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Brain Acetylcholine and Habituation*
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 e
x
~
PART 2
Scop. ~
~
DRINKING
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
I
Time to drink in test (sec) 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
a 1
Exposed (Soline)
Unexposed
Exposed
(Stop,)
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
51
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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P. L . C A R L T O N
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.
BRAIN ACETYLCHOLINE A N D HABITUATION
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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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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
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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
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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
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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 I
120
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.
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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.
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 e
x
~
PART 2
Scop. ~
~
DRINKING
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
I
Time to drink in test (sec) 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
a 1
Exposed (Soline)
Unexposed
Exposed
(Stop,)
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
51
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
52
P. L . C A R L T O N
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.
BRAIN ACETYLCHOLINE A N D HABITUATION
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
54
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
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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 I
120
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
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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.