Naunyn-Sehmiedebergs Arch. exp. Path. u. Pharmak. 248, 406--416 (1964)

~'rom the Departments of Pharmacology and Psychiatry, Harvard Medical School Boston, Massachusetts

Antagonism between Physostigmine and Atropine on the Behavior of the Pigeon*

By GEORGE ]~. VAILLANT **

With 5 Figures in the Text

(Received December 27, 1963)

Although for many years a significant role has been postulated for acetylcholine in the central nervous system, its function in integrated nervous activity remains obscure Relatively few studies using quantita- t ive behavioral techniques have investigated the effects of acetylcho- line-like componds on the central nervous system. FUNDERBURK and CASE (1947)found tha t in cats 0 .25mg/kg of physostigmine would abolish a conditioned avoidance response, but tha t if the cats were pretreated with 1.2 mg/kg atropine, the responding was not affected. However, in their investigation only one dose of atropine was used, and no effort was made to control for the profound peripheral effects produced by physostigmine. PFEIFFER and JE~NE:Z (1957) observed rats tha t had been trained to avoid a shock by climbing an insulated pole at the sound of a buzzer preceding the shock. They found 0.25 mg/kg of physostigmine abolished avoidance responding and tha t pre t reatment with 5 mg/kg atropine restored it. Doses of physostigmine tha t affected avoidance, however, also suppressed escape from the actual shock. Methylatropine 10 mg/kg did not protect against suppression of avoid- ance responding by physostigmine, a ter t iary amine, but did protect against suppression by 50 mg/kg methacholine, a quaternary amine. Methacholine in doses as small as 0.5 mg/kg produced obvious peripheral muscarinic effects.

Recently STARK and BOYD (1963) reported tha t when dogs with chronically implanted hypothalamic electrodes were given 0.1 mg/kg of physostigmine, a marked decrease in the rate of electrical self-stimula- t ion occurred but tha t 0.025 mg/kg physostigmine and 0.05 mg/kg neostigmine did not alter the response rate from control levels. 0.2 mg/kg

* Dedicated to Professor OTTO KRAYER On his 65th birthday. ** Thus study was supported by grants MH-02645, MH-02094 and 2M-7084

from the United States Public Health Service.

Antagonism between physostigmine and atropine 407

o f a t rop ine p ro t ec t ed the dogs f rom the physos t igmine effect b u t 0.2 m g / k g of m e t h y l a t r o p i n e d id not .

W i t h o u t knowledge of the effects of t he drugs a t o ther doses, i t is difficult to d r aw rel iable conclusions f rom the above exper iments , b u t t he suggest ion is c lear t h a t behav io r m a y be suppressed b y drugs ac t ing on cen t ra l receptors of a muscar ine - type . We have found t h a t p re t r ea t - m e n t wi th abou t 0.25 mg/kg physos t igmine p roduced a b r u p t cessat ion of food-ma in t a ined ope ran t behav ior of pigeons. The presen t r epor t is of a sys t emat i c s t u d y of some of the pharmacolog ica l p roper t ies of th is suppress ion of behavior .

The charac te r i za t ion of a d rug an tagon i sm usua l ly requires s t u d y of the effects of the agonis t and an tagon i s t over dosage ranges cover ing severa l orders of magn i tude . This is o rd ina r i ly impossible in behav io ra l s tudies. Ev idence of specif ic i ty m u s t therefore be sought a long different lines. I t has been shown r epea t ed ly t h a t different componen t s of a com- p lex p a t t e r n of behav io ra l responding charac te r i s t i ca l ly show grea t ly different sensi t ivi t ies to modif ica t ion b y a drug. I f a complex p a t t e r n of r e spond ing t h a t has been p ro found ly modif ied b y the effect of an agonis t can be re s to red essent ia l ly to no rma l in all i t s aspects b y a suit- able dose of an tagonis t , t hen this is evidence for specif ici ty of the an tagonism. I t is analogous to the f inding t h a t the agonis t is an tagon ized in a v a r i e t y of effects on a v a r i e t y of t issues b y the an t agon i s t ; such findings have been t a k e n in genera l pharmacolog ica l s tudies to be evidence for a pharmacolog ica l an tagonism.

Methods The experiments were conducted on 8 white male Carneaux pigeons maintained

at 800/0 of their ad lib. weights by partial food deprivation for the duration of the experiment. Their weights ranged from 390--520 grams with a mean of 428 grams. An apparatus for operant behavior similar to that previously described was used. (DEws 1955; F~RSTER and SKINNER 1957). The schedule in effect in all experiments was a mult FIFR. Two parameter values for the F I (interval component) were used--FI 180 seconds and FI 720 seconds.

When the FR 30 component of the schedule was in effect, the key was illumi- nated with a blue light and every thirtieth peck on this key resulted in immediate presentation of food. When the F I 180 seconds was in effect, the key light illumina- tion was red and the first peck on the key after 180 seconds (3 minutes) had elapsed resulted in the immediate presentation of food. When the F I 720 seconds was in effect, the key light was yellow, and the first peck after the elapse of 12 minutes produced food. The sequence of schedule components was: FR, FR, FI, FR, FR, FR, FI, FR, FR, FR, FI, FR, FR, FI, after which the sequence was repeated.

A limited hold of 80 seconds was employed. This meant that if, when the blue light was on, 30 pecks were not emitted by the bird in 80 seconds, or if, when the red or yellow light was on, a single peck was not emitted by the bird within 80 seconds after the elapse of the minimum interval, the next programmed schedule component came into effect. The mult F I 180 seconds FR 30 was terminated automatically

408 GEOROE E. VAILLANT:

after either 50 reinforcements or 3,600 seconds (1 hour), which-ever came first. The mult FI 720 seconds FR 30 was terminated after 19 reinforcements or 5,120 seconds (85 minutes). Birds 24, 212, 36, 37 were subjected to a mult FI 180 seconds FR 30 schedule throughout, Birds 239, 222, 150 to mnit FI 720 seconds FR 30 schedule throughout, and Bird 31 to both schedules.

Procedure. Before this series of experiments all of the pigeons had worked on other schedules. During the experimental period lasting 9 months, the birds were subjected to a standard session at the same hour 5--6 times a week. When the pigeon had developed a stable day to day performance, drugs were administred according to the following rules: every session during which a drug was administered was preceded by a normal session the day before. All control sessions were preceded by a normally executed session the day before.

Measurements. The number of responses during the FI and the FR components were recorded separately on digital counters. The total time elapsed during the FR components was recorded on an elapsed time meter. The overall rate for the mult FI 180 seconds FR 30 sessions was calculated on the basis of the maximum time allowed--3600 seconds. Birds could, however, complete the session in a mini- mum of about 3100 seconds. On the mult FI 720 seconds FR 30, the average rate on the FI (yellow) key was again calculated in terms of 3600 seconds, representing five minimum FI 720 seconds although if the bird did not respond the yellow light could be present as long as 4000 seconds during the session. Use of a constant time base served to decrease recorded differences in rate between drug and control sessions but to an insignificant degree.

Brain cholinesterase determinations were measured eleetrometrically by the method described by ]VIaeH~L (1949) for red ceils. The method was modified by substituting 0.02 ml. of brain homogenate diluted by 5 volumes of 0.025 M NattCO a for 0.02 ml packed washed red cells.

Drugs. Atropine sulfate, methyl atropine nitrate, neostignfine methyl sulfate, physostigmine salicylate, scopolamine hydrobromide and nicotine sulfate 1 were dissolved in 0.90/0 saline. Drugs were administered into the breast muscle. Atropine, scopolamine, and methyl atropine were admiTdstered 120 minutes before the mult FI 180 seconds FR 30 sessions and 90 minutes before the mult FI 720 seconds FR 30 sessions. In the antagonism experiments, physostigmine was administered 50 minutes before the mult FI 180 seconds FR 30 sessions and 45 minutes before the FI 720 seconds FR 30 sessions. Nicotine and neostigmine were given immediate- ly before each run. Doses are recorded as the total weight of salt given to each bird.

Results

The mul t F I 180 seconds F R 30 schedules produced pa t te rns of

behavior t h a t conformed with prior work (FERSTER and S K I ~ E R 1957),

(Fig. 1, Control). The bird responded a t a rapid uni form rate while the F i t 30 was in effect. Dur ing the F I 180 seconds the pigeon responded

a t a ra te t h a t was ini t ia l ly low bu t increased as the in te rva l progressed. Such a per formance gives a character is t ic scalloped appearance to the

1 The Drugs used in these studies were kindly given by the following companies: Atropine sulfate, Physostigmine salicylate, and Scopolamine hydrobromide from Merck, Sharp and Dohme Company; Neostigmine methyl sulfate from Hoffman La Roche Incorporated; and Methyl atropine nitrate from Strasenburgh Labora- tories.

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a b c d

Fig. l a - - d . Typica l cumula t ive records of per formance on mul t F I 180 seconds F R 30. :Responses are recorded cumula t ive ly by u p w a r d m o v e m e n t of the recorder pen ; downward deflection of the pen indicates re inforcement . All figures represent the first s ix th of a session (FR, F R , F R , F I , F R . F R , F I , F R , FR) . - - Fig. l a . control session. - - Fig . lc . p igeon p re t rea ted wi th 0.3 ml 0 . 9 % saline 2 hours before run, 0.1 m g physos t igmine 50 minu te s before run. H a t c h m a r k s a long line a t base of record indicate p r o g r a m m e d progress ion to n e x t schedule component wi thou t r e i n f o r c e m e n t . - Fig . lb . p igeon p re t rea ted wi th 0.03 m g a t rop ine 2 hours before run, 0 . l m g physos t igmine 5 0 m i n u t e s before run. Differences be tween th is and the control record are wi th in l imits o f normal var iab i l i ty . F ig . ld . pigeon p re t rea ted wi th 0.3 m g a t rop ine 2 hours before run, 0.1 m g physos t igmine 50 minu te s before run. This last record is s imi lar to records a f t e r 0.3 rag atropine, or 0.3 m g scopolamine alone. Note reduc t ion of responding in F I component wi th the ra te well ma in t a ined dur ing F R components

Effect of g,l~g PhFsosfigmine

B i r d 37 j ~ ~

B i r d 3 4

t i t i t < I r l f e l t / r t ¢ ¢ ¢ r ¢ l ~ t

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I / / / / ! / / I / / / / I / / I I / / I I I I I J I

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Fig .2 . Typical cumula t ive records of pe r fo rmance on mul t F I 180 seconds F R 30. The records are for full exper imenta l sessions s t a r t i n g 50 minu te s a f t e r pigeons had been pre t rea ted w i t h 0.1 m g physos t igmine . Upper record demons t r a t e s the fact t h a t bird 37 ab rup t ly r e sumed a near ly normal pa t t e rn of behav ior d a r i n g the course of the exper imen ta l session. Ha t ches on the lower line of each record indicate p r o g r a m m e d progress ion to the n e x t schedule component wi th no occurrence of reinforcing s t imul i . W h e n the exper imenta l session was ex tended , birds 24 and 36 also ab rup t ly

r e s u m e d normal pe r fo rmance

410 GEORGE E. VAILLANT:

cumulative record. The mult FI 720 seconds FR 30 produced a quali- tatively similar performance except more responses occurred du/ing the longer FI 720 seconds component than in the individual FI 180 seconds component.

In all pigeons 0.1 mg (about 0.25 mg/kg) physostigmine produced a period of 1 to 3 hours with no responses, and then a resumption of nearly normal appearing behavior (Fig. 2). Doses of physostigmine too small to suppress recorded behavior completely did not modify it from control values. Increasing doses of physostigmine produced progressively longer periods of suppressed behavior. Physostigmine suppressed be- havior on all three different schedule components (FR 30, FI 180 seconds, FI 720 seconds) for similar periods of time. Following 0.1 mg of physo- stigmine, two birds (37 and 212) recovered their normal pattern of respond- ing during the latter part of the standard experimental session, but 6 of the birds (24, 31, 36, 150, 239 and 222) emitted no responses for the entire standard session. After injection of 0.1 mg physostigmine, how- ever, 4 of these 6 birds would still eat at any time during the 1 to 2 hour post-injection period. Under experimental conditions, this is the period during which the food-maintained operant responding was abolished. Two birds (31,239) would eat only during the latter half of the period during which the experimental session would have been in effect.

A dose of 0.1 mg of physostigmine had grossly observable effects upon the birds. They salivated, defecated, and for a portion of the experimental period crouched nearly motionless in the corners of their cage. In pilot studies, it was found that for 3 pigeons the lethal dose of physostigmine was between 0.2 and 0.5 mg and for 3 pigeons the lethal dose of neostigmine ranged from 0.1--0.3 mg.

Abolition of responding by physostigmine could be completely prevented by pre-treatment with suitable doses of atropine (Fig. 1). For each bird, there was an optimum dose of atropine that restored control performance in all essential particulars. Not only was the overall rate restored, but also the pattern of the responding. Dose-effect curves were obtained by averaging values for 3 birds (Fig. 3). Responding could also be restored by scopolamine. At higher dose levels of atropine and scopolamine the mean rate of responding fell again. This apparent failure of antagonism of physostigmine effects at the higher dose levels of atropine or scopolamine is due at least in part to the complication of additional effects of atropine or scopolamine themselves. The effect of 0.3 mg atropine and 0.1 mg physostigmine shown in Fig. 1 is indis- tinguishable from the effect of 0.3 mg atropine alone in this bird. Unlike the case with physostigmine, responding was never totally abolished by the doses of atropine studied. There was, rather, a graded suppression of responding with increasing dose.

Antagonism between physostigmine and atropine 411

The optimum dose of atropine or of scopolamine that prevented the subsequently administered physostigmlne from moving the per- formance out of the normal range (see above; Fig. 1 and 5) differed from bird to bird. There was no one dose that maintained normahty in

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Fig. 3a. Dose-effect curves for the an tagonism by atropine, scopolamine and methyl-atropine of the suppression of responding under mu l t :FI 180 seconds leR 30 by physost igmine. Abscissa: dose of antagonist . Ordinate: responses in session /3600. o atropine alone; [] scopolamine alone;

methyla t ropine alone; • • physost igmine after a t ropine; • . . . . • physost igmine after scopolamine; • . . . . • physost igmine after methylatropine. Points represent means of determinations on 3 birds (24, 36 and 37). The hatched area covers the range from the highest to lowest of control rates for the three birds • round the mean marked X. The range was greater than 2 s tandard errors in

each direction. All values bu t those for scopolamine were in duplicate

F ig .3b . Dose-effect curves for antagonism by atropine and methylat ropine of the suppression of responding under mu l t F I 180 seconds F R 30 by neostlgmine. Symbols as in Fig. 3a. Points represent means of determinat ions on 4 birds (24, 31, 36 and 37). Hatched area covers 2 s tandard errors above

and below mean

Fig .3c . Dose-effect curves for " 'antagonism" by atropine and methyla t ropine of the suppression of responding under mu l t F I 180 seconds PR 30 by nicotine. Symbols as in :Fig.3a. Points represent means of determinat ions on 3 birds (24, 36 and 37). Hatched area covers 2 s tandard errors above and

below mean

412 GEORGE E. VAILLANT-"

all birds so that the average curves of Fig. 3 do not attain the normal range, l~ethylatropine was about one-tenth as potent as atropine in

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Fig. 5. Dose-effect curves for the an t agon i sm by a t ropine and methy la t rop ine of the suppress ion of responding under m u l t F I 180 seconds F R 30 by different doses of physost ig- mine. • * 0.1 m g physos t igmine a f te r a t ropine; o o 0 . 1 5 m g physos t igmine a f te r a t ropine; • . . . . • 0.1 m g physos t igmine a f te r me thy la t rop ine ; a . . . . a 0.15 m g physos t igmine a f te r methy la t rop inc . Po in t s represent single de te rmina t ions a t each dose combinat ion except tha t the points on the f a r lef t indicat ing physos t igmine alone represent ave raged dupl icate de terminat ions . X is the m e a n control value for b i rd 212 and the ha tched a rea represents

the r ange of one s t anda rd devia t ion

antagonizing physostig- mine (Fig.3a and 4). I t was also less potent in causing modification of responding when given alone.

Studies with addi- tional birds working under a schedule with a different parameter value (mult F I 720 seconds FR 30 instead of mult F I 180 seconds FR 30) are shown in Fig. 4. They serve to confirm the relationships shown in Fig. 3a and to show that they are not crucially dependent on particular parameter values.

For bird 212 raising the suppressant dose of physostigmine from 0.1mg to 0.15mg uni- formly increased the amount of methyl atro- pine and atropine needed to antagonize it (Fig.5). Antagonism by atropine of the behavioral effects of physostigmine appear- ed surmountable and the difference in potency be- tween methylatropine and atropine was not an artifact dependent on a single dose of physostig- m i n e .

Neostigmine produced changes in pattern of responding and in effects on grossly observable behavior that were similar to those of physostigmine but shorter in duration. A dose of 0.1 mg neostigmine

Antagonism between physostigmine and atropine 413

supressed responding for about 1 hour. Atropine was rather less potent in antagonizing the suppression caused by neostigmine than that caused by physostigmine, but methyl atropine was very much more potent against neostigmine than against physostigmine (Fig.3a and b). The relative positions of the curves for atropine and methyl atropine are reversed according to whether antagonism of physostigmine or neostig- mine is being studied. At higher doses of atropine (more than 0.3 mg) responding is better maintained ff neostigmine is given than if phy- sostigmine is given (Fig. 3a and b).

Nicotine caused suppression of responding under mult FI 180 seconds FR 30. A dose of 0.3 mg produced equivocal changes of behavior of a grossly observable kind, and the suppression of responding by this dose was less prolonged (about one-half hour) than with 0.1 mg of either physostigmine or neostigmine. There was, however, no suggestion of antagonism of the nicotine suppression by either atropine or methyl atropine (Fig. 3c).

The lethal dose of atropine in the pigeon has been reported as 200 mg/kg (WILBURG 1914) and the lethal dose of methyl atropine as 50 mg/kg (FLVRY 1935). Of three pigeons we studied, one was killed by 50 mg/kg and one by 75 mg/kg of methyl atropine; the third tolerated 120 mg/kg of atropine but was killed by 250 mg/kg. About 1/7 of the reported lethal dose of methyl atropine (7.5 mg/kg) had no effect on a pigeon's mult FI 180 seconds FR 30 behavior, but about 1/80 of the estimated lethal dose of atropine (2.5 mg/kg) had considerable effect. These findings support the belief that in general, the quaternary amine, methyl atropine, which is as or more potent peripherally than atropine (GRAHAM and LAZARUS 1940; FINK and C]~RVONI 1953) exerts much less effect centrally.

Determination of one pigeon's brain cholinesterase immediately after death from 0.5 mg physostigmine (given 25 minutes before) revealed a 670/0 reduction in brain enzyme activity. Immediate post mortem determination of the brain cholinesterase in 2 pigeons, one of which was killed by 0.4 mg neostigmine and one by 0.5 mg neostigmine (given 15 minutes before) revealed depression of brain chohnesterase of 10°/0and 200/o respectively. Because of dilution of the enzyme-inhibitor complex, such results have only qualitative significance. Nevertheless, they are in keeping with observations by FELDBERO (1961) and ECCLES (1956) that neostigmine, a quaternary amine, penetrates the blood brain barrier with much greater difficulty than does physostigmine.

Discussion

The main results of these studies are immediately apparent in Fig. 3. The suppression of behavior by physostigmine is effectively antagonized

414 GEO~OE E. V ~ L ~ T :

by atropine and scopolamine but not by methylatropine. The suppression of behavior by neostigmine, in contrast, is even more effectively antago- nized by methylatropine than by atropine. The antagonisms have been established as shifts of dose-effect curves. The restoration of responding by atropine and methylatropine is not due to non-specific physiological type antagonism, since neither drug had any effect on the suppression of responding by nicotine. The other figures show that the antagonismn of physostigmine by atropine can be complete in individual birds, in tha t in the presence of atropine, normal performance can continue following doses of physostigmine otherwise causing suppression, and that the antagonism occurs at other schedule parameters than those used in the main study.

All these results are in full conformity with present views on the accessibility of the brain to these drugs. Physostigmine, a ter t iary amine, enters the brain easily; its effects there can be antagonized by ter t iary amines, atropine and scopolamine, but by the slowly penetrating quater- nary amine, methylatropine, only when doses are very high. The effects of neostigmine, on the other hand, are confined to the periphery; so its effects are even more readily blocked by methylatropine, with its higher intrinsic activity, than by atropine. The concordance of all these findings lends support to the premise of main interest, that some of the behavioral effects of physostigmine--those seen in the presence of methylatropine-- are due to an effect on the central nervous system. That these effects are mediated through muscarine-type receptors is strongly supported by complete lack of antagonism of the effects of nicotine by atropine.

Other findings in the literature have led to this conclusion. Pr~,IF]~wR and J E ~ Y (1957) observing "conditioned avoidance" behavior in the cat, and STA~K and ]3OYD (1963) observing operant behavior maintained by electric self-stimulation in the dog concluded that these behaviors could be centrally suppressed by physostigmine. FELDB~RG and SHag- wood (1954) injecting DFP and physostigmine directly into the cat ventricle observed that after the first 20 minutes the outstanding drug effect was reduction of overall gross behavior. The fact, then, tha t food-maintained operant behavior is also suppressed lends support to the idea that a central pharmacological effect of acute anticholinesterase administration is to abohsh motor output maintained by many different conditions.

I f the premise that physostigmine has behavioral effects by actions on the central nervous system be accepted, the nature of the behavioral effects becomes of interest in offering clues as to the possible role of eholinergic systems in higher nervous activity. The most characteristic feature of the effect of physostigmine was that it was all-or-none. Ei ther the birds were responding according to normal patterns to the schedule,

Antagonism between physostigmine and atropine 415

or they were not responding at all. All components of the schedule performance showed equal susceptibility to physostigmine. This is in marked contrast to the effects of barbiturates, amphetamines, pheno- thiazines and other classes of drugs, including atropine and scopolamine. Here there are graded behavioral effects, distortions of pat terns of responding before abolition, and, characteristically, large differences in susceptibility of performances under different schedules. The effects of physostigmine were indistinguishable from the presumably peripheral actions of neostigminc. The obvious inference is tha t if there are cholin- ergic muscarine-type cholinoceptive systems in the central nervous system, they affect behavior in gross fashion and appear to be concerned more with occurrence or non-occurrence of responding, rather than with the dynamic determinants of patterns of behavior tha t are affected by drugs more commonly employed to modify behavior. There is no good evidence available in the literature to contradict this suggestion. The inference is based, however, on highly indirect evidence, and the possibil- i ty of a role for cholinergie systems in more subtle functions or in be- havioral functions less sensitive to modification by physostigmine is in no way ruled out.

Summary

The effects of physostigmine upon food-maintained operant behavior of the pigeon were investigated. Physostigmine completely suppressed responding for a period up to 180 minutes and small doses of atropine could antagonize this suppression. Neostigmine produced a similar sup- pression of behavior of shorter duration. Methylatropine, much less potent than atropine in antagonizing behavioral suppression by phy- sostigmine, was more potent in antagonizing neostigmine. Nicotine produced a suppression of behavior similar to tha t produced by the two anticholinesterases, but neither methylatropine nor atropine antagonized this effect. These findings support the general supposition tha t suppres- sion of behavior by physostigmine is ia par t mediated by muscarinic receptors in the central nervous system.

Acknowledgements. The author is deeply indebted to Prof. P. B. D~ws and Dr. W. H. MORSE for valuable advice and to ~V[rs. CATt~ERINE JACKSON for conscientious assistance throughout the experiments.

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416 GEORGE E. VAILLAI~T: Antagonism between physostigmine and atropine

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Dr. GEORGE m. VAILLANT, USPHS Hospital, Lexington, Kentucky/U.S.A.