AND MAGOUN,H. W. (1949) Brain stem reticular formation and activation of the EEG. EEG Clin. Neurophysiol., 1, 455473. 28 PAVLIN, R. (1965) Cholinesterases in reticular nerve cells. J. Neurochem., 12, 515-518. 29 RIEHL,J. L., PAUL-DAVID, J. AND UNNA,K. R. (1962) Comparison of the effects of arecoline and muscarine on the central nervous system. Int. J . Neuropharmacol., 1, 393-401. 30 RINALDI, F. (1965) Direct action of atropine on the cerebral cortex of the rabbit. Horizons in Neuropsychopharmacology, Vol. 16, Progress in Brain Research, W. A. Hirnwich and J. P. Schadk, Eds., Elsevier, New York-Amsterdam, pp. 229-244. 31 RINALDI,F. AND HIMWICH, H. E. (1955a) Alerting responses and actions of atropine and cholinergic drugs. Arch. Neurol. Psychiat., 73, 387. 32 RINALDI,F. AND HIMWICH, H. E. (195513) Cholinergic mechanism involved in function of mesodiencephalic activating system. Arch. Neurol. Psychiat., 73, 396. 33 ROSENBERG, P. AND ECHLIN,F. (1965) Cholinesterase activity of chronic partially isolated cortex. Neurology, 15, 700-707. C. H., EVERETT, J. W. AND GREEN, J. D. (1954) The rabbit diencephalon in stereotaxic 33a SAWYER, coordinates. J . Comp. Neurol., 144, 253-259. 34 SCHWEIGERDT, A. K. AND HIMWICH, H. K. (1964) An electroencephalographic study of bufotenin and 5-hydroxytryptophan. J. Pharmacol. Exper. Therap., 114, 253-259. 35 SHUTE,C. C. D. AND LEWIS,P. R. (1963) Cholinesterase-containing systems of the brains of the rat. Nature, 199, 1160-1164. 36 SMIRNOV, G . D. AND ILYUTCHENOK, R. V. (1962) Cholinergic mechanism of cortical activation. Sechenov Physiol. J. USSR, 49, 127-139. 37 SPEHLMANN, R. (1963) Acetylcholine and prostigmine electrophoresis at visual cortex neurons. J. Neurophysiol., 26, 127-139. 38 STEINER, W. G. AND HIMWICH, H. E. (1962) Central cholinolytic action of chlorpromazine. Science, 136, 783-875. 39 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effects of adrenergic drugs. J. Pharmacol. Exper. Therap., 125, 339. 40 WHITE,R. P., SEWELL, H. H., JR. AND RUDOLPH, A. S. (1965) Drug-induced dissociation between evoked reticular potentials and the EEG. EEG Clin. Neurophysiol., 19, 16-24. 41 WHITTAKER, V. P. (1964) Investigations on the storage sites of biogenic atnines in the central nervous system. Biogenic Amines, Vol. 8, Progress in Brain Research, H. E. Himwich and W. A. Himwich, Eds., Elsevier, New York-Amsterdam, pp. 9CL117. 42 WHITTAKER, V. P. AND SHERIDAN, M. N. (1965) The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles. J. Neurochem., 12, 363-372.

40

Influence of Atropine, Scopolamine and Benactyzine on the Physostigmine Aroussl Reaction in Rabbits Z . V O T A V A , 0. B E N E S O V A , Z . B O H D A N E C K Y *

AND

0. G R O F O V A * *

Department of Pharmacology, Medical Faculty of Hygiene, Charles University, Prague (Czechoslovakia)

Acetylcholine is one of the most important mediators of nervous transmission not only in the peripheral, but also in the central nervous system. Research on the catecholamines and serotonin has progressed more quickly than that on acetylcholine because of technical difficulties in the determination of this unstable substance. Recently, intensive research on the effect and mode of action of drugs influencing central cholinergic structures has been undertaken using electrophysiological, behavioural and biochemical techniques. As many authors have stated, both cholinomimetic and cholinolytic drugs change the EEG traces in cortical and subcortical structures, the former provoking the arousal pattern, the latter the resting pattern. These results have been recently reviewed by Long0899 and Votaval5. Wiklerlg, using dogs was the first to notice the “dissociation” between the EEG record and the behaviour of animals after the administration of atropine. The EEG patterns resembled those of sleep, whereas the animals were awake or even excited. These results have been later confirmed by many authors. In more detailed studies, it was found that memory was impaired when cholinolytic drugs provoked “sleep” patterns in EEG13J4. The EEG changes as well as behavioural impairment could be restored by administration of physostigmine10111. In previous paperslJ6, we showed that the EEG arousal reaction provoked by administration of physostigmine in rabbits with chronically implanted electrodes could be used as a tool for the evaluation of the central effects of anticholinergic drugs. In the present paper the effects of atropine, scopolamine and benactyzine are compared. METHODS

The techniques employed in these experiments follow closely those previously describedlJ6. Twelve male Chinchilla rabbits each weighing approximately three kilograms were employed throughout the study. The animals were implanted with four cortical and two subcortical electrodes under local tetrocaine anesthesia.

* **

Research Institute of Pharmacy and Biochemistry, Prague. Pepartment of Anatomy, Faculty of Medicine, Prague,

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

4I

Nickelplated screws of 3 mm diameter were used as cortical electrodes; two were implanted into the skull in the frontal area (one on the right, one on the left hemisphere) and two similarly in the occipital area. The subcortical electrodes were bipolar (tip separation 1.5 mm), made of enamelled constantan wire, 0.17 mm diameter, fixed in a miniature electric plug and implanted into the hippocampus and thalamus. All electrodes were attached to the skull by dental phosphate cement. The positions of the subcortical electrodes were determined according to the stereotactic atlas of rabbit brain by Fifkovh and Margalas. During the experiments, the electrodes were connected to an 8-channel VEB Dresden electroencephalograph by means of miniature plugs and fine cables. The fixation of the non-anesthetized rabbit during the experiment in the special metal cage is shown in Fig. 1. Details of the implanted electrodes and the plugs for the EEG records are given in Fig. 2.

Fig. 1. Fixation of the rabbit in a special metal cage during experiment.

After completing all experiments (in about three months), the rabbits were killed under thiopental anesthesia, the brains were perfused with 10 % formol, removed and fixed in 10% formol. After two months, the brains were embedded in celloidin, sectioned at 40 micromillimeters and stained with cresyl violet according to Nissl. The location of the electrodes of our group of twelve rabbits in two anterior-posterior levels, i.e. in the level AP 0.5 and AP 4.0 is shown in Fig. 3. The correct localization within the structures of hippocampus (pyramidal layers, fascia dentata) was found in peferences p . 46-47

42

z. V O T A V A et 01.

Fig. 2. Detailed view of the head with chronically implanted electrodes and miniature plugs to be connected to EEG device.

ten out of the twelve rabbits. In two cases the electrodes were placed in the white matter upon the hippocampus. The localization of the electrodes in the thalamus was correct in all twelve rabbits; their tips being located in the anterior nuclei (in AP 0.5 level) or in the posterior nuclei of thalamus (in AP 4.0 level). At the beginning of the experiments, the rabbits were placed in the cage for about one hour to habituate them to the procedure. The tests were started about two weeks after implantation of electrodes and performed at weekly intervals. All drugs were administered into the auricular vein over a two minute period. Prior to this normal (isotonic) saline solution was injected to determine the effect of injection procedure. After an interval of one week, the effect of physostigmine at the dose level of 0.1 mg/kg was tested. This dose of physostigmine evoked a cortical desynchronization and subcortical synchronization (regular sinusoidal waves of 4-7 c/sec, theta activity) for a period of 20 to 40 minutes. No autonomic and/or behavioural effects were observed after this dose of physostigmine. During the following weeks, the drugs to be tested were administered intravenously; (atropine 0.5 and 1.0 mg/kg, scopolamine 0.05,O.l and 0.5 mg/kg, benactyzine 0.1 and 0.5 mg/kg) with again physostigmine at the same dose, i.e. 0.1 mg/kg after a 20 minute interval. The previous administration of anticholinergic drugs curtailed the duration of the EEG arousal reaction, as demonstrated in Figure 4. For the quantitative evaluation of the duration of the EEG arousal reaction, the hippocampal and/or thalamic theta activity was used, as its presence is easily deter-

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

43

Fig. 3. Scheme of a typical location of the electrodes in two anterior-posterior levels according to the stereotactic atlas of Fifiovk and Margala (1960). The upper part of the figure represents the cut in the level AP 0.5, the lower part in the level AP 4. AD = nc. anterodorsalis thalami; AM = nc. anterornedialis thalami; AV = nc. anteroventralis thalami; GL = corpus geniculatum laterale; GLV = corpus geniculatum laterale pars ventralis; LA = nc. lateralis anterior; LP = nc. lateralis posterior; M D = nc. rnediodorsalis thalami; PT = nc. parataenialis; R = nc. reticularis thalami; RE = nc. reuniens; SN = substantia nigra; TMT = tractus marnmillothalamicus; VA = nc. ventralis anterior; VL = nc. ventralis lateralis; VM = nc. ventralis medialis; VPL = nc. ventralis posterolateralis; VPM = nc. ventralis posteromedialis; ZI = zona incerta.

mined. Changes in the cortical EEG traces (fast frequency, low voltage waves) were used only for judging the general state of the rabbit. At the end of the experiment, the effect of physostigmine alone was established to determine whether the reaction of the rabbit was changed in the course of the experiment. To eliminate the influence of the sequence of drugs, the rate of the administration was fixed individually for each rabbit according to random analysis. The change in the duration of the theta activity after the pretreatment with test drugs was compared with the effect of physostigmine alone at the beginning and end of the experiment and expressed as the percentage of the duration. The results for the group of animals were statistically evaluated and the significance of differences was determined. References p . 46-47

44

Z. V O T A V A

et nt.

RESULTS

After a short latency time of about one to two minutes, the intravenous administration of physostigmine at the dose level of 0.1 mg/kg evoked an irregular fast-frequency low voltage activity in the cortical leads and regular slow frequency (five to six per second) high voltage (200 to 300 pV) activity in the hippocampus and thalamus, so-called theta activity. This reaction lasted between 20 to 40 minutes in individual rabbits, and was relatively stable for each rabbit (see Fig. 4 upper part). No behavioural reactions were observed, in accordance with the findings described by Bradley and Elkes2. Thus, physostigmine produced a “dissociation” between EEG and behav-

Fig. 4. EEG record of a rabbit as an example of the experimental procedure. The upper part: On the left is the control record, other records are taken after physostigmine administration in 2, 5, 15, 18 and 20 minute intervals. The lower part: On the left is the record after the administration of atropine. Physostigmine was given 20 minutes after atropine injection. Note the curtailing of the duration of theta activity, evoked in the hippocampus and thalamus by the injection of physostigmine, after pretreatment with atropine.

INFLUENCE OF ANTICHOLINERGIC DRUGS ON PHYSOSTIGMINE REACTION IN RABBITS

45

ioural arousal reaction, as described by Wiklerlg for atropine. Blood pressure and heart rate were not changed by this dose of physostigmine, as was stated earlier. The effects of atropine, scopolamine and benactyzine on the EEG traces were qualitatively similar and they differed only in the intensity of their actions. Very soon after the administration of anticholinergic drugs, a consistent shift to low voltage slow activity was noted and a diminution of the 8-10 clsec waves, which formed the alpha activity of the EEG. This pattern was very similar to that observed in the state of drowsiness, although the rabbits were awake. The intensity of this EEG change was independent of the dose administered. After a period of 20 minutes following physostigmine administration, the EEG arousal reaction was shortened, and started after a longer latency time. With higher doses, the EEG arousal reaction was completely eliminated. The patterns in the cortical leads were sometimes changed in shape, but the theta activity remained unchanged (Fig. 4, lower part). The quantitative comparison of the effect of atropine, scopolamine and benactyzine is shown in Figure 5. It is clear that all the drugs tested significantly reduced the EEG theta activity in the hippocampus and thalamus, evoked by administration of PHYSOSTIGMINE

0.1 mg kg1i.v. 10000-

SCOPOLAMINE PHYSOSTIGMINE BENACTYZINE

PHYSOSTlGMlNE

PHYSOSTIGMINE

6040-

'"1 0

0.5

1.0

ILJL

--0.1 0.5

0.05 0 3

0.5 rng/kg iv.

Fig. 5. Shortening of the duration of EEG theta activity evokcd in rabbits by i.v. administration of physostigmine (first shaded column, duration is taken as 100%) 20 minutes after pretreatment with atropine, benactyzine and scopolamine in doses indicated on the abscissa. The straight lines in the columns indicate the fiducial limits for probabilityp = 0.15.

physostigmine. Scopolamine produced the greatest effect, while atropine had the least effect. The quantitative differences among the drugs tested were statistically significant. If the effect of atropine is taken as 1 .O, then the effect of benactyzine is 2.0 and that of scopolamine 12.0. It should be stressed that it is valid only for one time interval, i.e. 20 minutes after intravenous administration. DISCUSSION

As has been stated by Bradley and Elkesz, physostigmine, in a suitable dose, evokes EEG arousal without changing the general behaviour of animal or provoking excitaReferences p. 46-47

46

Z.V Q T A V A et al.

tion. We have used this effect of physostigmine for the evaluation of drugs with central cholinomimetic or anticholinergic action. In the present paper we compared the effect of three anticholinergic drugs, atropine, scopolamine and benactyzine. In accordance with other authors2,7J7Ja we established that the effect of scopolamine exceeded that of atropine more than ten times. Also, benactyzine proved to be about twice as effective as atropine. These ratios of the effects of these three drugs is also in good agreement with the results obtained in behavioural experiments4~5~6~16 or with different central pharmacological effectslz. If the ratio for the central and peripheral anticholinergic effects of atropine, scopolamine and benactyzine is evaluated12 the specificity of the central effect of scopolamine and especially that of benactyzine is evident. SUMMARY

EEG recordings were made in a group of twelve uiianesthetized rabbits with electrodes implanted in cortex (frontal and occipital), hippocampus and thalamus. Physostigmine (0.1 mg/kg i.v.) evoked EEG arousal reaction lasting 20 to 40 minutes, without any change in general behaviour, blood pressure or heart rate. The effects of atropine (0.5 and 1.0 mg/kg), scopolamine (0.05, 0.1 and 0.5 mg/kg) and benactyzine (0.1 and 0.5 mg/kg) were compared against the EEG arousal evoked by physostigmine. The drugs were given i.v. 20 minutes before administration of physostigmine and the shortening of the duration of the EEG arousal was evaluated. The degree of central anticholinergic effect in the drugs studied in this investigation was: atropine 1.O, benactyzine 2.0, scopolamine 12.0.

REFERENCES 1 BENESOVA, O., BOHDANECK+, Z. AND GROFOVA, I. (1964) Electrophysiological anslyses of the neuroleptic and antidepressant actions of psychotropic drugs in rabbits. Int. J. Neuropharmacol., 3,479488. P. B. AND ELKES,J. (1953) The effect of atropine, hyoscyamine, physostigmine and 2 BRADLEY, neostigmine on the electrical activity of the brain of the conscious cat. J. Physiol. (Land.), 120, 148-149. 3 FIFKOVA, E. AND MARSALA, J. (1960) Stereotaxic atlases for the cat, rabbit andrat. In the book: BureS, Petraii and Zachar: Electrophysiological methods in biological research. Czechoslovak Academic Press, Praha, pp. 426467. 4 HERZ,A. (1959) Ueber die Wirkung von Scopolamin, Benactyzin und Atropin auf das reaktive Verhalten der Ratte. Arch. exp. Path. Pharmak., 236, 110-1 12. 5 HERZ,A. (1960) Die Bedeutung der Bahnung fur die Wirkung von Scopolamin und ahn1i:he Substanzen auf bedingte Reaktionen. 2. Biolog., 112, 104-1 12. 6 HOLTEN,C. H. AND SONNE, E. (1955) Action of a series of benactyzine-derivatives and other compounds on stress-induced behaviour in the rat. Actapharmacol. toxicol. ( K b h . ) , 11, 148-155. 7 LONGO, V. G. (1956) Effects of scopolamine and atropine on electroencephalographic and behavioural reactions due to hypothalamic stimulation. J. Pharmacol., 116, 198-208. 8 LONGO, V. G. (1965) Analyse de la dissociation entre les effets des medicaments anticholinergiques sur le comportement et sur I'activite electrique ckrebrale. AclualifPsPharmacologiques, 18,289-308. 9 LONGO,V. G. (1966) Mechanisms of the behaviour and electroencephalographic effects of atropine and related compounds. Pharmacological Reviews, 18, (in press).

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47

10 LONGO,V. G. AND SILVESTRINI, B. (1958) Contribution a l’etude des rapports entre le potentiel rtticulaire evoque, l’etat d’anesthesie et l’activitk electrique ctrebrale. Electroencephal. clin. Neurophysiol., 10, 111-120. 11 MIKHEL’SON, M. YA. (Ed.) (1957) The physiological action of acetylcholine and the search of new drugs. Leningrad, 1957. 12 PARKES, M. W. (1965)An examination of central action characteristic of scopolamine: Comparison of central and peripheral activity in scopolamine, atropine and some synthetic basic esters. Psychopharmacologia, 7, 1-19. 13 ROUGEL, A., VERDEAUX, J. AND GOGAN,P. (1965) Limits of the dissociation between EEG and behaviour. Int. J. Neuropharmacol., 4, 265-272. 14 SADOWSKI, B. AND LONGO,V. G. (1962) EEG and behaviour correlates of an instrumental reward conditioned response in rabbits. A physiological and pharmacological study. Electroenceph. clin. Neurophysiol., 14, 465-476. 15 VOTAVA, Z. (1966) Pharrnacologie des structures cholinergiques centrales. Actualit& Pharmacologiques, 1, (in press). Z., BENE~OVA, O., METYSOVA, J., AND SOUSKOVA, M. (1963) Drug-induced changes of 16 VOTAVA, higher nervous activity in experimental animals. In the book: Psychopharmacological Methods, Votava, Horvath and Vinai (Eds.), Pergamon Press, Oxford, pp. 31-40. 17 WHITE,R. P. AND BOYAJY,L. D. (1960) Neuropharmacological comparison of atropine, scopolamine, benactyzine, diphenhydramine and hydroxyzine. Arch. int. Pharmacodyn., 127, 260-273. 18 WHITE,R. P., NASH,C. B., WESTERBEKE, E. J. AND POSSANZA, G. J. (1961)Phylogenetic comparison of central actions produced by different doses of atropine and hyoscyamine. Arch. int. Pharmacodyn., 132, 349-363. 19 WIKLER, A. (1952) Pharrnacologic dissociation of behaviour and EEG sleep patterns in dogs: morphine, n-allylnormorphine and atropine. Proc. SOC.exp. Biol. ( N . Y . ) , 79,261-265

48

arain Acetylcholine and Habituation * P. L. CARLTON Rutgers, The State University, New Brunswick, New Jersey (U.S.A.)

Habituation is evidently a relatively primitive form of learning and refers to the fact that the effects of a stimulus disappear under certain conditionsll. Thisloss of stimuluseffectiveness follows two simple rules. First, relatively protracted exposure to the stimulus is required. Second, the stimulus must not itself be a biologically significant one nor can it be associated with a second stimulus having such significance. What do I mean by “biological significance”? A stimulus can have such significance in two different senses. It can be demonstrably significant in determining, first, the survival of the individual organism or in determining, second, the survival of the species. For that minority species that can learn instrumental behaviors, such biologically significant stimuli act as rewards. I will return to the relation of habituation and reward later in the paper. In thinking about habituation, an obvious question arises: what events in the brain control habituation? 1 have preferred to ask the question in a slightly more restricted form : what chemical events in the brain underlie the process? An alternative version of the same question asks about the areas of the brain involved. The “chemical” question about habituation can be re-phrased to read :The activity of what naturally occurring substance is required for habituation to take place? About three years ago2, I made the guess that the normal muscarinic activity of brain acetylcholine might be critical in the process. The experiments I want to describe now suggest that there may be something to that guess. The first problem in investigating the idea was to develop a scheme that would provide a behavioral index of habituation. To get such a measure, we utilized the fact that the ongoing behavior of a n animal is disrupted in a novel environment, but that this disruption wanes as habituation takes place; as the environment becomes less novel. The second problem was to find a means for altering the normal activity of brain acetylcholine. Well established pharmacological techniques are, of course, available ; the action of anticholinergics like scopolamine and atropine is to attenuate the muscarinic activity of acetylcholine (ACh). If ACh activity is, in fact, required for habituation, then habituation should be drastically attenuated when ACh activity is attenuated by the anticholinergics. The particular experimental set-up we have used involves two parts. These are

* Research supported by USPHS Grant MH 08585.

49

BRAIN ACETYLCHOLINE A N D HABITUATION PART 1 ___

Saline

PART 2

Scop.

DRINKING e

x

~

~

~

2days c i -+~ ANIMALS D E ~ PlR l ~ 1~ day~ +(allTEST animals In chamber) No druqs

--e

Exposed I n j e c t ion

Fig. 1. Summary of habituation procedure.

summarized in Fig. 1 . (This experiment and all others used Sprague-Dawley rats as subjects.) In the first part, some animals (Group D) were given 15 min exposure to the chamber 15 min after an injection of 0.5 mg/kg scopolamine hydrobromide (i.p.). Other animals (Group B) were not exposed to the chamber, but were removed from their home cages and given a drug injection. Two other groups were also involved. Both received a control injection of saline; one (Group C) was exposed to the chamber, the other (Group A) was not. The second part of the experiment began two days after this treatment. All animals were water-deprived. A day later (72 h after exposure), all animals were placed in the chamber to which only some had prior exposure. One change was made in this chamber: a water bottle was introduced. We recorded the time it took the thirsty animals to start drinking. This procedure has been described in detail elsewheres. Let me stress that no animal was injected before this test; injections were given only during the first part of the experiment. The results of this procedure are shown in Fig. 2. The animals that had not been exposed took a considerable amount of time to start drinking when tested in the absence of a drug. The values for these two groups are not reliably different. In contrast, prior habituation to the test chamber led, not surprisingly, to much faster initiation of drinking in animals given saline before prior exposure. But animals given the same opportunity to habituate at the time of reduced ACh activity failed to show the effect of this exposure. Rather, they behaved as if they had not been exposed at Time to drink in test (sec) I

1

Saline

Not exposed Scop.

Saline

Exposed

=

Scop. 1

Fig. 2. Mean times to make initial contact with a water bottle in a 3-min session. The results indicate that prior exposure decreases initial contact time but that scopolamine, only at the time of prior exposure, attenuates this effect. References p. S940

50

P . L. C A R L T O N

Exposed (Soline)

Unexposed

Exposed

(Stop,)

a 1

3 160

Fig. 3. Time to initiate drinking in a 3-min test session. Each bar denotes the result obtained from a single rat. Animals tailing to contact the drinking tube were assigned a criterion score of 3 min.

all; as if habituation had not taken place. Fig. 3 is a plot of the data for individual animals (each bar denotes the value obtained from a single rat). The two unexposed groups did not differ; their data are in the center of the figure. The data from exposed animals are at the left. The vertical, dashed line indicates the maximumtime todrink in this group. There is virtually 110 overlap between unexposed and previously habituated animals. The differential effect of prior habituation is a substantial one. The effect of scopolamine is of an equally large order of magnitude. Animals given a prior opportunity to habituate following scopolamine behaved much as if they had not had that opportunity. There is virtually no overlap of their data with those from exposed (saline) animals. (There is one exception, as there is i n the unexposed group). Furthermore, these data are well within the distribution of values obtained from unexposed animals. This effect suggests an involvement of acetylcholine in habituation; if normal ACh activity is blocked, so is habituation. Before this suggestion can be taken seriously, however, there are several questions that need to be answered. First, are we talking about a result of attenuated ACh activity, or are we talking about some idiosyncratic and unknown effect of one anticholinergic, scopolamine? A direct way to answer the question is to evaluate the effects of atropine. If both drugs produce comparable results, we are almost certainly dealing with a muscarinic action of ACh. In the study described below, we used a dose of atropine (10 mg/kg, i.p.) that, on the basis of a wide variety of studies, would be expected to produce about the same effect as the dose of scopolamine used in the experiment I just described. The potency ratio of the central effects of atropine to scopolamine is about 15 or 20 to 1. This brings up a second question. If attenuated habituation is indeed related to ACh activity, is it related to brain ACh? ACh acts as a transmitter in both the peripheral and the central nervous systems. But scopolamine has profound peripheral as well as central effects. Are the effects I have described central? A direct answer to the question i s provided by comparing the action of scopolamine with that of methyl scopolamine. Methyl scopolamine and methyl atropine are at least equipotent with their parent compounds in terms of peripheral action. But the methyl compounds pass the blood-brain barrier very poorly. Thus, equimolar doses of scopolamine and

BRAIN ACETYLCHOLINE AND HABITUATION

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methyl scopolamine will produce comparable peripheral effects, but vastly different central effects. Therefore, any effect of scopolamine that is not produced by an equimolar dose of methyl scopolamine can most reasonably be attributed to an action in the central, not the peripheral, nervous system. This approach has been discussed and documented elsewhere2. A third question has to do with the specificity of the habituation deficit produced by scopolamine. If we are truly dealing with an effect involving ACh, the phenomenon should be produced only by drugs that can attenuate the action of ACh. On the other hand, is the effect a non-specific one? Will any centrally active drug produce the same deficit in habituation? As a start toward answering this question, I selected two common drugs that are known to have profound behavioral effects. These are the stimulant, amphetamine sulphate ( I .O mg/kg, i.p.) and the depressant, sodium pentobarbital (8.0 mg/kg, i.p.). These doses were used in the study described below and were selected because of their clearly established effects on the learned behavior of rats. These doses are not sub-threshold; they are extremely active in altering certain classes of behavior2.4. In the experiment I want to describe now, we used the same technique as that in the first experiment. We studied the effects of scopolamine, methyl scopolamine, atropine, amphetamine, pentobarbital and a control saline injection : six different kinds of injections in all. In all cases, the drug was given to one squad before exposure and to a second squad that was not exposed. Thus, there were twelve groups of animals, six given an injection before exposure and six given the same injection but no exposure. Again, injections were given only before exposure; no injections were given before the subsequent test. The results of this test are summarized in Fig. 4. The data from Time to drinkin test (sec)

Saline

scop. ALi-op

M-Scop. P.Barb.

Arnph.

=

I

I I I

I I I

Not exposed I I I

I I

Fig. 4. Mean times to drink in a 3-min test session. Relative to unexposed animals, exposure following saline results in shorter times, scopolamine and atropine reverse the normal effect of prior exposure, whereas methyl scopolamine, pentobarbital and d-amphetamine are relatively inactive.

the various unexposed groups did not reliably differ; these data were therefore pooled. The overall effect of lack of exposure and consequent habituation is indicated by the vertical, dashed line. The various bars indicate the average amounts of time to drink References p . 59-60

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52

taken by the animals that were exposed following an injection. The exposed animals given saline were faster than unexposed rats; scopolamine again washed out this habituation effect. Atropine had an effect comparable to that of scopolamine. The lack of habituation seems to be related to an attenuation of ACh activity. A dose of methyl scopolamine equimolar to that of scopolamine had no effect on habituation. The phenomenon can therefore be most reasonably supposed to reflect an action on brain ACh. Neither amphetamine nor pentobarbital produced a shift in the effect of prior habituation despite the fact that the doses used have profound effects in other behavioral situations. Although dose-response data are certainly required, these results at least suggest a specificity of action peculiar to the centrally active anticholinergics. This lack of effect of amphetamine and pentobarbital, at these doses, does not mean that a deficit might not be obtained at substantially higher doses. A high, hypnotic dose of pentobarbital would, of course, produce a total deficit. Also, it seems likely that higher doses of either pentobarbital or amphetamine would produce a deficit because of dissociationg. The phenomenon of dissociation is discussed more fully below. The point to be made on the basis of the data in Fig. 4 is that other centrally active drugs, at dose levels that do have some behavioral effects, are evidently inactive in this situation. This brings up another question: Are we talking about a deficit in memory‘!’ Is a particular process, called habituation, reduced in exposed animals given anticholinergics - or, do such animals simply not “remember” that they have been exposed? If the phenomenon is truly a more general memory deficit, it should be possible to demonstrate that deficit in a situation in which habituation does not play a role; that is, one involving biologically significant stimuli. I n one experiment of this type, we first trained thirsty rats to drink from a water bottle in a response chamber. On the next day, they were returned to the chamber, but the water bottle had been removed. Four groups, two given saline and two given 0.6 mg/kg scopolamine (i.p.), were involved. Two of these groups (one saline and one scopolamine group) were presented four moderate intensity, 10-sec tones. The inter-

5001

Mean time to criterion licks Pre.tone (100 licks) Tone (10 licks) I I

DOSE

1

Fig. 5 . Effects of prior conditioning following saline or scopolamine injections. Times to complete 100 licks are plotted at the left; time to complete 10 licks in the presence of the tone are at the right. Relative to unshocked controls, animals that had had the tone paired with pain-shock show reliable suppression of drinking in both periods; drug at the time of conditioning had no effect.

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53

tone interval was one minute. The other two groups were given the same treatment except that, coincident with the termination of each tone, they also received a brief, inescapable painful shock applied through the grid floor of the chamber. All injections were given 20 min before conditioning. Two days later, all animals were returned to the chamber; the water bottle had been replaced, N o animal was injected. We recorded the time it took the animals to take 100 licks of water. With the occurrence of the 100th lick, the tone came on. We recorded the amount of time it took each rat to take 10 more licks. The results of this test are summarized in Fig. 5. Animals that had been shocked showed a generalized fear of the chamber, as indicated by the relative suppression of drinking in the pre-tone period. Scopolamine treatment before the prior fear conditioning had no effect. These animals evidently remembered that the chamber was a “dangerous” place just as well as the controls. The same kind of effect was found in the tone period (at the right of the figure). Fear, based on prior pairings of tone and shock, led to substantial suppression; again there was no differential effect due to drug treatment at the time of conditioning. There was no evidence of a memory deficit due to scopolamine. It could be argued that scopolamine had no effect because the event to be remembered was such a “vivid” one. That is, the degree of conditioning was so great that no other variable could reasonably be expected to affect it. This possibility seems unlikely for two reasons. Firstly, reliable evidence of conditioning was obtained in the pre-tone period. But scopolamine, at the time of conditioning, had no effect on this relatively low-level fear. That generalized fear of the chamber was indeed less than that elicited by the tone is indicated by the fact that, in the pre-tone period, the animals took less time to make 100 licks than they took to make only 10 in the presence of the tone. Tf scopolamine had produced a deficit in memory, it seems likely that some indication of it would have appeared in the pre-tone data. There was not, however, even a hint of such an effect. Secondly, we have tried to obtain some evidence of a memory deficit due to scopolamine in several different conditioning experiments. No such evidence was obtained in any of them. We have also evaluated the effects of atropine using the same technique as that used in the experiment summarized in Fig. 5. In the atropine experiment, we also examined the possibility that a memory deficit might appear against a relatively weak base-line of fear conditioning. To do this, we varied the number of conditioning trials. One group received 4-tone presentations, but no shock; a second received a single tone-shock pairing and a third received 4 pairings. These animals were given a saline injection 20 min prior to conditioning. A second set of three groups was given the same treatment except that atropine (8.0 mg/kg, i.p.) was injected. In the pre-tone period, the effect of tone-shock pairings was a re1iable:one. Prior fear conditioning did produce suppression; this suppression increased as a function of the number of pairings, but no effect of the drug was obtained. Comparable effects were obtained in the tone period, as shown in Fig. 6. Again, a reliable increase i n suppression was obtained with increased numbers of prior conditioning trials; atropineat the time ofconditioning had no effect. If anything, atropine produced slightly improved memory. Furthermore, the relatively lower level Refeiences p.;59-60

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P. L. C A R L T O N

Training trials

Fig. 6 . Effects of prior conditioning following saline or atropine. The number of shock presentations was varied between groups. No effect of drug was obtained.

of fear obtained with a single pairing was unaltered by drug treatment. This extends the results obtained in the pre-tone period. Although anticholinergics may produce amnesia i n mans, they do not appear to do so in the rat; at least not at the dose levels we have used. This uniform lack of effect suggests that the results we have interpreted as reflecting a deficit in habituation cannot reasonably be attributed to a more general deficit i n memory. Although the lack of effect of prior exposure appears to be due to attenuated activity of brain ACh, but not due to a deficit in general memory, there is still another question as to the validity of interpreting the deficit in terms of habituation. The effect could be due to dissociation. The term dissociation refers to the fact that animals can discriminate a drug-state from a no-drug state; the presence or absence of a drug's effect can have a discriminative control of behaviorg. Thus, the effects I have been describing could be due, not to a deficit in habituation, but toa stimulusdi fference between prior exposure (with drug) and subsequent test (without drug). An analogous situation would be to expose animals witha buzzer sounding and to test them without the buzzer. Longer times to initiate drinking would be expected in tests simply because of the difference in stimuli between exposure and test sessions. Interpretation of the data I have described in terms of dissociation, or stimulus change, seems unlikely for several reasons. First, centrally active doses of amphetamine or pentobarbital might be expected to produce effects like those produced by scopolamine if stimulus change were the only factor involved. But they do not. There is, of course, the unlikely possibility that the change due to scopolamine (or atropine) vs. no scopolamine (or no atropine) is large, whereas the change due to amphetamine (or pentobarbital) vs. no amphetamine (or no pentobarbital) is negligible. A second consideration bearing on the stimulus change interpretation is that no evidence for such a factor was obtained in the conditioning experiments summarized in Figs. 5 and 6. To the extent that stimulus change is a potent variable, there should have been

BRAIN ACETYLCHOLINE A N D HABITUATION

55

less evidence of conditioning in a subsequent no-drug test when prior tone-shock pairings were given following drug injection. No evidence of such an effect was obtained. Still a third result weighs against the stimulus change interpretation. Overton (personal communication) has found that dissociation can, in his experimental set-up, be obtained with atropine, but the lowest dose that produced reliable dissociation was about 20 times greater than that used in our experiments. Thus, a direct test of dissociation due to atropine suggests that the phenomenon I have been discussing is a league apart from that leading to dissociation. Another direct test of dissociation has been carried out by Meyerss. Meyers used low doses of scopolamine roughly comparable to those we have studied. No evidence of dissociation, at these doserevels, was found. We have independently replicated the essential features of Meyers’ experiment. All of these findings indicate that the deficit I have been describing cannot leasonably be attributed to dissociation. Although both memory deficit and dissociation do not seem to be likely candidates for explaining the basic phenomenon, still another factor might be involved. Suppose scopolamine blocked the “extinction of fear” during the initial exposure; in the later test, animals that had had scopolamine might be slow to drink because they were more fearful. That fear does suppress drinking is amply demonstrated by the experiments summarized in Figs. 5 and 6. One qualification of this point is in order. Extinction typically refers to a shift from reward to non-reward: e.g., an animal is first trained to get reward by emitting some response; reward is then discontinued. In the experiments I have described, an animal may indeed fear the novel environment, but he does so, not because of his previous training, but because of his presumably innate reaction (in the rat, at least) to “strangeness”. Thus, if we are, in fact, talking about extinction, we are talking about the waning of unconditioned rather than conditioned responses. Viewed in this way, there seems to be a rather thin line between habituation and “extinction of fear”. In both cases, an animal comes to a new situation with a set of responses to stimuli. As a consequence of exposure to these stimuli, the initial responses disappear. A distinction can, however, be made. A rat may do one of two antagonistic things in a novel environment; he may suppress behavior (because of fear), or he may move about the environment and thereby explore it. As a consequence of this exploration, the animal finds that some stimuli are neither biologically significant themselves nor correlated with others having such significance. These stimuli thus lose their initial control of behavior; the animal no longer explores them. It is in this sense, rather than one having to do with suppression due to fear, that I have used the term habituation. An animal that has already habituated to an environment starts drinking promptly because he does not explore the environment to the extent that the unhabituated animal does. But is this restriction of usage justified? My best guess at the moment is that it is. First, observation of the animals during their initial exposure to the chamber reveals little, if any, suppression of behavior. In fact, we have consistently used an unemotional strain of rats and, in addition, typically gave them a preliminary period of handling and “gentling” before beginning the experiments themselves. If our animals are fearful during initial exposure, they give very little References p.k59-60

56

P. L. CARLTON

evidence of it. What they do do is explore the chamber; animals given scopolamine are, if anything, hyperactive, hyperexploratory. In addition, a direct evaluation of the effects of scopolamine on extinction of fear indicates that the rate of extinction under drug is the same as that in normal animals. In this study, animals were first given tone-shock pairings like those used in the experiments summarized in Figs. 5 and 6. They were then given a single test trial in the drinking situation. All animals showed high levels of suppression in the tone. No drugs had been given up to this point. The animals were then divided into two groups. Both groups were given several extinction sessions in which the drinking tube was not available and in which the tone, but not shock, was presented; one group received scopolamine before each extinction session, the other received saline. All animals were then given a test in which a single tone was presented, the animals were drinking, no shock was delivered and no injections were given. The extent of suppression of drinking was, as usual, taken as an index of conditioned fear. Cycles of multiple extinction sessions (following injection) plus single test (without injection) were continued until there was no suppression of drinking in the tests. No difference due to scopolamine was obtained. Three points should be made about this study. First, the experiment is a very preliminary one based on few animals; a dubious basis for accepting a negative result. Second, the test sessions without injection were also extinction sessions. It could, therefore, be that this extinction, common to both groups, attenuated any difference that might have been produced by scopolamine. Detailed extension and replication of the study are required on both counts. There is a final qualification. This experiment involved conditioned fear, whereas the fear, if any, that could operate in our earlier studies is of the unconditioned variety. Thus, if one is to accept the lack of effect of scopolamine on extinction of fear as indicating that fear is not an important factor in the earlier work, it must be assumed that rules about one kind of fear will apply to the other. The best that one can say at this point is that interpretation of the effect of scopolamine in terms of habituation (as I am using the term) is at least not contradicted by the lack of effect of scopolamine on the extinction of conditioned fear. Let me change the subject somewhat. I began this discussion by describing a largeorder effect of scopolamine. One interpretation of this effect is that the normal activity of brain ACh is required for habituation; if ACh activity is blocked, so is habituation. But is this interpretation reasonable? This question was answered by asking a series of other questions: Is the effect actually peripheral rather then central? Can the effect be obtained with any centrally active drug? Can the effect be interpreted as being due, not to habituation, but to memory deficit or dissociation or fear? The negative outcome of attempts to answer these questions all supports the original interpretation. But there is a serious gap in such a conclusion. If attenuated ACh activity in the brain retards habituation, accentuated AChactivity :(with a cholinesterase inhibitor like eserine) should accelerate it. The gap in the story I have been telling is that we have, as yet, no data bearing on the effects of accentuated ACh activity. Until such data are in hand, the presumed relation of ACh to habituation

BRAIN ACETYLCHOLINE AND HABITUATION

57

can be accepted only with considerable caution. There is, however, some evidence that indicates heightened ACh activity may increase habituation. This evidence is, unfortunately, rather circumstantial. It is based on effects seen in learning situations. What role might habituation play in learning? As others7 have pointed out, the role of habituation is not negligible. Various experiments support the idea that one aspect of learning a complex problem, a maze for example, is the minimization of the behavioral control exerted by stimuli that do not lead to reward. That is, minimization of the impact of stimuli that are not associated with another having biological significance. Put crudely, successful performance hinges, in part, on suppressing the tendency to explore novel aspects of the environment. The animals must habituate to certain aspects of the situation. An experiment by Whitehouse12 will illustrate the point. This study involved the learning of a discrimination problem. Some animals were given atropine before each learning session; others were given eserine and still others, saline. The results are shown in Fig. 7. I have plotted the numbers of correct responses made by drugged Total correct Rs ( % normal) 100 110

120

I

I

Atropine

b I

I

Replotted from

1

Whttehouse,1959

Fig. 7. Overall numbers of correct responses /relative to controls - 100%) in a series of learning trials. Some animals received atropine before each trial, others received eserine, controls received saline. Atropine tended to impair performance, eserine tended to enhance it.

animals as a percentage of those made by normal controls. The numbers of correct responses made in the course of the series of trials reflect the rate of learning. As the figure indicates, atropine retarded learning, whereas eserine accelerated it. This result makes sense if two assumptions are made. First, there is the very reasonable one that habituation is involved in the successful performance seen in complex learning situations. Second, one must grant what the previous experiments have suggested; that the normal activity of brain ACh is involved in habituation. Thus, reducing ACh activity with atropine should lead to reduced habituation (and poorer performance), whereas increasing ACh activity with eserine should lead to increased habituation (and better performance). Although these expectations are generally borne out, Whitehouse found that only the deficit due to atropine was a statistically reliable one. In an experiment preliminary to the one described here, he had, however, found a reliable improvement due to eserine. This divergence is not surprising if one considers some aspects of actions of a cholinesterase inhibitor like eserine. Cholinesterase inhibition can result in accentuated References p . 59-60

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ACh activity5. But higher levels of ACh can also lead to less neural activitys. Thus, the action of a drug like eserine will be a biphasic one; heightened cliolinergic activity followed, at higher levels of esterase inhibition, by lower levels of activity. This relationship of ACh and effective, aggregate neural activity is schematized in the upper left portion of Fig. 8.

Replotted from ADrison

XI201

-100 I

20 40 6060.80 *I00 ACHE. % normal

Fig. 8. Schematic representation of relations between ACh activity, as a percent of normil, and neural activity (upper left), cholinesterase activity and ACh activity (upper right), and the consequent relation between cholinesterase activity and neural activity (bottom). T h e figure at the upper right is based on data reported by Aprison'. The arrows indicate the relative values at which functionally heightened activity would be obtained.

What is the relation of cholinesterase inhibition and ACh activity? Aprison' has found that ACh activity in brain increased only after levels of enzyme inhibition fell to about 40-60"/, of normal. At lower levels, ACh activity in brain increased sharply. This relationship (based on Aprison's data) is shown at the upper right of Fig. 8. These two relationships generate the curve shown at the bottom of Fig. 8. Cholinesterase inhibition should have no effect until levels of inhibition reach 40-60% of normal. At that point, there should be an increase in functional ACh activity. With still greater enzyme inhibition, there should be an abrupt decline. The decline will be an abrupt one because of the very rapid rise in ACh activity at levels of inhibition below the 40-60% level. That is, heightened ACh activity should occur only within a very restricted range of cholinesterase inhibition. This will be the case because inhibition in excess of this range should rapidly lead to high levels of ACh and, consequently, a functionally lower level of aggregate cholinergic activity (see the schematic at the upper left of Fig. 8). Because dose-response curves differ between animals, it should be a simple matter to obtain only marginal effects due to cholinesterase inhibition; the performance of some animals may be unaffected, facilitated in some, and depressed i n others. Thus, the effect on a group of animals might be minimal. Furthermore, slight variations between experiments could have very substantial and different effects.

BRAIN ACETYLCHOLINE A N D HABITUATION

59

These relations suggest that in learning situations increased cholinesterase inhibition should facilitate performance in the 4 0 4 0 % range, and depress it at lower levels. RusselP has summarized the results of his own experiments i n just these terms : “. . . when behavior is affected it appears to piss through four phases as ChE activity is reduced.

From 60 to 100 per cent activity no significant eyects hzve bsm observed. Thzre is a suggestion in our data that between 40 and 60 per cent activity the bshsvior mpy show a phase of heightened efficiencv .... Further reduction is associated with a rapid loss in efficiency, which might for convenience be referred to as a phase of ‘bzhavioral toxicity’.”

The role of brain ACh in the control of habituation that I have suggested, coupled with the role of habituation in maintaining performance, is thus indirectly supported. Direct evaluation of the effects of cholinesterase inhibition are, nonetheless, required. One way of summarizing what 1 have been suggesting is to think of the organism as being on the inside of a large balloon filled with stimuli. One problem facing the organism is to handle this profusion of stimuli. Nervous systems seem to have evolved so that, rather than processing everything at once, only certain hunks of the stimulus population are selected, and therefore control the organism’s behavior. It appears that the balloon gets selectively deflated to manageable proportions. This filtering process is called habituation, and follows the rule that biologically significant stimuli do not get filtered. Such stimuli are called rewards in certain contexts and for certain species. Habituation may thus play a part in controlling the behavior seen in such situations. A number of experiments support the guess that directly measured habituation requires the normal activity of brain ACh. Furthermore, results obtained in learning situations appear to support this possibility. I should add that I d o not feel that these data are as yet overwhelmingly in favor of the suggestions I have made. The data are only suggestive. What they suggest is that one of the most important things a brain must do, functionally cancel those stimuli that are not to have an impact on the animal’s behavior, involves the action of brain ACh. This possibility seems to account for a reasonable amount of available data; how much more experimental mileage can be got out of it, remains to be seen. REFERENCES 1 APRISON, M. H. (1962) On a proposed theory for the mechanism of action of serotonin in brain. Recent Adv. Biol. Psychiatry, 4, 133-146.

2 CARLTON, P. L. (1963) Cholinergic mechanisms in the control of behavior by the brain. Psycho/. Rev., 70, 19-39. 3 CARLTON, P. L. AND VOGEL,J. R . (1965) Studies of the amnesic properties of scopolamine. Psychon. Science, 3, 261- 262. 4 GELLER, 1. AND SEIFTER, J. (1960) The effects of meprobamate, barbiturates, &hetamine and promazine on experimentally induced conflict in the rat. Psychopharmacol., 1, 482-492. 5 GOODMAN, L. S. AND GILMAN, A. (1960) The pharmacological basis of experimental therapeutics. New York: Macmillan. 6 MCLENNAN, H. (1963) Synaptic Transmission. Philadelphia: Saunders. 7 MEEHL, P. E. AND MACCORQUODALE, K. (1954) In: W. K. Estes et a/. (Eds.), Modern Learning Thcory. New York: Appleton-Century-Crofts. 8 MEYERS, B. (1965) Some effects of scopolamine on a passive avoidance response in rats. Psychopharmacol., 8, 111-1 19.

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9 OVERTON, D. A. (1964) State-dependent or “dissociated” learning produced with pentobarbital. J. Camp. Physiol. Psychol., 51, 3-12. 10 RUSSELL, R. W. (1958) Effects of “biochemical lesions” on behavior. Actu fsychohgica, 11, 28 1-294. I I THORPE, W. H. (1963) Lenrningadinsrinct inanitnals. Cambridge: Harvard Univ. Press. I2 WHITEHOUSE, J. M. (1959) The effects of physostigmine and atropine on discrimination lenrning in the rat. Unpuhlished doctoral dissertarion, University of Colorado.

61

The Effect of Physostigmine and Atropine on some Behavioral and Electrophysiological Functions in Rats J. B U R E S Instilute of Physiology,

Czechoslovak Academy of Sciences, Prague (Czechoslovakia)

The chemical aspects of brain organization become more and more important for the neurophysiological analysis of learning. Accumulating evidence about the diversity of central transmitters present in the same brain areas leads logically to the assumption that synaptic chemistry may link neurons to circuits subserving specific behavioral functions. In this case, blockade of a certain type of synaptic transmission may eliminate the corresponding behavior rather than induce a diffuse impairment. Although little is known so far about the exact nature of the central transmitter s u b ~ t a n c e s l 4cholinergic ~~~, transmission i n several brain regions has been established beyond any doubt. The distribution of acetylcholine as well as related enzymatic systems (choline-acetylase, acetylcholinesterase) has been thoroughly described. Drugs interfering with the activity of cholinergic synapses either by anticholinergic or excessive cholinomimetic action are readily available and the basic mechanisms of their effects are well understood. The dissociation of EEG and behavior4.26 raised an important question about the correlation of electrophysiological and behavioral events. These are obviously the reasons why so much attention is paid to the behavioral role of the cholinergic systems. The purpose of the present paper is to test some of the current hypotheses about their role. METHODS

All the experiments were performed in rats aged three months. Assuming that cholinergic transmission can be impaired by both acetylcholinesterase blocking agents and by anticholinergic substances, attention was concentrated on the use of physostigmine salicylate and atropine sulphate. Drugs were applied in concentrations the effects of which on the EEG of rats were thoroughly described in our earlier papers6p7. After intraperitoneal injections of physostigmine or atropine 10 and 15 min respectively were allowed for the full development of the drug effect. The testing period did not usually exceed 30 min.

Rcfermres p

,

71-72

62

J.

RURES

RESULTS

Recent rneniory

Interference with cholinergic transmission was repeatedly shown to impair the acquisition of new memory traces without adversely affecting retrieval of overtrained conditioned reactions6~7~12~19. Although the validity of this statement is limited to certain types of behaviour, similar findings lead to the assumption that cholinergic transmission may be involved in short-term storage of the incoming information. Two experiments were performed to verify this hypothesis. In the first the technique of Blodgett and McCutchanz was modified. An H-shaped apparatus was used (Fig. I )

I

I

The animal was alternately started from the points S1 or SZ while the entrance into the opposite alley was closed by the sliding wall W1 or WP respectively. Intermittent electric shocks were applied with a 5 sec delay until the animal escaped into one of the two goals. Under control conditions most animals displayed a clear-cut tendency to alternate the goals when the starting points were alternated. Usually (80 %) the rats preferred Gz when started from S1 and GI when started from SZ (Fig. 2). The habit to alternate left and right turns is well known from maze studies and is usually attributed to factors like “forward-going tendency” and “centrifugal swing”. It involves some kind of short-term storage of kinesthetic, somesthetic and visual signals influencing the behavior of the animal at the choice point. In physostigmine-injected animals, the alternation was significantly impaired (Fig. 2). The choice did not become random but the rat systematically preferred one of the goals irrespective of the start from which it was released. This is illustrated i n Fig. 2b comparing the preference for one of the goals in the ten drug influenced trials. There was no difference in the effect of 0.5 and 0.25 mg/kg physostigmine. Different results were obtained with 6 mg/kg and 15 mgikg atropine which did not significantly impair the goal alternation in spite of dosages eliciting clear-cut EEG synchronization. 111the second experiment, a left-right alternation was elaborated in a group of

B E H A V I O R A L S I G N I F I C A N C E O F C H O L I N E R G I C SYSTEMS

%

63

I

v

0.25-05

C

Ph

,10 9 0 , 0 1 2

6-15

C

At

7

6 3

4

5 5

Fig. 2. The elTect of 0.25-0.5 mg/kg physostigmine (Ph) or 6-1 5 mg/kg atropine (At) on spontaneous alternation. C control conditions. Above : percentage of alternations. Below : percentage of animals displaying various degrees of preference (abscissa) for one of the goals.

10 hooded rats. The animal was placed on the start of a simple T-maze and required to avoid or escape electric shocks by reaching one of the two goals GI and G2. Both doors were opened during the first run. The rat was allowed to stay in the goal compartment for 10 sec and then was returned to the start again. The opposite goal was then accessible. The goals were regularly alternated. A correction technique was used throughout. Entering the alley leading to the incorrect door was considered as an error. Only a sequence of two correct choices was classified as an alternation reaction. The rats mastered the alternation task to a criterion of 9 alternations in 12 consecutive trials during 32 trials of the first day. After daily training the number of criterion trials dropped to 5.8 on the fifth day, at which point the pharmacological experiments were started. On each day the criterion was reached first. Then the drug was applied and after an interval of 10 min (with physostigmine) or 15 min (with atropine) the training was continued until the criterion was attained again. The interruption alone did not adversely affect the performance in control experiments in which saline was applied instead of drugs (Fig. 3). Both physostigmine (0.5 mg/kg) and atropine (6 mg/kg) caused a clear impairment of the alternation habit, while response latency was only slightly reduced. The rats could be retrained to criterion before the drug effect subsided, however, after an average of ten trials (Fig. 4). Similar results were obtained when the alternation delay was prolonged to 30-90 sec. The performance dropped to chance level during the first 5 trials with physostigmine. The atropine effect was similar for the 30 sec delay but considerably less definite for References p. 71-72

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Fig. 3. The effect of 0.5 mg/kg physostigmine (Ph) or 6 mg/kg atropine (At) on delayed alternation. Columns indicate number of criterion trials under control conditions and after administration of drugs. 10, 30 and 90 sec - the delays used.

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Fig. 4. The eRect of 0.5 rng/kg physostigrnine (Ph) or 6 mg/kg atropine (At) on delayed alternation. The percentage of correct alternation responses (blocks of 5 trials) increases with continued training. The horizontal dashed line corresponds to the pre-drug performance, From left to right 10, 30 and 90 sec delays.

the 90 sec delay. The average number of criterion trials is shown in Fig. 3. Although the dynamics of the drug action must be taken into account, especially when using the 90 sec delay, in most experiments the alternation responding was perfect before the decline of the drug effect. Two additional factors must be considered when interpreting the above results. As the same rats were used throughout, the habit gradually became more and more overtrained. Furthermore the alternation was repeatedly learned under physostigmine or atropine. When the same dosage and delay was used again, the alternation was less impaired by the second drug application than by the first one. The less pronounced effect of drugs in the later phase of the experiment can be explained therefore

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by better fixation of the alternation habit, by state-dependent learning and by drug tolerance. The relative significance of these factors must be elucidated by further research. While the spontaneous alternation of left and right turns in the H-shaped maze is disrupted by physostigmine for periods corresponding well to the electrophysiological changes, the more difficult alternation (with delays of up to 90 sec) could be mastered even at the height of the drug effect. Even higher doses of physostigmine (1 mg/kg) or atropine (15 mg/kg), causing a decrement of avoidance reactions from 80% to about 20-30%, did not eliminate the alternation in highly overtrained animals. This points to the conclusion that cholinergic systems are not indispensable for delayed reactions of the above type, when the animal is adequately motivated and when the habit is sufficiently fixed. These experiments do not answer the question about the cholinergic nature of recent memory. Learning of new habits is impaired both by atropine and physostigmine, but this does not necessarily imply that the deficit is due to loss of recent memory. Cholinergic systems are perhaps engaged in normal learning as well as in short-term storage of information not subject to long-term retentions. A decrease of spontaneous goal alternation after scopolamine w d S reported by Meyers and Dominolx in rats. Bradley and Roberts5 found delayed responses in monkeys considerably impaired by atropine but also by atropine methyl nitrate, which does not penetrate the blood brain barrier. When the cholinergic storage is eliminated by drugs, a noncholinergic trace can be formed which is more or less resistent to cholinergic blockadel7. Overtraining has a similar effect, probably because the non-cholinergic system becomes more and more important with the continuing fixation of the engram.

Locus ojaction In spite of many attempts to determine the brain structures primarily affected by cholinergic and anticholinergic compounds the evidence is still inconclusive. As shown by Bradley and Elkes4 and by Rinaldi and HimwichZ4,physostigmine induces cortical desynchronization in cerveuu isole' rats but not in the isolated hemisphere preparation. This points towards involvement of thalamocortical and limbic mechanisms. Participation of the lower brainstem is not ruled out by the above experiments. The reticular threshold for EEG arousal is raised by atropinez4, but not decreased by physostigminel5>20.Behavioral arousal is altered neither by cholinergic nor by anticholinergic drug+. However, the dissociation of EEG and behavior limits the value of electrophysiological evidence for analysing the cholinergic mechanisms of learning. Structures primarily responsible for behavioral symptoms due to cholinergic and anticholinergic substances can also be identified by comparing the results of pharmacological experiments in normal animals and in animals with circumscribed brain lesions. As pointed out by Meyersl7 the atropine effect resembles the behavioral deficits caused by hippocampal lesions. No systematic study of the cholinergic and anticholinergic influences on the behavior of lesioned animals is available, however. An additional difficulty in such experiments is the sensitivity of denervated structures References p . 71-72

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which h a y considerably distort the usual relationships between the remaining brain centers. We attempted, therefore, to provide further electrophysiological and behavioral data characterizing the mechanism of physostigmine and atropine influences from the above aspects. Electrophysiological experiments The excitability of the cerebral cortex of unanesthetized curarized rats (BureS and Herink, unpublished data) was examined using two techniques. In the first experiment, a penicillin focus was evoked in the frontoparietal cortex by local application of a minute quantity of G-penicillin onto the exposed brain surface. The EEG was recorded from the area of penicillin application and from the symmetrical point in the opposite hemisphere. Within a few minutes high voltage spikes appeared in the region of the focus as well as in the symmetric contralateral cortical area. After the frequency was stabilized (about 25-40/sec), 1 mg/kg physostigmine or 6 mg/kg atropine were injected intraperitoneally and the recording was continued for 10 or 25 min respectively. An injection of the antagonistic drug followed. The results of these experiments are summarized in Fig. 5. Whilst under control conditions the maximum frequency of the penicillin spikes attained at the beginning of the experiment

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Fig. 5. The effect of physostigmine and atropine on the rate of penicillin spikes generated from a cortical focus. The average rate before drug application is taken as 100 %. Abscissa = time in minutes.

exponentially decayed with a half time of 30-40 min, atropine prevented the decline after a few minutes and later caused an increase of spike frequency. In 50% animals trains of spikes appeared, raising the spiking rate to 50-100/min. Physostigmine, injected 25 min after atropine, further enhanced the seizure-like bursts, the average spike frequency increasing’to 250% of the control level. In experiments in which physostigmine was injected first, the spike frequency slowly decreased in most animals with trains of spikes occurring in only one animal. After atropine injection there was

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---

Fig. 6. Cortical after-discharge before (A) and 5 (B) or 10 (C) min after administration of 1 mg/kg physostigmine. The inset brain scheme illustrates the arrangement of the recording (0, I , 2) and stimulatingelectrodes (S). ECG = electrocardiogram.Time after stimulation in minutes is shown over the record samples in C.

a short-lasting decrease in the discharge rate followed by activation. Spike grouping was observed in 60% of animals after atropine. Somewhat different results were obtained in the second series of experiments in which cortical afterdischarge was evoked by electrical stimulation (lO/sec, 2 msec, 10 sec) of the frontoparietal cortex. After the average duration of the afterdischarge had been determined, physostigmine or atropine was injected and electrical stimuli were applied at 5 min intervals. While the afterdischarge duration was not much changed by atropine it was strikingly prolonged in the physostigmine injected animals (Fig. 6). In most, the afterdischarge developed into continuous paroxysmal activity lasting for the rest of the experiment (Fig. 7), i.e. over several hours in some cases. The seizure continued even after injection of 6 mg/kg atropine which seems to be inadequate to antagonize the physostigmine effect. Only in one case (Fig. 8) did the afterdischarge stop 8 min after atropine administration and its duration returned to References p. 71-72

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Ph

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Fig. 7. Average duration of cortical afterdischarge after application of drugs. Physostigmine does not influence the afterdischarge when applied after atropine, whilst atropine does not stop the afterdischarge prolonged by previous application of physostigmine.

normal values; it could be prolonged again, however, by a fresh injection of physostigmine. Physostigmine administered after preliminary treatment with atropine evoked a moderate increase in the afterdischarge duration but no continuous paroxysmal activity developed (Fig. 8). The above results indicate that atropine and physostigmine affect the excitability of a localised cortical area less than the excitability of the corticothalamolimbic circuits mediating the cortical afterdischarge. The atropine effect is rather inconspicuous, indicating that noncholinergic neurons are probably involved in the afterdischarge mechanism. Similar results were recently obtained with atropine by Berryl. On the contrary, the physostigmine effect was striking, as only EEG activation without any symptoms of seizure activity is induced by lmg/kg3*6.The extremely prolonged afterdischarge is evidently due to a tendency to maintain reverberative activity started by an intense stimulus. This indicates that blockade of acetylcholinesterase interferes with the mechanism causing abrupt cessation of the seizure activity, characteristic in the normal animal. Formation of secondary epileptic foci may explain the extremely long duration of the seizure, far outlasting the physostigmine effect. Curarization may be considered as another factor involved since acetylcholine and d-tubocurarine were shown to act cumulatively in facilitating cortical afterdischargeslO. Hyperventilation has the same effect22. Further research is required in order to reveal the relative significance of the above factors and to determine the structures primarily involved.

Behavioral experiments The significance of cortical mechanism in the behavioral effects of cholinergic and antich olinergic drugs was analysed by comparing their effects on active avoidance learning in normal and functionally decorticated animals. The naive rats were allowed at

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Fig. 8. Examples of the differential effect of atropine and physostigmine on cortical afterdischarge.

0C 0

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Fig. 9. The effect of functional decortication and of physostigrnine and atropine on ..arning of a simple avoidance reaction. Above : number of criterion trials (total columns) and of spontaneous return reactions (shaded columns). Below : Time spent on the grid floor during the first (I) and second (11) exploratory test. For details see text.

first to explore for five minutes a rectangular runway with an electrifiable grid floor in one half and a wooden floor in the other half ofthe apparatus. The times spent in the two parts of the runway were measured and crossings were recorded. An avoidance reaction was then elaborated: the animal was placed on start and required to reach within 5 sec the wooden floor, otherwise intermittent electric shocks were applied until a successful escape reaction was made. The rat was left for 40-60 sec in the goal compartment and then placed on start again. Training continued to the criterion of 9 avoidances in References p . 71-72

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10 consecutive trials. Twenty-four hours later the exploration test was repeated. Bilateral cortical spreading depression was evoked by local application by 25 % KCl onto the exposed cerebral surface 10 min before the first exploration. Drugs were applied at the same time. The results are illustrated in Fig. 9. The exploratory behavior of normal rats was not changed either by physostigmine or by atropine. The animals preferred to stay on the grid floor where they spent the major part ofthe 300 sec exploratory period. Learning the avoidance reaction was slower after physostigmine but not after atropine. The retention test revealed decreased preference for the grid floor in the control group but not in the animals learning under atropine. Although learning was considerably impaired under bilateral cortical spreading depression, most rats were able to reach the criterion in less than 50 trials. While physostigmine did not affect the learning ability of the decorticated animals, the atropine effect was very peculiar. The animals learned rapidly to run from the start to the goal within 5 sec, the avoidance criterion being attained with nearly the same speed as in normal animals. After reaching the goal, the animals often returned back to the grid floor, however, where they were shocked again until they finally spent at least 40-60 sec in the goal compartment. Spontaneous returning from the goal area to the electrified grid occurred in all spreading depression groups, but was most pronounced in the atropinized animals. The low number of criterion trials after atropine does not indicate that the learning ability was improved but that the procedure raised the overall activity of the animal and thus increased the probability of avoiding shocks as well as of receiving them by reaching or leaving the goal. This conclusion is also supported by the results of the exploratory test performed 24 hours later: the preference for the grid floor remained preserved and did not differ from that in normal naive animals. Atropine acts as a psychomotor stimulant in mice16 and rat$. Conversely, cholinergic stimulants reduce spontaneous activity11 and decrease amphetamine toxicityla. As the general stimulating effect of atropine is considerably enhanced in functionally decorticated rats, its site of action must be sought mainly at the subcortical level. On the contrary the behavioral impairment due to functional decortication is not further increased by physostigmine, which probably affects the regions directly or indirectly eliminated by the spreading depression process. It can be conceived that functional decortication produces an imbalance between the remaining parts of the two antagonistic systems, resulting in cholinergic predominance, which can be counteracted by atropine. DISCUSSION

In spite of the accumulating evidence, that the projxtion of the reticular arousal system to the cerebral cortex is cholinergic23, and that the principal cholinoceptive structures include the hippocampus, caudate and thalamus21, the results of our experiments indicate that in the rat other mediators can maintain even complex behavioral functions after cholinergic transmission has been effectively impaired. It seems justified to assume, therefore, that noncholinergic systems duplicate most behavioral functions of the cholinergic ones. No definite behavior can be claimed to be

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specifically dependent on cholinergic transmission, which diffusely participates in activities of different CNS structures and is perhaps most clearly expressed in the corticothalamolimbic system. In general, excessive stimulation of cholinergic synapses tends to abolish the motor output and results in some degree of sedation while administration of central cholinergic blockers has an opposite effect. As participation of cholinergic systems in organized neural activity is impaired in either case, the above differences are evidently due to the effect on the synaptically connected noncholinergic neurons, the bombardment of which is increased by physostigmine and decreased by atropine. The relative significance of the primary interference with the cholinergic transmission and of the secondary effects on the noncholinergic system is different in various CNS regions and can be revealed by electrophysiological experiments as well as by behavioral tests performed in lesioned animals. SUMMARY

The behavioral significance of cholinergic systems was examined in rats injected with physostigmine salicylate (0.25-1 .O mglkg) or atropine sulphate (6-1 5 mg/kg). Physostigmine but not atropine impaired spontaneous alternation of left and right turns in an H-shaped maze. Both drugs disrupted delayed alternation in a T-maze (delays from 10-90 sec). With continued training, however, the correct responding returned, before the drug effect started to decline. With repeated drug applications the effect became less marked. Atropine increased more than physostigmine the discharge rate of high voltage spikes elicited by local application of penicillin on the motor cortex of unanesthetized curarized rats. Physostigmine caused an extreme prolongation (up to several hours) of cortical afterdischarge, which could be prevented by atropine. Both physostigmine and atropine slightly impaired the acquisition of a simple avoidance reaction in normal rats. The same avoidance reaction required more criterion trials in functionally decorticated animals, the learning ability of which was unaffected by physostigmine and seemingly improved by atropine. The latter drug increased the overall activity of the animal and thus raised the probability of correct as well as of incorrect responding. It is concluded that cholinergic systems are paralleled by noncholinergic ones, which can take over many initially impaired behavioral functions. The mechanism of the cholinergic drug action is discussed and the differential influencing of various CNS levels is stressed. REFERENCES 1 BERRY, C. A. (1965) A study of cortical afterdischarge in the rabbit. 4rch. int. Pharmacodyn., 154, 197-209. 2 BLODGETI-, H. C. AND K. MCCUTCHAN (1944) Choice point behavior in the white rat as influenced by spatial opposition and by preceding maze sequence. J. Comp. Psychol., 37, 51-70. 3 BOHDANECK~, Z., T, WEISSAND E. FIFKOVA (1963) Influence of neocortical and hippocampal spreading depression on “theta rhythm” elicited by physostigmine. Arch. int. Pharmacodvn., 143,23-33. 4 BRADLEY, P. B. AND 5. ELKES(1957) The effects of some drugs on the electrical activity of the brain, Brain, 80, 77-1 17,

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5 BRADLEY, P. B. AND M. H. T. ROBERTS (1967) Studies on the effects of drugs on recent memory in animals. Physiol. Behav. In press. 6 BURES,J., Z. BOHDANECK? AND T. WEISS(1962) Physostigmine induced hippocampal theta activity and learning in rats. Psychopharmaccl., 3, 254-263. 7 BURESOVA, O., J. BURES,Z. BOHDANECK~ AND T. WEISS(1964) Effect of atropine on learning, extinction, retention and retrieval in rats. Psychophurmacol., 5, 255-263. 8 CARLTON, P. L. (1963) Cholinergic mechsnisms in the control of behavior by the brain. Psychol. Rev., 70, 19-39. 9 CARLTON, P. L. AND P. DIDAMO (1961) Augmentation of the behavioral effects of amphetamine by atropine. J. Pharmacol. exp. Ther., 132, 91-96. AND G . MOLNAR (1965) The effect of neuromuscular blocking agents on 10 FOHBR,O., G. KLITINA the electrical activity of cats cerebral cortex. Arch. irit. Pharrnacodyn., 158, 277-285. 11 HARRIS,L. S. (1961) The effect of various anti-cholinergics on spontaneous activity of mice. Fed. Proc., 20, 395. 12 HERZ,A. (1960) Die Bedeutung der Bahnung fur die Wirkung von Scopolamin und ahnlichen Substanzen auf bedingte Reaktionea. 2. Biol., 112, 104-1 12. 13 KILLAM, E. K. (1962) Drug action on the brain-stem reticular formation. Pharmacological Reviews, 14, 175-210. K. (1965) Transmitters in the cerebral cortex. Zr.t. Congress, Tokyo, Lectures and 14 KRNJEVIC, Symposia, PIoc. int. union physiol. sci., 23, 435443. (1957) Action of eserine and amphetamine on the electrical 15 LONGO,V. G. AND B. SILVESTRINI activity of the rabbit brain. J. Pharmacol., 120, 160-170. 16 MENNEAR, J. H. (1965) Interactions between central cholinergic agents and amphetamine in mice. Psychophurmacol., 7 , 107-1 14. 17 MEYERS, B. (1965) Some effects of scopolamine on a passive avoidance response in rats. Psychopharmacol., 8, 11 1-1 19. 18 MEYERS, B. AND E. F. DOMINO (1964) The effect of cholinergic blocking drugs on spontaneous alternation in rats. Arch. irt. Pharrnacodyn., 150, 525-529. 19 MEYERS, B., K. H. ROBERTS, R. H. RICIPUTI AND E. F. DOMINO (1964) Some effects of muscarinic cholinergic blocking drugs on behavior and the electrocorticogram. Psychopharrnacol., 5,289-300. 20 MONNIER, M. (1960) Actions klectro-physiologiques des stimulants du systltme nerveux central. 1. Systtmes adrknergiques, cholinergiques et neurohumeurs serotoniques. Arch. int. Pharmucodyn., 124, 281-301. 21 MONNIER, M. AND W. ROMANOWSKI (1962) Les systkmes cholinoceptifs cerebraux - actions de I’acetylcholine, de la physostigmine, pilocarpine et de GABA. Electroenceph. clin. Neurophysiol., 14,486-500. 22 OLIVER, K. L. AND W. H. FUNDERBURK (1965) Possible role of hyperventilation in the CNS effects attributed to tubocurarine. Electrmnceph. clin. Neurophysiol., 19, 501-508. 23 PHILLIS, J. W. AND G. C. CHONG(1965) Ace+ylcholinerelese from the cerebral and cerebellar cortices: its role in cortical arousal. Nature, 207, 1253-1255. 24 RINALDI, F. AND H. E. HIMWICH (1955) Alerting responses and actions of atropine and cholinergic drugs. Arch. Neurol. Psychiat., Chicago, 73, 387-395. 25 ROEERTIS, E. DE (1965) Subcellular localization of transmitter substances and related enzymes in the CNS. Znt. Congress, Tokyo, Lectures and Symposia, Proc. int. union physiol. sci., 23,411418. 26 WIKLER, A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: morphine, n-ally1 normorphine and atropine. Proc. SOC. exp. Biol., N . Y., 79, 261-265.

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Some Actions of Cholinergic and Anticholinergic Drugs on Reactive Behaviour A. HERZ Deutsche Forschungsaristaltfur Psychiatrie, Max-Planck-Institut, Munich (Germany)

The obvious lack of grossly visible behaviour changes following application of cholinomimetic and cholinolytic drugs led to the term of a ‘dissociation’ between EEG-pattern and behaviour4.39. Further investigations of the correlates of physostigmine- and atropine-induced EEG-patterns at the behavioural level, showed that stimulation as well as inhibition of central cholinoceptive structures is followed by distinct but more subtle changes of behaviour. The investigation of reactive behaviour proved to be very suitable for the detection of such subtle behaviour changes caused by substances acting on central cholinoceptive structures. In the case of cholinolytic drugs, the investigation of the acquisition of the conditioned responses proved to be very suitable and allowed the assumption that cholinolytic drugs interfere with memory processes, whilst in investigating cholinergic stimulants, outlines of a more general concept of the meaning of cholinergic stimulation for responding behaviour became evident. 1.

THE D I F F E R E N T I A L EFFECT OF C H O L I N O L Y T I C D R U G S O N A C Q U I S I T I O N A N D R E T E N T I O N O F C O N D I T I O N E D RESPONSES

In 1959 we demonstrated that the action of cholinolytic drugs on a conditioned avoidance response is highly dependent upon the establishment of this reaction and contrasting effects can be obtained whether the experiments are performed on rats in the state of acquisition of the response or on overtrained animals. Fig. 1 shows the performance of the conditioned pole jump in the first session of training. Every minute the conditioning signal, consisting of a tone of 5 sec duration, was given it was followed by an electric shock. After about 10 min the first conditioned avoidance responses (CAR) occurred and 30 min later a performance level of about 80% was reached. An injection of saline was without any effect and did not impair any further acquisition of the CAR, but the application of scopolamine (0.2 mg/kg) completely disrupted acquisition of the conditioned response. At the height of the action the unconditioned response was also partly abolished. Following scopolamine the animals showed a distinct pattern of excitement, especially when the acoustic signal was given: they walked about in the box, searching and sniffing. When the pole was in their field of view, they sometimes jumped onto it. One had the impression that the animals still References p . 54-85

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Fig. 14. Modification of neocortical EEG effects of HC-3 by various cholinergic agonists. HC-3 induced EEG slow waves were antagonized by intravenous arecoline and pilocarpine, but not nicotine. Methyl atropine was given prior to these drugs to prevent hypotension.

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2. EfJpcts qf hemicholinium on drug-induced EEG activation in the dog As might be expected from the highly cationic character of hemicholinium (see Fig. 4), intravenous injection of this agent did not produce consistent EEG changes in dogs maintained with adequate artificial ventilation. Enormous doses, 5-10 mg/kg, given intravenously in animals occasionally produced marked EEG slowing and blockade of activation to all afferent stimuli. It was assumed that the marked variability was due to individual differences in blood-brain barrier permeability, brain and/or blood levels of choline, etc. Therefore the drug was given intraventricularly. Via this route, remarkably small amounts of HC-3 produced consistent EEG changes in neocortical and limbic areass.9. As illustrated in Fig. 13, HC-3 (5 mg total dose) caused within 40 min spiking in the limbic areas and abolition of the hippocampal theta rhythm. However, neocortical activation still persisted. After 4 hours both neocortical and limbic areas showed diffuse slow waves and spiking. These persisted for about two days with partial recovery 45 h after injection. During this period both neocortical and limbic system activation was blocked. We have previously reported8, that these effects are partially reversible with choline, and are not produced with intraventricular administration of sodium chloride, sodium bromide, d-tubocurarine, and gallamine indicating the specifity of HC-3 action. Furthermore, following HC-3 i n total doses of both 50pg and 5 mg, brain acetylcholine levels in subcortical structures near the ventricle are reduced by about 50% in 4 h9. These changes in brain acetylcholine content qualitatively parallel the distribution of **C-labelledHC-3 (Domino et a]., unpublished observations). Inasmuch as 5 mg of HC-3, given intraventricularly, does not deplete brain acetylcholine completely, it would be expected that some cholinergic agonists would antagonize the functional deficits of lowered brain acetylcholine. This indeed was found to be the case. The H I cholinergic agonists arecoline (40 pg/kg i.v.) and pilocarpine (500 pglkg, i.v.) reversed the HC-3 induced EEG neocortical slow waves but the ii cholinergic agonist nicotine did not (see Fig. 14). Physostigmine (IOOpg/kg, i.v.)also antagonized HC-3 induced slow waves, but epinephrine (5 pglkg, i.v.) and damphetatnine (2 mg/kg, i.v.) did not (see Fig. 15). These findings are reminiscent of those of White and Boyajy25 and White and D a i g n e a u P with atropine. CONCLUSIONS AND SUMMARY

Pharmacological data on the importance of cholinergic mechanisms in EEG activation and behavioral arousal as well as sleep are impressive. The gross behavioral consequence of the initial EEG activation is clearly a wake-up or arousal state. It has previously been reported by Wikler27, Bradley and Elkesl, Bradley and Nicholson2 and others, that cholinergic agonists and antagonists produce EEG dissociation from gross behavior. Similar findings have been made by us using large doses of atropine and physostigmine. However, it should be pointed out that effective doses of physostigmine and other cholinergic agonists produce initial behavioral arousal that is associated with neocortical and limbic activation. The emphasis in the literature on EEG dissoc-

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iation from gross behavior may have been overstated, particularly in relationship to the awake-sleep cycle of the chronic cat. The findings of Bradley and Elkes and others with cholinergic agonists were made at a time when the stage of fast wave sleep as described by Dement3 and Jouvet15 was not generally known. It would appear that in some instances investigators may have been observing fast wave sleep and did not recognize it as such. By use of various m and n cholinergic antagonists with differential abilities to penetrate the blood-brain barrier, it has been possible to determine if the actions of various cholinergic agonists given intravenously were primarily central or peripheral in origin. Four predominantly muscarinic (m) cholinergic agonists (acetylcholine, arecoline, pilocarpine, and physostigmine) and two nicotinic ganglionic (n) cholinergic agonists (DMPP and nicotine) were studied on the awake-sleep cycle of cats. The animals had chronic indwelling brain electrodes in various neocortical and limbic areas. The effects of these compounds were compared before and after the following m and n cholinergic antagonists: atropine, methyl atropine, mecamylamine and trimethidinium. Atropine pretreatment blocked EEG activation induced by acetylcholine, arecoline, pilocarpine and physostigmine, but only reduced that produced by DMPP and nicotine. Atropine also blocked nicotine induced hippocampal theta wave activity. Methyl atropine, an m cholinergic antagonist with predominant peripheral effects, markedly antagonized EEG activation by acetylcholine, but did not block EEG activation induced by other m or n cholinergic agonists. The n ganglionic cholinergic antagonists, mecamylamine and trimethidinium, had no significant effects on EEG activation induced by m cholinergic agonists. On the other hand, the actions of n cholinergic agonists such as DMPP and nicotine were completely blocked by mecamylamine. Trimethidinium blocked EEG activation of DMPP but reduced slightly that of nicotine. In general, the gross behavior of the cats paralleled the initial neocortical EEG effects of these drugs when given in low doses. Another pharmacological approach to studying central cholinergic mechanisms was with the drug hemicholinium (HC-3) which decreases acetylcholine synthesis by interfering with choline transport. Acute dog preparations were used to study the effects of HC-3. The actions of the drug were unpredictable on intravenous administration, but highly reproducible when given intraventricularly in total doses up to 5 mg. HC-3 produced initially amygdala spiking and blockade of hippocampal theta wave activity, but did not affect neocortical activation. This demonstrates a dissociation between the neocortical and limbic activating systems. Eventually neocortical slow waves appeared. The EEG effects of HC-3 are related to lowered brain levels of acetylcholine, because subcortical acetylcholine was reduced approximately 50 % 4 h after drug administration. Exogenous choline produced a delayed and transient reversal of the HC-3 effects. Arecoline, pilocarpine, and physostigmine caused EEG activation following HC-3, whereas nicotine, epinephrine and d-amphetamine were either much less effective or their EEG actions were completely blocked.

References p . 132-133

132

E. F. D O M I N O . K . Y A M A M O T O A N D A. T. D R E N

REFERENCES 1 BRADLEY, P. B.

A N D ELKES, J. (1957) The effects of some drugs on the electrical activity of the brain. Brain, 88, 77- 1 17. 2 BRADLEY,P. B. AND NICHOLSON, A. N. (1962) The effect of some drugs on hippocampal arousal. Electroenceph. cliw. Neurophysiol., 14, 824-8 34. 3 DEMENT, W. (1958) The occurrence of low voltage, fast electroencephalogram patterns during behavioral sleep in the cat. Electroerrceph. cliw. Neurophysiol., 10, 291-296. 4 DENISENKO, P. P. (1961) Cholinergic and adrenergic systems in the reticular formation of the midbrain and the reaction of activation in the cortex. Sechenov Fiziol. Zh. USSR (Big.), 47,609-61 6. 5 DENISENKO, P. P. (1962) Influence ot pharmscological agents upon cholinoreactive and adrenorcactive systems of the reticular formation and other regions of the brain. Proc. 1st I t d . Pharmacol. Meetings. Phartnacolngical Analysis of Central Nervous Action, Paton, W. D. M. Ed, Pergamon Press. 6 DOMINO, E. F. (1955) A pharmacological analysis of the functional relationship between the brain stem arousal and diffuse thalamic projection svstem. J . Pharmacol. Exptl. Therap., 115, 449463. K. (1966) Pharmacologic evidence for cholinergic 7 DOMINO, E. F., DREN,A. T. A N D YAMAMOTO, mechanisms in neocortical and limbic activating systems. Hakone Symposium held in Japan 1965. Progr. Brain Res. In press. 8 DREN, A. T. AND DOMINO, E. F. (1965) Someeffects of hemicholinium (HC-3) on EEG desynchronizating mechanisms in the dog. Pharmacologist, 7 , 143. 9 DREN, A. T. AND DOMINO, E. F. (1966) Effects of Hemicholinium (HC-3) on EEG activation and brain acetylcholine in the dog. Pharmacofogist, 8, 183. 10 H E R N ~ N D E Z - PR. E ~AND N , CHAVEZ-IBARRA, G. (1963) Sleep induced by electrical or chemical stimulation of the forebrain. In “The Physiological Basis of’ Mental Activity.” R. Hernandez-Pebn Ed. Electroenceph. Clin. Neurophysiol. Suppl., 24, 188-198. 1 1 HERNANDEZ-PE~N, R., CHAVEZ-IBARRA, G., MORGANE, P. J . AND TIMO-TARIA, C. (1963) Limbic cholinergic pathways involved in sleep and eniotional behavior. Exptl. Neurol., 8, 93-1 I!. 12 ILYUTCHENOK,R. 1. (1962) The role of cholinergic systems of the brainstein reticular formstion in the mechanism of central effects of anticholinesterase and cholinolytic drugs. Proc. 1st lnt. Pharmacol. Meetiws. Paton, W. D . M. Ed. Pharmacological Analysis of Central Nervous System, 8, 21 1-216. 13 ILYUTCHENOK,R. I. A N D MASHKOVSKII, (1961) Electrophysiological data on cholinereactive elements of the reticular formation of the brain stem. Sechenov Fiziol. Zh. USSR.,47,1352-1359. 14 ILYUTCHENOK, R. I. AND OSTROVSKAYA, R. U. (1962) The role of mesencephalic cholinergic systems in the mechanism of nicotine activation of the electroencephalogram. Bull. Exptl. Biol. and Med., 54, 753-757. 15 J O U V ~ TM. , (1961) Telencephalic and rhombencephalic sleep in the cat. Ciha Foundatiorr Symposiun~on the Nature of Sleep. pp. 188-206. 16 KNAPP, D. E. AND DOMINO, E. F. (1962) Action of nicotine on the ascending reticular activating system. Int. J . Neuropharmacol., 1, 333-351. 17 LONGO,V. S. (1966) Behavioural and electroencephalographic effects of atropine and related compounds. Pharmacol. Rev., 18,965-996. 18 MICKELSON, M . J. (1961) Pharmacological evidences of the role of acetylcholine in the higher nervous activity of man and animals. Activ. Nerv. Super., 3, 2. 19 RINALDI, F. AND HIMWICH, H. E. (1955) Alerting responses and actions of atropine and cholinergic drugs. AMA Arch. Neurol. Psychiat., 73, 387-395. 20 RINALDI, F. AND HIMWICH, H. E. (1955) Cholinergic mechanism involved in function of mesodiencephalic activating system. A MA Arch. Neurol. Psychiat., 73, 396-402. A. V. (1961) Thepharmacology of reticular fortnutior! andsynaptic transmission. pp. 432. 21 VALDMAN, Leningrad. 22 VALDMAN, A. V. (1963) Problems of pharmacology ofreficular formalion and synaptic transmission. pp. 416. Leningrad. 23 VELLUTI, R. AND HERNANDEZ-PEON, R. (1963) Atropine blockade within a cholinergic hypnogenic circuit, Exptl. Neurol., 8, 20-29. 24 VILLARREAL, J. E. A N D DOMINO, E. F. (1964) Evidence for two types of cholinergic receptors involved in EEC desynchronization. Phavmacologist, 6, 192. R. P. A N D BOYAJY, L. D . (1959) Comparison of physostigmine and amphetamine in anta25 WHITE, gonizing the EEG of CNS depressants. Proc. SOC.Exptl. Biol. N . Y., 102, 479-483.

CHOLINERGIC MECHANISMS I N SLEEP A N D WAKEFULNESS

I33

26 WHITE,R. P. AND DAIGNEAULT, E. A. (1959) The antagonism of atropine to the EEG effeects of adrenergic agents. J . Pharmacol. Exptl. Therap., 125, 339-346. 27 WIKLER,A. (1952) Pharmacologic dissociation of behavior and EEG “sleep patterns” in dogs: morphine, n-allylnormorphine, and atropine. Proc. Soc. Exptl. Biol. N. Y. 79, 261-265. 28 YAMAVOTO, K. AYD DOMINO, E. F. (1965) Nicotine-induced EEG and behavioral arousal. Znt. J. Neuropharmacol., 4, 359-373. 29 YAMAMOTO, K. AND DOMINO, E. F. (1967) Cholinergic neocorticil and hippocampal EEG activation, Int. J . Neuropharmacol. (In press)

134

Cholinergic Brain Mechanisms and Behaviour R.YU. TLYUTCHENOK Insritute of Cytology arid Gertetics ( U S S R ) *

The problem of cholinergic mechanisms in the brain is very complicated. In the present study we have tentatively ignored all the complexities and have focused our attention on some of the problems related to the participation of cholinergic mechanisms in behaviour. In what behavioural reactions do cholinergic mechanisms play an important role? Is behaviour correlated with changes i n bioelectrical brain activity and stress reactions? What is the possible mechanism of behavioural changes under the effect of cholinergic drugs? Certainly, it is difficult to give an exhaustive answer to all these questions. Evidence obtained during the past few years has enabled us to gain an understanding of the possible role of cholinoreactive structures in behaviour. CHOLINERGIC MECHANISMS A N D EMOTIONAL BEHAVIOUR

In previous studies it has been demonstrated that the most characteristic changes produced by cholinergic drugs in experimental animals are those of observed emotional behavi ou r. Our very first observations on the effect of tropazine, an anticholinergic drug relieving fear neurosis, focused our attention upon the study of the role of cholinergic mechanisms in behaviour27. At this early stage, it was already hypothetically presumed that cholinergic mechanisms play an important role in the emotional fear reaction. Experiments using an anti-acetylcholinesterase (galanthamine) provided additional data on the participation of cholinoreactive mechanisms in defensive reactions46. This work has shown that in animals galanthamine administration enhances passive defensive reactions. A number of workers have also demonstrated the participation of cholinergic mechanisms in behavioural reactions3.16,".37.47,65. During the past few years A. G . Yeliseyeva, in our laboratory, has studied in detail the role of the cholinoreactive structures in the mechanisms of the emotional fear reaction. Food conditioned reflex

* Pharmacological Laboratory, Experimental Biology Dept., Siberian Branch, Academy of Sciences of the USSR, Novosibirsk, 90, USSR,

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

135

Fig. 1. Inhibition of the emotional fear reaction by muscarinic anticholinergic agent (amysil). When the conditioned food reaction was elaborated (A) the dog was given electric shock (B). After that it did not leave the box for a long period of time to respond to the presentation of the conditioned stimulus then it attempted to run out of the room (D-I?), resisted when attempts were made to bring it to the food cup (F). After the administration of amysil “fear” reaction disappeared. On the next day at the presentation of the conditioned stimulus the dog came near the food cup and ate (G-H). The arrow on the light background marked the action of the conditioned stimulus.

conditioning was carried out with unrestrained dogs (Fig. 1 A). After establishing conditioned responses, the dogs received an electric shock the moment they came into References p . 146-148

I36

R. Y U . I L Y U T C H E N O K

contact with their food cup. After one or two electric shocks a conditioned fear reaction was produced (Fig. I B). This fear reaction was manifested each time the conditioned stimulus was present, overlapped by the electric shock (Fig. 1 C-F). Fear reaction of this type can be observed lasting for a few months. This is confirmed by a number of other authors'. Another series of experiments was performed on cats. An auditory signal was combined with an electric shock delivered through the grid floor. Fear reaction was well established after 3 or 4 combined stimuli and was expressed by the following symptoms: the animal stood stock still with the head drawn in, eyes closed and ears retracted. Urination and defecation were commonly observed. Sometimes the response was o f another type: the animal hissed, snarled, raised its paw threateningly and jumped. This aggressive reaction, however, was noted only at the very beginning of the conditioned response elaboration. With the increasing number of trials the aggressive reaction was replaced by a passive-defensive one. In our experiments on dogs, the blockade of muscarinic cholinergic brain structures by amysil (benactyzine) or bensazine immediately after electrostimulation or on the next day (0.5-1 .O mg/kg intramuscular 2-3 times daily for 1-3 days), considerably inhibited the conditioned responses. Fear reaction disappeared at the same time. Thus, immediately after anticholinergic drug administration it was possible to note its inhibitory effect on fear reaction, in spite o f general inhibition of conditioned reflexes. It should be emphasized that an anticholinergic drug does not inhibit the unconditioned food response. During the following days the conditioned food response was restored completely, but the fear reaction was not re-established (Fig. I G, H). In a series of experiments performed on cats we have also noted an isolated inhibition of the fear reaction due to the effect of anticholinergic drugs. In cats, intravenous administration of amysil in a dose of 0.5 mg/kg abolished the conditioned fear reaction after a few minutes. At the same time no change in the unconditioned reaction to electrostimulation appeared. Components of the aggressive reaction, i.e. the raising of paws, sniffing and hissing (in cases where they were noted previously) were also maintained. Concerning the blocking of the adrenoreactive brain structures, it has not been possible to reveal its isolated influence on the emotional fear reaction in animals. When aminazine (chlorpromazine) was administered intravenously in a dose of I mg/kg, the fear reaction was not abolished. When the intravenous dose was increased to 3-5 mg/kg, the response was attenuated with progressive deepening of the sedative state and parallel decrease of the unconditioned defensive response, aggressive response, loss of motor co-ordination and the presence of muscular relaxation. When the unconditioned fear reaction and the capacity to react to other stimuli were maintained to some degree, the fear reaction was not abolished. It may be supposed that chlorpromazine does not block the mechanism of fear reaction, but inhibits the mechanism through which the motor-vegetative response is realized as a result of general reactivity reduction, including the attenuation of response to noxious stimuli. These facts are completely consistent with our previous observations31~",46, according to which the stimulation of the central cholinoreactive brain structures by

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

I37

cholinesterase inhibitors induces and intensifies the fear reaction. Thus, when the central cholinoreactive structures are stimulated these reactions appear, whereas their blockade abolishes these responses. It is therefore possible that the activity of the central cholinereactive structures is included in the specific mechanisms of the fear reaction19,31133, Complete and stable inhibition of an emotional reaction takes place not only when the muscarinic cholinoreactive structures of the brain are blocked not only immediately but also when the anticholinergic drugs are administered during the following days. This effect is not related to the blockade of conduction of nerve impulses under the effect of anticholinergic drugs since the fear reaction, having disappeared at the moment when the muscarinic cholinoreactive structures were blocked, is not restored after normalization of their activity. Moreover, the administration of the same dose of this agent after an interval of 14-1 5 days, does not inhibit the emotional fear reaction. To obtain this effect considerably higher doses are required. Thus, when the muscarinic cholinoreactive structures are blocked, not only are recent memory traces vulnerable (minutes and hours after the establishment of the fear reaction), but also those that are more prolonged (in the first days following their fixation). The present data indicates the important role of muscarinic cholinoreactive brain structures in the mechanisms of emotional memory34. The hypothesis we suggest is directly opposed to the generally accepted notion of the adrenergic nature of passive-defensive reactions. As early as 1945, Arnold6 reported that fear is mainly due to sympathetic activation, as the state of excitement and elation are related to moderate parasympathetic activity. Later Solomon and Wynne, Boward, Anokhin and others4,9>59demonstrated the participation of adrenergic mechanisms in defensive reactions. What was the reason for such a ready and widespread recognition of the adrenergic nature of defensive reactions? It is possible that this attitude is the result of a deep-rooted concept according to which stress is considered to be a reaction related to the sympathetic adrenal system. At the same time it is generally known that the defensive reaction to a pain stimulus is one type of stress response. EMOTIONAL BEHAVIOUR A N D STRESS

Bowardg considers that a system related to positive emotions has a parasympatheti function and inhibits the neuroendocrinal response to stress. The stimulation of the “negative system” produces fear, rage, aggression and promotes stressful neuroendocrinal responses and releases a sympathetic effect. in our laboratory have However, experiments performed by Ye. V. Naumenk05~9~~ shown that stimulation of the central adrenoreactive structures by piridrole (pipradol) has no marked effect on the function of the adrenal cortex and does not alter the response of the pituitary-adrenal system to stress (Table I). The stimulating effect of phenamine (amphetamine) on the adrenal system is abolished following mesencephalic section, whereas EEG activation is maintained (Table 11). The level of corticosteroids in the peripheral blood did not change under the effect of phenamine both References p. 146-148

I38

R. Y U . I L Y U T C H E N O K

TABLE I T H E C H A N G E S OF ~ 7 - H Y D R O X Y C O R T l C O S T E R O I D S L E V E L P R O D U C t l ) H Y V A R I O U S S T I M U L I I N G U I N F A PIGS WITH I N l A C T H R A I N .

~~

Ti,eatrrrent

No. of atiinia/~

~

~~

-

~~~

Level of I7-hyctroxycort ico rteroih 111

jig 04

M

tvi

+

~

P -

~~~

Distilled water Naphtyzin (1 mg/kg)

15

36.81 i 4.32 114.26 1 6.1 I

0.001

Distilled water Naphtyzin (3 nig/kg)

13

47.12 4 6.88 161.61 & 12.02

0.001

Distilled water Amphetamine (5 mg/kg)

12

41.54 4 4.68 106.43 & 9.43

0.001

Distilled water Amphetamine (I0 mg/kg)

14

42.45 I 138.69

3.52 9.00

0.001

Dktilled water Piridrol (5 mg/kg)

10

37.30 44.97 i

4.90 6.42

0. I

Distilled water Piridrol (10 mg/kg)

31

49.28 55.90

6.19 5.92

0.1

Distilled water Galanthamine ( 5 mg/kg)

27

35.16 f 3.94 65.14 4.24

0.001

Distilled water Galanthamine (10 mg/kg)

19

24.13 & 3.45 97.17 & 6.92

0.001

~~

+ j

+ +

~~

T A B L E 11 I

Hr

CHANGES O F 17-HYDROXYCORTICOSTEROlDS LEVEL PRODUCED B Y VARIOUS STIMULI I N GUINEA PIGS WITH MIDBRAIN S t C T l O N

Level of Treatriient

No. of

T i i w of

cteterminatioti

I@ O h

ariimalt ~-

Control

17-liy~troxicortico~trroid~ P

M f nz ~ - _ _ _ -

10

I h after section 2 h after section

65.75 70.63

Saline solution into ventricle

24

I h after section 2 h after section (1 h after injection saline solution)

61.95 & 3.83

Amphetamine (10 mg/kg)

21

Galanthamine (10 m g / W

1 h after section

2 h after section (1 h after amphetamine injection) 14

I h after section 2 h after section (1 h after galanthamine injection)

7.33

t 6.68

0. I

0.001 119.86 & 7.52 63.22 4 4.72 0. I

74.74 1 8.01 66.57 & 5.14

0.1 67.80 t 6.00

CHOLINERGIC BRAIN MECHANISMS A N D BEHAVIOUR

139

following pretrigeminal section and in the cerveau isole'. It is therefore reasoned that the changes observed in the functional state of the adrenal cortex under the effect of phenamine are connected with the stimulation of the peripheral adrenoreactive structures. This is confirmed by the rise in the level of 17-oxycorticosteroids in the blood following intravenous administration of naphtyzine, an agent mainly stimulating the peripheral adrenoreactive structures (Table I). The available data allows us to assume that adrenomimetics exert an effect on the pituitary-adrenal system by stimulating the peripheral adrenergic structures. Thus, it becomes apparent that an hypothesis involving the participation of the central adrenoreactive structures in stress reactions lacks sound evidence. On the contrary, the data cited above prove that this hypothesis is inconsistent. At the same time this suggestion turns out to be a significant argument in the formation of a hypothesis on the role of adrenergic mechanisms in the realization of defensive reactions. It should be noted that the administration ofgalanthamine, a cholinesterase inhibitor, also raises the level of corticosteroids in the blood (Table I). But with pretrigemina1 section or in the cerveau isole'the stimulation of adrenal cortex is eliminated under the effect of anti-cholinesterases (Table rT). It may be supposed that the activating effect of the anti-choline sterases and acetylcholine on the hypothalamic-pituitaryadrenal system is also connected with its primary effect on peripheral cholinoreactive structures. The stimulation of these structures is transmitted through the brain stem to the hypothalamus. The hypothalamic regions contain serotonino-reactive structures through which it is probable that the whole hypothalamic-pituitary-adrenal system is activated. The acceptance of such a mechanism as regulating stress responses casts some doubt on the obligatory relations between behaviour and the endocrine reactions. Tn ordinary environments, fear and rage i n animals are usually accompanied by stress reactions. But in experimental conditions it is possible to differentiate these two sets of reactions. A rise in thelevel of 17-oxycorticosteroidsintheblood following naphtyzine administration may take place without concomitant behavioural changes. On the contrary, a marked behavioural change without stress response is observed under the effect of piridrole. Thus, it is possible to produce isolated stress reactions and behavioural changes separately. Tt seems that the functional systems governing behavioiir and stress reactions are not components of one and the same mechanism, although they are intimately interrelated in the organism as a whole. BEHAVIOUR A N D ELECTRICAL ACTIVITY O F THE BRAIN

A correlation between behavioural changes and brain electrical activity is established

most commonly when two such functionally opposed states as wakefulness and sleep are compared. The correlation between EEG and behavioural changes is more readily revealed under the effect of drugs acting on the adrenoreactive structures. Our data confirm the well known fact that adrenergic stimulation in animals is accompanied by EEG activation. In contrast, when the adrenergic mechanisms are blocked, the animal is sedated and slow EEG waves appear. However, it is hardly expedient to draw such Refevences p . 146-148

140

R. YU. I L Y U T C H E N O K

sharp distinctions between the presence of behavioural changes in adrenergic wakefulness responses and their absence in cholinergic responses. The behavioural result of adrenergic drug administration is undoubtedly more clearly demonstrated than that of cholinergic drug administration. But in certain experimental conditions after the use of drugs acting on cholinergic structures, peculiar behavioural changes are observed. Ln open space conditions, under the effect of a tertiary anticholinesterase (galanthamine), altertness gradually changing into restlessness was observed in cats. Sometimes, even in the absence of environmental stimuli, the cat arched its back and piloerection took place. Occasionally the cat jumped back as if something had loomed before it, and a tendency to squeeze itself into all kinds of small spaces was noted. In small doses, these drugs accelerate the elaboration ofconditioned reflexes and intensify them, whereas large doses inhibit conditioned reflexes. Central muscarinic-anticholinergic agents block the EEG activating effect of anticholinesterases as well as behavioural responses in animals. This paper has already dealt with the characteristic effect of anticholinergic drugs on the emotional fear reaction. Thus, chemical stimulation and blocking of cholinergic brain structures are characterized not by the complete absence of behavioural reactions but by the peculiar forms in which they manifest themselves29~46.These changes, however, do not satisfy the usual correlation of EEG and behaviour when excitation is accompanied by EEG activation and a sedative or drowsy state is in its turn accompanied by EEG synchronisation. It is known that these EEG patterns tis appear with activity changes in the ascending reticular activating system. Thus, it is a correlation between behaviour and an EEG pattern that characterizes the changes in the function of the brain-stem reticular formation. The following question then arises : why under the effect of cholinergic and adrenergic drugs is there only one type of correlation between EEG and behaviour? Adrenergic and cholinergic drugs produce different types of EEG activation@; moreover, the mechanism of adrenergic and cholinergic EEG activation is not identical. These structures do not have one and the same location in the reticular formation12,'4,17,20,25,44,45,49,54,65,66.

It has been demonstrated in experiments conducted in our laboratory that adrenoreactive structures are limited mainly to the caudal regions of the midbrain and pons. The activating effect of adrenomimetics in the cerveau isolb is attenuated, while in the premesencephalic section it disappears completely. Serotonino-reactive structures are also found in the caudal regions of the ponto-mesencephalic reticular formation30~31. A summary of recent contributions on serotonin is presented by Garattini and Valzelli22. Cholinoreactive structures are widely distributed throughout the ponto-mesencephalic reticular formation28J1J5. It has been established that in the cerveau isolP (when a portion of the midbrain remains above the section level), galanthamine and eserine not only inhibit acetylcholinesterease activity but also produce a marked EEG activation. This effect was more obvious in the asymmetric section (Fig. 2). When the midbrain was isolated completely (premesencephalic section), the acetylcholinesterase activity ofthe brain areas situated above the section was inhibited to the same extent as in intact animals. In spite of this, EEG activation did not take place (Fig. 3). This

CHOLINERGIC BRAIN MFCHANISMS AND BEHAVIOUR

141

Fig. 2. The influence of galanthamine o n the EEG of a cat with non-symmetrical brain section (on the left - cerveau isole, on the right - premesencephalic section). From above downwards: lef frontal and occipital, right frontal and occipital cortex. A - before, B - 1 min after intravenous administration of 3 mg/kg galanthamine.

allows us to conclude that the presence of EEG activation is dependent on the degree of acetylcholinesterase inhibition, in the ponto-mesencephalic region of the brain"?". Thus, the brain stem reticular formation is a biochemically heterogeneous system. I t has varying neurochemical regulatory mechanisms. The physiological implication of its chemical heterogeneity has not been investigated and is not as yet very clear. Undoubtedly, different chemically sensitive components of the brainstem reticular formation are responsible for the manifestation of the different reactions in the central nervous system. They participate in the mechanisms of electrical activity of the brain and to a certain extent in mechanisms of behaviour. But it is unlikely that the behavioural pattern under the effect of pharmacological agents is the result only of changes in the activity of the brain stem reticular formation. Probably, in the complex behavioural situation, the components correlating with a spontaneous EEG pattern must have a reticular mechanism. Consequently, one may suppose that correlation takes place i n the case when the changes of both electrical activity of the brain and of certain behavioural components result from changes in the activities of the same functional systems of the brain. References p . 146-148

1 42

R. Y U. I L Y U T C H E N O K

L -

.> ._

I

It

.@

U

0

U

cortex

thalamus hypothalamus midbroin mtdulta

r Jintuct brain

a prcmcscncephalic

section

Fig. 3. Galanthamine's influence 011 EEG and acetylcholinesterase of the brain. I

-

intact brain

11 - premesencephalic section. From above downwards: left frontal and occipital, right frontal and

occipital cortex. A - before and B - 4 min. after the administration of 9 mg/kg galanthamine. I11 - The change of acetylcholinesterase in cats after the administration of 9 mg/kg galanthamine. Expressed on 0.001 M of acetic acid per 0.2 ml of brain homogenate.

The activating influence of anticholinesterases is maintained against a background of previous antiadrenergic drug e f f e ~ t s ~ ~ , ~ ~ , 3 5 , ~ 1 , 6The 4 1 6blocking 7. by high doses of chlorpromaLine of EEG activation caused by anticholinesterase is accounted for by the anticholinergic effects of chlorpromazine01. According to our experiments31 this effect of chlorpromazine is related to the blocking of the central muscarinic cholinergic structures. At the same time, signs of chlorpromazine depression prevail in the behaviour of the animals with a background of motor anxiety and fear reaction which are characteristic of the effect of the anticholinesterases. Simultaneously, the conditioned reflexes are inhibited. EEG activation resulting from the excitation of adrenoreactive structures of the mesencephalic reticular formation is easily blocked by small doses of central muscarink anticholinergic agents"'131~~5~~"66. These agents do not attenuate motor activity induced by adrenomimetics. In some experiments, anticholinergic agents somewhat intensify this activity, at the same time considerably inhibiting the orientation reaction. Thus, some of the effects are removed, whilst others are maintained. In order to have some understanding of the role of different cholinoreactive brain

CHOLINERGIC BRAIN MECHANISMS AND BEHAVIOUR

143

structures in the mechanisms of EEG and behavioural changes, the presence of a cholinergic mechanism at the level of the cortex in synaptic transmission from the brainstem reticular formation must be taken into account. EEG changes characteristic of reticular cortical activation are related to these mechanisms. The investigations we have performed in collaboration with G. D. Smirnovs7 have shown that the terminal pathway of the ascending activating system forms cholinoreactive synapses at the cortex. This is most markedly expressed in the antagonistic effects of anticholinergic drugs and anticholinesterases on reticular cortical arousal potentials which have been shown in our laboratory by V. S. Zinevich. The cortical neurones of the ascending activating reticular system have muscarinic proper tie^^^,^^. This is confirmed by the data obtained in our laboratory by M. A . Gilinsky who has demonstrated the presence of an antagonistic effect of anticholinesterase and muscarinic anticholinergic drugs in relation to spike activity of cortical neurones. The data on the role of the cortical cholinergic neurones is confirmed in the works of a number of author~3*~39~~0.~6,60. Thus, the activity of the ascending reticular activating system must be considered as proceeding from the chemical heterogeneity of the brain-stem reticular formation to the homogeneity of cortical neurones of the ascending activating system. We consider that there exist mechanisms for transmitting impulses arising from the mesencephalic reticular formation (when different chemoreactive systems are stimulated) to muscarinic cholinergic cortical n e u r o i i e ~ 3 ~ , ~ ~ . Proceeding from the results on the chemical heterogeneity of the ascending reticular activating system at the brain stem level and on the cholinergic properties of the cortical neurones of this system, the reason for the blocking of adrenergic and serotoninergic EEG activation by anticholinergic drugs is clear. Thus, when central chemoreactive systems are stimulated or blocked, complex mechanisms are involved correlating EEG and behavioural responses. It is necessary to note the orientation reaction changes under the effect of anticholinergic drugs. Surely, it should not be considered that reticular mechanisms alone participate in the production of the orientation reaction. Sokoloff58 is possibly right when he suggests that the mechanism of the orientation reflex cannot be limited to any single part of the brain. However, there is adequate evidence indicating that the brain stem reticular formation plays an important role in the mechanisms of the orientation response. There is no doubt that changes in the activity of the brain-stem reticular formation may to some degree alter the manifestation of the orientation reaction. It is a fact that when anticholinergic drugs block the arousal reaction, the inhibition of the ascending reticular activating system attenuates the orientation response. It is hard to say at this moment whether all the components of the orientation response are attenuated, but the motor components are notably inhibited. Thus, in relation to the orientation response and the spontaneous EEG under the effect of anticholinergic drugs, we obtain a correlation showing that the same reticular mechanisms participate in both cases. But behavioural changes are not only determined by the stimulation or blocking of the ascending activating system. Behaviour is largely influenced by activity changes of the limbic, neuroendocrine, vegetative and other systems under the effect of neurotropic substances. Thus, a correlation should be found between the various Rriferenirs p . 146-148

I44

R. Y U . I L Y U T C H E N O K

behavioural components, together with other electrical changes, having non-reticular mechanisms ; for example, the correlation with changes of electrical activity in the limbic system. Further investigations will possibly establish the presence of correlations between various behavioural components and other electrical physiological phenomena (neuronal activity, impulse transmission to different systems etc.). SOME NEUROC’HEMICAL M E C H A N I S M S A N D B E H A V I O U R A L C H A N G E S

Most or the existing hypotheses on neurohumoral behavioural mechanisms are based mainly on changes in food and defence conditioned reflexes under the effect of adrenergic and cholinergic drugs. Data on the changes in conditioned reflexes under the effects of these agents cannot serve as proof that defensive responses are adrenergic or that food responses are cholinergic in nature. All the drugs stimulating the chemoreactive brain structures in low doses intensify conditioned responses. Large doses inhibit conditioned reflex activity and depression of food-, as well as defencen-coditioned responses is observed. In intermediate doses, agents stimulating cholinergic and adrenergic brain structures raise the conditioned reflexes but do not modify the EEG or behaviour. It may be presumed that either the functional state of the brain cortex alone is changed, or that the sensitivity of the methods of registering the activity of subcortical structures is inadequate when compared to the sensitivity of the conditioned reflex method. With a gradual increase of dose, we can observe simultaneously an intensification of conditioned responses and characteristic behavioural changes, and EEG activation. Finally, owing to the effect of high doses of agents stimulating the chemoreactive brain structures, considerable inhibition of conditioned responses, behavioural changes and a marked excitation of the neurones of the brain-stem reticular formation takes place. There is a prolonged EEG activation which is not observed in normal conditions when it is usually short term. Even electrical stimulation of a nerve induces changes for a few seconds, whereas under the effect of large doses of neurotropic agents, EEG activation is maintained for hours. This by itself is enough to cause marked behavioural changes in the animals. Behavioural changes cannot be looked upon as insignificant intensifications or reductions of conditioned responses. They are disruptions of conditioned reflex activity. The brainstem reticular formation i s blocked under the effect of agents inhibiting cholinergic and adrenergic brain structures. A consequence of this partial chemical deafferentation of the cortex is the reduction of the excitability of cortical neurons. At the same time, the association of afferent signals is disturbed, the animals lose contact with their environment and do not react adequately to environmental stimuli. Thus, under the effect of high doses of agents stimulating the cholinoreactive structures, as well as after the use of agents blocking these structures and exerting a strong influence on reticular mechanisms, we observe very similar changes in conditioned responses with sharply distinct EEG patterns. Behavioural changes will also be different because they depend not only on reticular mechanisms but also upon the mechanisms of activity changes in other functional systems of the brain.

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145

One cannot as yet the judge role of one or tis other humoral mechanisms in complex emotional responses on the basis only of the changes of simple conditioned responses and the EEG. It is necessary to study the role of different chemoreactive structures and the influence exerted upon them by pharmacological agents in different emotional states. At the same time, various alterations of behavioural patterns under the effect of cholinergic agents are not in themselves proof of cholinergic mechanisms. These alterations could be the result of activity changes in any other functional brain system which is not included in the intimate mechanism of the given pattern of behaviour. It may be only reflected action, realized through a wide interrelationship, between the different brain functional systems. A more convincing proof is an isolated change of one of the brain functions, although it is quite difficult to obtain such an isolated effect. It was during the study of fear emotional reaction that a precise difference in the change of this reaction under the effect of drugs blocking the adrenoreactive and cholinoreactive brain structures was observed. The above experiments have shown that antiadrenergic agents do not block the fear reaction in isolation, whereas anticholinergic drugs produce an isolated blockade of this reaction. Such a blockade of the fear reaction by anticholinergic drugs and an intensification of it by anticholinesterases led us to suggest a hypothesis concerning the cholinergic mechanism of this reaction. What are the brain structures to which the chemical differentiation of different biological reactions are related? Some investigators attach great importance to the brainstem reticular formation in mechanisms of defensive reactions14.42. Undoubtedly the reticular formation plays an important role in the mechanisms of emotional behaviour. The effect of neurotropic drugs on the reticular formation cannot explain the emotional reaction as a whole, it can only account for some behavioural components. It seems that the primary area of the c.n.s. involved in emotional behaviour is mainly the limbic ~ystern~~~~23J6.53, whereas at the level of the reticular formation and hypothalamus only primitively organized, relatively indifferent, types of emotions (in the sense of Bradyls) can be formed. It would seem that hypothalamic structures are mainly responsible for the somatic-vegetative components of emotional reactions. It can be deduced, therefore, that the effect of pharmacological agents in altering emotional defensive reactions is related to the mechanisms of the limbic system. Unfortunately, the mechanism of cholinergic drug action upon the limbic system is not quite clear as yet, except for the fact that these substances, besides evoking EEG activation in the cortex and mesencephalic reticular formation, also produce marked EEG changes in the archaecortex and paleo~ortex2~*J3~6~. The presence of cholinergic neurones has been demonstrated by direct registration of neuronal activity in the septum and hippocampus under the effect of eserine or nicotine13~49,52.62~63. But it is still unknown whether the limbic system includes muscarinic neurons. As indicated above, an isolated inhibition of the emotional fear reaction is possible only under the effect of muscarinic anticholinergic drugs. That is why it is important to determine the presence of muscarinic cholinoreactivse tructures in the limbic system. References p . 146-148

I46

R. Y U . I L Y U T C H E N O K

I n the experiments performed in our laboratory by Yu. Ph. Pastukhoff a n intensification of hippocampal spike activity has been shown when muscarinic cholinergic structures were stimulated byarecoline. It is important to note that previous administraation of drugs blocking the central nicotinic cholinergic structures (gangleron) had no effect on spike activity of the hippocampal neurones following subsequent arecoline or galanthamine administration. The presence of muscarinic neurones in the limbic system and the changes in their activity under the effect of amysil or bensazine confirm our hypothesis concerning the important role of niuscarinic cholinergic structures of this system in mechanisms of emotional behaviour and memory. It is possible that, not only behaviour in general, but also, various patterns of emotional behaviour have different neurochemical mechanisms. However, further investigation of the mechanisms of action of these drugs and, consequently, of the role of cholinergic structures in mechanisms of emotional reaction, should be centred in the first place not on the study of their effect on brain regions and nuclei, but on definite functional brain systems. Analysis of data on the ascending reticular activating system allowed us to suggest the hypothesis3lP3Jthat each functional brain system has a characteristic set of mediators. Possibly it is not the anatomical structures that possess chemical specificity. We assume that there are functional brain systems which include functionally integrated neurones of different anatomic structures.

REFERENCES ADEY,W. K. (1959) Intern. Rev. of Nrurobiology, 1, 1-44. ALLIKMETS, L. KH. (1964) Zhurn. Nevropatologii i Psikhiutrii, 61, 1241-1248. ALLIKMETS, L. KH. (1964) Uchenyye zapiski Tartusskogo Cos. Uiziv., Tartu, vip. 163, 123-127. ANOKHIN, P. K . ( I 958) Vnutrenneye rormozhenie kak probleiiia fiziologii, Moskva, Medgiz. ANOKHIN, P. K . (I958) Elektroeticepplialograficheskiy analiz uslovnogo refleksa, Moskva, Megdiz. ARNOLD.M. R. (1945) Physiol. Rev., 52, 35-48. BERITOV,I . S. (1961) Nervnyye mekhanismi povetler.i,ya vi.rshikh pozvoizochnykh zhivotnykli. Moskva, izd-vo AN SSSR. 8 BOROUKLN, Yu. S. (1965) Material; 1 pribaltiyskoiy konferentsii 7kNIL’ov Metl. institiitov i fakul’tetov, Kauns. 50-52. 9 BOWARD, E. W. (1962) Perspect. in Biol. M e d ? 6, 116-127. 10 BRADLEY, P. B. A N D J . ELKES,(1957) Brain, 80, 77-1 17. 1 I BRADLEY, P. B. AND A. J. HANCE,EEC Cliri. Neurophysiol., 1957, 9, 191-215. 12 BRADLEY, P. B. A N D B. J. KEY,(1958) EEC Clin. Neurophysiol., 10, 97-110. 13 BRADLEY, P. B. AND A . N. NICHOLSON, (1962) EEC Clin.Neurophysiol., 14, 824. 14 BRADLEY, P. B. AND J. H. WOLSTENCROFT (1965) Brit. Med. Uull., 21, 15-18. 15 BRADY,J. V. (1958) In: Biologicaland Riocheniical Bases of Behaviour. Ed. by H. F. Harlow and C. N. Woolsey. The University of Wisconsin Press, 193~-235. 16 BUR&, J., 0.BURESOVA, Z. BOHDANECKq AND T. WEIS (1964) Ciba Foundation SyiTipObiUni jointly with the Co-ordinating Committee foi Syrnposis on Animal Behaviour and Drug action, 1 2 3 4 5 6 7

134.142. 17 COURVILLE, J., J . WALSH,A N I ) J. P. CCRDEAU, (1962) Science, 138, 973--974. 18 DELGADO, J . M. R. (1965) In: Pharmacology of Cmditioning, Learning and Retention. Oxford-

London-Edinburg-New-York-Paris-Frankfurt, Pergamon Press, Praha, Czechoslovak Medical Press, 133-156. 19 YELSEYEVA, A. C . (1965) V sb.: “Voprosi eksperimental’tzoy psikhiatrii”, Novosibirsk, 69-70.

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EXLEY, K. A., M. C. FLEMING, A N D A. D ESPELtEN, (1958) Brit. J. Pharmacol. Chem., 13,485-492. FINK,M. (1960) EEG Clin. Neurophysiol., 12, 359-369. GARATTINI, s. AND I*. VALZELLI,(1965) serotonin, Elsevier, Amsterdam. GREEN,J. D. AND A. ARDUINI(1954) J. Neurophysiol., 17, 533. HERNANDEZ-PEON, R., G. CHAVEZ-IBARRA, P. J . MORGANE AND C. TIMO-IARIA (1963) Exp. Neurol., 8, 93-1 1 1. 25 HIEBEL, G., M. BONVALLET A N D P. DELL,(1954) Sernaine HGpit., Paris, 30, 2346. 26 HIMWICH, H. E., A . MORILLO AND W. G. STEINER (1962) J . Neuropsychiat., 3, suppl. I, 15-26. R. Yu. (1957) Zhurnal vysshey nervnoy deyarel’nosti, 7,2, 254-262. 27 ILYUTCHENOK, R. Yu. ( I 962) In : First International pharmacological meeting “Mode ofaction of 28 ILYUTCHENOK, drugs”. Vol. 8 : Pharmacological analysis of central nervous action. Oxford-London- New-YorkParis, Pergamon Press, 21 1-216. 29 ILYUTCHENOK, R. Yu. (1963) In: Biochemical pharmacology. Prague, Pergamon Przss, suppl. to 12, 270. 30 ILYUTCHENOK, R. Yu. (1961) Psychopharmacological Methods, Symp. Efect Psychotr. Drugs, Prague, Pergamon Press, 115-1 22. R. Yu. (1965) Neyro-gumoral’nye mekhanizmi reticulyarnoy formazii stvola mozga, 31 ILYUTCHENOK, Moskva, izd-vo Nauka. 32 ILYUTCHENOK, R. Yu. (1965) Clinical neurophysiology. EEG-EMG. 6th International Congress of electroencephalography and clinical neurophysiology, Vienna, 5 13-5 1 5. 33 ILYUTCHENOK, R. Yu. (1965) V sb. : Voprosi eksperimentalnoy psykhiatrii. Novosibirsk, 66-68. 34 ILYUTCHENOK, R. Yu. A N D A. G. YELISEYEVA (1966) X v I I l Infern. Psychol. Congress, Biological bases of memory traces, Moscow, pp. 62-64. 35 ILYUTCHENOK, R. Yu. AND M. D. MASHKOVESKIY (1961) Fiziologicheskiy zhur. SSSR, 47, 13521359. 36 ILYUTCHENOK, R.Yu. AND L. N. NESTERENKO (1965) Fiziol. zhuriz. SSSR, 51, 1177-1181. 37 JACOBSEN, E. (1964) In: Psychopharinacological agents. New-York, Academic Press Tnc., pp. 287-300. 38 JUNG,R. (1958) Klin. Wochenschri/t., 36, 1153. 39 KANAI,T. A N D J. C. SZERB(1965) Nature, 205, 4966, 80-82. 40 KRNJEVIC, K. ( I 964) Neuro-Psychopharmacol., 3, 260-264. 41 LABORIT, H.,C. BARONA N D B. WEBER (1965) Agressologie, 6, 655-720. 42 LINDSLEY, D. B. (1951) In: Handbook ofexperimentalpsychology.Ed. S. S. Stevens, New-York J. Wiley & Sons, pp. 473-516. 43 LONGO,V. G. (1962) EEG atlas for pharmacological research. Elsevier, Amsterdam. 44 LONGO,V. G. AND B. SILVESTRINI (1957) J. Pharmacol. Exp. Ther., 120, 160-170. H. W. (1958) The waking brain, Springfield, Illinois, Charles C. Thomas Pub],. 45 MAGOUN, 46 MASHKOVSKIY, M. D. AND R. Yu. ILYUTCHENOK (1961) Zhurn. Nevropath. iPsikhiat.,61, vip. 2, I 66-1 73. 47 MIKHELSON, M. YA. ( I 957) Fiziologicheskaya rol’ atsetilkholina i iziskanye novykh 1ekar.rtvennykh veshchestv, Leningrad, izd-vo Len. rned. ins-ta. 48 MITSKENE, V. P. AND A. M. MITSKIS (1965) Fiziol. zhurit. SSSR, 51, 544-546. M. AND W. ROMANOWSKI (1962) EEG Cliiz. Neurupkysiol., 14, 486-500. 49 MONNIER, 50 NAUMENKO, YE. V. (1965) Problemi endokrinologii i gormonoterapii, 4, 99-104. 51 NAUMENKO, YE. V. AND R. Yu. ILYUTCHENOK (1964) Pharmacol. i toksikol., 6, 670-672. 52 PETSCHE, H. AND CH. STUMPF(1962) Physiologie de I’Hippocampe. Colloq. Int. du CNRS, Paris, 107, 121-141. 53 PRIBRAM, K. H., W. A. WILSON,JR. AND J. CONNORS (1962) Exp. Neurol., 6 , 3 6 4 7 . (1 955) Diseases of the Nervous System, 10, 133- I4 1 . 54 RINALDI,F. AND H. HIMWICH 55 ROTHBALLER, A. B. (1956) EEG Clin. Neurophysiol., 8, 603-621. (1964) Science, 144, 3618, 493-499. 56 SALMOIRAGHI, G. C. AND F. E. BLOOM 57 SMIRNOV, G. D. AND R. J. ILYUTCHENOK (1962) Fiziol. Zhurn. SSSR, 48, 1141-1145. 58 SOKOLOV, TE. N. (1958) Vospriyatiye i uslovniy rejeks, Moskva, izd-vo Mosk. Univ., 59 SOLOMON, P. AND WYNNE (1950) Amer. Psychologist., 5, 264. R. (1963) J. Neurophysiol., 26, 127-139. 60 SPEHLMANN, 61 STEINER, W. G. AND H. E. HIMWICH(1962) Science, 136, 3519, 873-875. 62 STUMPF, CH. (1 964) In: Neuropsychopharmacology, Amsterdam, Elsevier, 241-244. 63 STUMPF, CH., H. PETSCHE AND G. GOGOLAK 1962, EEG Clin. Neurophysiol., 14, 212-219. 20 21 22 23 24

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64 VOTAVA, Z., 0. BENESOVA, Z. BOHDANESK+, J. METYSA N D J . METYSOVA (1964) Poutery Hygieizyi Medycyny Do3 wiadczalnej, 18, 925-943. 65 VOTAVA,Z. AND M. V A N I ~ E(1956) K Physiol. Boheino>loven., 5, 460-467. R. P. AND L. D. BOYAJY (1959) Proc. Soc. Exptl. Biol. Med., 102, 479-483. 66 WHITE, 67 WHITE,R. P. AND E. A. DAICNEAULT (1959) J . Pharni. Exptl. Ther., 125, 339-346.

149

EEG and Behavioral Aspects of the Interaction of Anticholinergic Hallucinogens with Centrally Active Corn pounds T. I T I L

AND

M. F I N K *

Departmeut of Psyc’liairy at the Missouri lnvtiiute of Ps.vchiatr.v, ffniver.rii.v of Mi~soiiriSchool o f Merliciize, 5400 Arsenal Street, St. Lwis, Missouri 63139 (U.S.A.)

The changes in EEG and behavior after administration of anticholinergic drugs have been an intriguing problem for mor? than two decades. The EEG alterations induced by atropine, scopolamine, and a wide range of experimental compounds have been difficult to describe and the conclusions have been controversial. Although some investigators could detect but little change in the EEG after giving anticholinergic d r ~ g s ~ , ~most ~ J *agreed , that anticholinergics, especially those with psychotomimetic effects, do produce systematic EEG changes in animals and in man. Both increase of fast activity with desynchronization7,10~21and the increase of slow wave activity with synchronization have been describedspl3J4Js. But most interesting have been the observations of sleeplike patterns with high voltage slow waves and spindle activity in animals3~12,27,~*~35~36 and in man11g25. The discrepancy between sleeplike EEG patterns and apparent waking behavior of animals led to the term “dissociation of EEG and behavior” which has bem postulated to be a special feature of cholinergic mechanisms of the central nervous system. Clinical and EEG correlations after anticholinergic drugs as well as the differences between the anticholinergic-induced sleeplike state and natural sleep have been described in man in earlier reports11,16~17~1x,1g. To provide additional information concerning anticholinergic-induced EEG and behavioral changes, the present investigation was designed to study the interaction of Ditrant and atropine with a variety of centrally active agents, using quantitative methods for the measurement of the EEG changes.

-

* Present Address: Department of Psychiatry, New York Medical College. Aided, in part, by MH-I 1380 and the Psychiatric Research Foundation of Missouri. 7 Ditran (N-ethyl-3-piperidyl-cyclopentyl-phenyl-glycolate-hydrochloride) supplied by Lakeside Labs., Milwaukee. References p . 166-168

I so

T. I T I L A N D M. F I N K MATERIAL AND METHODS

A total of 291 investigations were carried out i n 84 male and female subjects between 17 and 57 years of age. Sixty-five were classified as schizophrenic ctates and nineteen as affective, emotional or personality disorders. For at least two months prior to the acute investigation, these subjects had received no psychotropic medication. The dosages, rate of administration and number of investigations for Ditran and atropine are shown i n Table 1. Second compounds were administered intravenously 30 to 40 min after the anticholinergic drugs. Eye movements, electrocardiogram, electromyogram and pulse rate were recorded simultaneously with the EEG and continuously up to one hour after each drug. Blood pressure was measured for periods of up to three hours. TABLE I COMPOUNDS, D O S A G b \ AND NUMBER O F T R I A L S

_

_

~

~

~

t h a g e (nrglkg) --

-~

Fin/ clriig Ditran Atropine

~

.~

-

Tittle of Adminiwatiotr

( m ~r. .v.)

_

_

Number of Trialr

0.0054.30 0.04 -0.50

5

171

5

77

0.02 -0.25 0.01 -0.50 1-2 llgglkg 0.15 -0.30 0.10 -0.30 0.02 -0.05 0.5 -1.5

10 5 5 5

53 10

10

4 5

Secnird or third tirug

Chlorpromazine Yohirnbine LSD-25 Amphetamine sulfate lmipramine Neostigmine methylsulfate Tetrahydroaminoacridin (THA)

10-20 10-20

5 4

41

EEG records (right occipital to ear or right occipital to right frontal leads) were analyzed quantitatively using an electronic resonant-filter frequency analyzer. The electronic frequency analysis (power spectral density analysis) consisted of the measurement of the mean pen deflection in millimeters in each of 24 frequency bands from 3 to 33 cjsec for each epoch of 10 sec. Six artefact-free samples, each of 10-sec duration, were selected from the record before the iiijection of the anticholinergic drug and between 25 to 35 min after each injection. In several investigations, the EEG records were analyzed by digital computer methods using both period analytic and powcr spectral density methods. The programs used were those developed for the TBM 1710 system, using digitizing rates of 320 samples per second and epoch lengths of 10 to 30 sec. Behavior ratings were done before and 30 to 40 min after each injection, using our psychopathological rating scale16 of 95 single items divided into 15 symptom clusters. ln addition to the behavior rating scale, psychological tests such as the BenderGestalt, carpet sign test, drawing and neurological examinations were carried out.

_

References p. 166-168

CHANGES IN

EEG A N D BEHAVIOR

0

W

Fig. 1. Low voltage slow EEG after Ditran administration

152

T. I T J L A N D M. FINK

RESULTS

Efiect of anticholinergic drugs The EEG and behavioral effects of atropine and Ditran varied with the dose given and could be classified into three distinct patterns. Relatively low doses of Ditran (0.005-0.05 mg/kg) or atropine (0.04-0.30 mg/kg) produced changes of consciousness and a drowsy state. At the same time, subjects exhibited restlessness, sometimes with agitation and poor coordination. Affective and emotional changes of anxiety, fear, depression or euphoria were frequent. Perceptual disturbances and visual hallucinations were occasionally reported. These clinical alterations were associated with a decrease in alpha-activity and increases in low voltage 5-7 cjsec theta-activity and superimposed fast beta-waves in the EEG (Fig. 1). In comparison to Ditran, atropine induced more slow waves and less superimposed fast activity (Fig. 2). These EEG patterns showed some similarity to the drowsiness or low voltage natural sleep EEG pattern (stage B, Loomis et or stage 1, Dement and Kleitmang).

RO RE

:

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RF - R E

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w

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inno

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yser

-

-

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-

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-

-

!

-

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13 mins after Atropme I v. ( 0 3 r r g / k g )

H T Age 48 Record N o 4176

Behavior

drowsy, restless and ogllated

-schizophren o

Diagnoe

I

Fig. 2. Low voltage slow EEG after atropine administration.

a

C H A N G E S IN

EEG

153

A N D BEHAVIOR

RO - RE N

-

L

E

L

R F -RE

EOM

EMG

/ /

B e f o r e Drug

5

O

~

k

24mins after Atropine i v (0.3rng /kg)

Behavior stupor-like state with restlessness H T Age 48 Record No 4:76 Diagnosis schlzophrenx

Fig. 3. Slow wave spindle pattern after atropine administration. frequency onolyser chonnei

RO - RE ,

$

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,

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23mins: a f t e r Ditran i.v. (0.3mg./kg.) Behavior : stupor. like s1o t e , psyc homo tor

H

Age 48 Record No 4374 Diagnosis schizophrenia T

Fig. 4. Slow wave spindle pattern after Ditran administration, References p. 166-168

agitation

154

T. I T I L A N D M. F I N K

With the highest doses of Ditran (0.04-0.30 mg/kg) or atropine (0.25-0.50 mg/kg) in some subjects, states of deep slecp and stupor developed and persistcd for sevei-a1 hours. They exhibited marked changes i n consciousness and impaired orientation. Communication was difficult, memory function was impaired and psychomotor activity was decreased. Accompanying these behavioral changes were markcd alterations i n EEG patterns. After atropine, alpha-activity decreased and 12-14 cjsec spindle activity dominated (Fig. 3). I n contrast to atropine, Ditran produced in the same subject less slow wave activity, more superimposed fast beta-activity and less spindle activity (Fig. 4). Although these EEG patterns are similar to the deep sleep pattern of natural sleep (stages C and D, Loomis et ~ 1 . ~ 3or 3 ; stages 2 and 3, Dement and Kleitmang), a study of the two states in the same patient showed significant differences. Sleep-like patterns induced by anticholinergic drugs are characterized by less spindle activity and more superimposed fast activity than natural sleep (Fig. 5). Also, during anticholinergic sleep-like activity, the EEG responses to acoustic stimulation were inhibited or abolished. Patients could open their eyes when told to do so, but no significant changes i n the EEG were seen (Fig. 6). With thc administration of Ditran in doses bctween 0.04 and 0.25 mg/kg or with atropine between 0.20 and 0.40 mg/kg i n most subjects, states of confusion and delirium

iye movement

--

-

27rnins after 136rng Ditran

I V

(behavior. stupor, sleep-like state with restlessness and confusion)

Natural Sleep

50uv

(behavior: deep sleep ) NN

Aqe 27

Dx S c h zophrenia Record Nos 1368, 1418

Fig. 5. Comparative EEG changes between Ditran-induced sleep-like pattern and natuml sleep in the same patient.

CHANGES I N

EEG

155

AND BEHAVIOR

were observed. Consciousness fluctuated and thought, association, memory and communication were disturbed. Subjects often reported visual hallucinatory phenomena. Motor activity, restlessness and athetoid movements were seen. Heart rate was

RO

Pre Drug Resting E E G

LF

RF

50 .uv

q

w

y 30rnins. after 13.6mg. Ditran

d I.V.

(0.21rng /kg.) N.N

Age 27

Dx.: Schizophrenio Record No.: 1368

Fig. 6. Response on eyes opening before and after Ditran administration. References p. 166-168

T. I T l L A N D M. F I N K

156 R

o

w

Re Drug Resting EEG

X

I

p

m

2 5 m i m after 1.4mg Ditran i.v.

i.,. I ,

+-

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WE DRUG

24mIns. after 1.4mg. Ditran i.v,

M M A o . 42 Dr Schizophralno RbCOrd No I432

Fig. 7. The number of “carpet signs” in 60 sec pre- and post-Ditran administration in relation to the EEG alterations.

Eye mavement

EMG

V

-Ivc-_

3

LF frequency onoiyzer

+

RF

R

0

F

-

w

Resting EEG

50,~-

IBmins after 68mg Ditron I v (behovior confusional - delirious state)

NN

Age 27

Diagnosis. Schizaphrenm Record No 1480

Fig. 8. Ditran-induced (0.10 mg/kg) disorganization of the EEG.

CHANGES IN

EEG

157

A N D BEHAVIOR

Before Difron

.- - - - - - - - 1 5 - 2 5

I1210O l

Dilron

100 -

-i:

90

mins after I v , 2 4 m g ovg

Recurd Nos 26?2,1818. 1246,560, 2699, 580. 638,920, 2545, 2637,1286

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11'0 I I 4 5 I I $ ? I 2b0 I 2;s I 3 6 0 I 120 150 180 220 270 330

FHEPUENCIES

Fig. 9. Changes in EEG frequency spectra with Ditran administration in patients with delirium. (Average of 1 1 investigations.)

increased. The skin became dry, pupils dilated and the subjects complained of difficulty in speech and in near vision. Performance on the Bender-Gestalt drawing tests was impaired and the number of figures completed i n a symbol reproduction task was severely reduced (Fig. 7). The increase in psychomotor activity with hallucinations was more pronounced after Ditran while the changes in consciousness were dominant after atropine. In association with these behavioral changes after Ditran, the electroencephalogram exhibited a reduction of alpha-activity and the appearance of high voltage delta- and theta-waves with superimposed 20-40 clsec desynchronized fast betaactivity (Fig. 8). Epileptic activity in the form of high voltage spikes and sharp waves was occasionally observed. In a group of subjects with induced delirium, the frequency analyzer data showed decreases in alpha-activity and increases in slow wave activity (Fig. 9). The increase in superimposed fast beta-activity was poorly reflected i n the analog power spectral density analysis but was evident on visual inspection and in the digital computer period analytic methods (Fig. 10).

Refrrmrrs p. 166-168

158

T. I T I L A N D M. F I N K

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MINUTES DITRAN = 3.7mq / 5 m i n s . THA = 6 0 m g / 9mins Leads = R FRONTAL- OCCIPITAL

-

Digitized 3 2 0 S P S DlTRAN SERIES MIP - 7 / 6 4 Fleming - 00 I

Fig. 10. Period analysis. First derivative - percent time.

Ititeruction of anticholinergic drugs with oilier compounds The relation of the EEG patterns to changes in behavior are more clearly defined in the changes induced by the subsequent administration of centrally active drugs to patients during experimental deliria. When intravenous chlorpromazine was given in very small doses (0.02-0.25 mg/kg) 30 to 40 min after Ditran or atropine, the psychomotor aspects of the anticholinergicinduced delirium were reduced and the alterations i n consciousness intensified. The subjects exhibited stupor or coma with a lack of response to acoustic and painful stimuli. Pupils were dilated, deep tendon reflexes diminished, respiration became more shallow and heart rate slowed. Associated with these behavioral changes were further alterations of anticholinergic-

CHANGES IN

22mins a f t e r Chlorpromozine i v (05mg/kg/ 159n11nsa f t e r D i t r o n )

...................................................

.

Behavior: __

EEG

AND BEHAVIOR

I59

sub-coma

H I Age 4 2 Record No. 5 2 0 Diagnosis. !chizophrenia

50-

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FAEOUENCIES

Fig. 11. Changes in EEG frequency spectra with Ditran and following the addition of chlor promazine.

induced EEG changes with increases in amplitude, marked enhancement of slow wave activity, decreases of fast beta-waves and the predominance of 8 to 12 cjsec spindle patterns (Fig. 11). The response to acoustic stimulation, which could not be evoked prior to chlorpromazine administration, reappeared i n the EEG. After yohimbine (0.0 1-0.50 mgjkg) the changes in consciousness induced by Ditran were lessened. Patients became alert and their speech more relevant, but motor restlessness and irritability persisted or even increased. Perceptual distortions were not altered. In the EEG, the slow delta-activity decreased and theta-activity increased. In some instances, beta-activity also increased and some alpha-waves recurred (Fig. 12). With LSD 25 (1-2 pgikg), the Ditran-induced changes in consciousness decreased and psychomotor activity and hallucinatory phenomena increased. Patients became more alert and communicated more easily. In the EEG, Ditran-induced slow wave activity was reduced, amplitudes decreased and fast activity (over 22 cjsec) was potentiated (Fig. 13). With dextro-amphetamine (0.15-0.30 mg/kg), the Ditran-induced changes of consciousness and psychomotor activity both decreased slightly. Patients became alert and responsive, and less irritable. There was a flattening in the amplitude of all frequencies in the EEG with a decrease in theta, alpha and beta activity (Fig. 14). References p . 166-168

T. I T I L A N D M. F I N K

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FREOUENCIES

Fig. 12. Changes in t E G frequency spectra with Ditran and following the addition of Yohirnbine.

Imipramine (0.10-0.30 mg/kg) given intravenously 30 to 40 min after Ditran reduced the delirium in some subjects. While thought disorder and hallucinatory phenomena were still prominent, the subjects were more alert, better oriented and more responsive. Psychomotor activity persisted. In some subjects, the EEG exhibited a decrease of slow wave activity, a marked increase of alpha-waves and a persistence of beta-activity (Fig. 15). Intravenous neostigmine (0.02-0.05 mg/kg) did not significantly alter the Ditraninduced psychotic state. The changes in consciousness seemed less and psychomotor activity and agitation were occasionally increased. These central changes were masked by the severe peripheral gastro-intestinal and vascular symptoms. In the EEG there were no significant changes (Fig. 16). Tetrahydroaminacrin (THA) (0.5-1.5 mg/kg) blocked almost completely the Ditran-induced confusional-delirious state. Vigilance was restored, motor restlessness and agitation diminished, the disturbances i n thought and association ameliorated and hallucinatory phenomena were inhibited. In the EEG also, the Ditran-induced changes were reduced after TH A. Slow activity decreased markedly, and fast a-activity recurred (Fig. 17).

CHANGES I N

EEG

AND

BEHAVIOR

Resting EEG

50,uv

161

1"'".

____________.

15mins. acter Ditran i.v. (4.8mg.l

15 rnins. after LSD-25 i.v. (0.2mg.)

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80

100

120

150

180

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270

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FREQUENCIES

Fig. 13. Changes in EEG frequency spectra with Ditran and following the addition of LSD-25.

DISCUSSION

The behavioral and EEG changes induced by Ditran and atropine depend on the dosage and the pre-drug resting EEG17. Quantitative analyses have shown that the administration of both these anticholinergic drugs is associated not only with an increase in slow wave activity, but also with a concurrent increase of superimposed fast beta-activity. The ratio of slow activity to fast activity is related to the type of clinical syndrome observedlg. When alpha-activity was replaced by 2 to 5 c/sec slow waves and 30 to 50 c/sec fast activity, a confusional-delirious state with increased psychomotor activity occurred. The appearance of 1 to 4 c/sec slow waves and 12 to 18 c/sec fast activity was associated with a stuporous state, characterized by severe changes in consciousness, but less psychomotor activity than in the delirious state. Compared to atropine, Ditran induced greater increases in fast activity but fewer slow waves. This difference was clinically related to greater psychomotor activity but lesser degrees of stupor. References p . 166-158

162

T.ITIL AND M.FINK

/ -

Before Drug

50pv 14mins. a f t e r D i t r a n i v ~ 0 0 5 m p / k g l --'p'q p. Behavior. confusional delirious state

14 mins after Dexlrc- Amphelamine iv ( (43mlns after Ditranl

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FREOUENCIES

Fig. 14. Changes in EEG frequency spectra with Ditran and following the addition of dextroamphetamine.

The relationship between changes in behavior and their EEG correlates became even more distinct when the syndrome induced by anticholinergic drugs was modified by the subsequent administration of various adrenergic or cholinergic blocking or sensitizing agents. Chlorpromazine, given after anticholinergic drugs, even in very low doses (1 to 2 mg), altered the anticholinergic-induced syndrome with extreme inhibition of psychomotor activity and a further decrease in the level of consciousness. Delirium was converted to a state of coma. The EEG counterpart of this state was a marked potentiation of slow wave activity. A similar potentiation of anticholinergic (atropine) induced slow waves by chlorpromazine was observed in animals by Bradley and Hance4, but they reported no behavioral association. Various degrees of alerting occurred with the administration of several compounds subsequent to anticholinergic drugs. Alerting was associated with decreased psychomotor activity after amphetamine, yohimbine and imipramine, increased hallucinatory phenomena and motor activity after LSD, and almost complete blocking of hallucinations as well as motor activity after THA. These behavioral alterations were accompanied mainly by decreases in slow and increases in fast wave activities. After neostigmine, neither behavioral nor EEG changes were observed.

~

~

CHANGES IN

EEG

163

A N D BEHAVIOR

PRE E E G 50pv

38mins after lmipromine i v 2 0 m g ( 7 9 m i n s a f t e r Ditran)

.. .. ... ...................

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C.B. Age:39 Rec No 1636 Dx.: Personolily Disorder

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FREQUENCIES

Fig. 15. Changes in EEG frequency spectra with Ditran and following the addition of imipramine.

These findings agree only in part with the earlier investigations in animals2Jp5. They do not support the suggestion that the correlation of EEG and behavior changes after cholinergic potentiating and blocking agents is poor in contrast to the relation of EEG and behavior changes after adrenergic sensitizing and blocking agents29. Our results do confirm, however, the reports which emphasize the differences between cholinergic and adrenergic activating me~hanisrns~5.~4,~~.~8.29. These investigations suggest that anticholinergic drugs, particularly those with psychotomimetic properties, exhibit a dual activity. They appear to stimulate two nonspecific subcortical mechanisms with opposite functions. It is possible that the concurrent activation of the medial ascending reticular activating system which exercises mainly cortical inhibition (and is associated with slow activity in the EEG), and the medial thalamic diffuse projection system, which has predominantly a facilitating influence on the cortex (and is associated with fast activity in the EEG), causes confusional delirious behavior. After low doses of anticholinergic drugs, central excitatory mechanisms appear to predominate, while after high doses, inhibitory functions seem to take precedence. In the modification of the anticholinergic syndrome by other compounds, the kind and speed of the functional alterations of these mechaD o & ~ o n , ~ on c

I66-IAR

T. I T I L A N D M. F I N K

164

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..............................

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...........

"

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A -

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