The cholinergic system, amnesia and memory

Physiology and Behavior, Vol. 8, pp. 511-514, Brain Research Publications Inc., 1972 Printed in Great Britain.

The Cholinergic System, Amnesia and Memory' DONALD

J. L E W I S A N D N O R M A N

J. B R E G M A N ~

Department of Psychology, University of Southern California, Los Angeles, California, 90007, U.S.A. (Received 11 N o v e m b e r 1971) LEWIS, D. J. ANDN. J. BREGMAN. The cholinergicsystem, amnesia, and memory. PHYSIOL.BEHAV. 8 (3) 511--514, 1972.Three experiments are reported which attempt to manipulate memory through the cholinergic system. All animals learned a single-trial passive avoidance response followed immediately by ECS or Sham ECS. In the first, prostigmine was used to rule out the possibility that effects could be due to peripheral response mechanisms. In the second, physostigmine was administered either one or 24 hr or 7 days after ECS. Physostigmine had no effect on the ECS-produced amnesia, but when administered alone, 24 hr following learning, it produced a partial amnesia. In the third experiment, scopolamine was administered prior to learning and ECS. No drug effect was detected. All tests were conducted 24 hr following injection. When the effects of the drugs have dissipated at the time of test, no effect is noted. This suggests that previous effects, found with contemporary drug effects still present, are not relatively permanent and therefore not on memory.

Amnesia

Memory

ECS

Prostigmine

Physostigmine

DEUTSCH, Hamburg and Dahl [5], Deutsch and Leibowitz [6], Hamburg [7], and Weiner and Deutsch [13] have indicated that the presence and absence of memory can be manipulated by chemicals which have an effect upon the cholinergic neurotransmitter system. Their results have encouraged Adams, Hoblit and Sutker [1], Davis, Thomas and Adams [4], and Weiner [12] to look, with some success, for a chemically produced memory return following amnesia produced by ECS. Adams, et al. [I ] gave active avoidance training followed by three ECSs within 20 min. Three and one-half hr after ECS his animals (rats) received either an IP injection of physostigmine (an anticholinesterase) or of scopolamine (an anticholinergic). Testing occurred 30rain after injection. They found that the scopolamine attenuated the amnesia and that the physostigmine enhanced it. Weiner [12] using a Y-maze avoidance situation injected the same compounds 23½ hr after a single ECS with test 30 min after injection. He found that the physostigmine produced a greater decrement than the ECS alone when ECS was given shortly after learning. The scopolamine reduced the amnesia produced when ECS was given 14 clays after learning but not at 5 min, 3 or 31 days. Davis, Thomas and Adams [4] gave ECS immediately following a one-trial passive avoidance task and tested four hr later. Scopolamine or physostigmine was injected 30 min prior to learning or 30 min prior to the retention test. Physostigminewhen injected prior to learning tended to protect memory from the amnesic effect of ECS, and scopolamine had the same effect when injected prior to retention. Also, scopolamine alone disrupted retention if given before learning, and physostigmine alone disrupted retention if given prior to test. From these data it would seem that a possible chemical means exists to dissipate amnesia.

Scopolamine

Cholinergic

EXPERIMENT 1 The first experiment was a test of the possibility that the interactive effects of IP injections of physostigrnine with ECS are not upon memory but upon a peripheral response mechanism. Prostigmine was used, a compound with similar properties to physostigmine except that it crosses the bloodbrain barrier with difficulty. METHOD

Animals Thirty-two naive Sprague-Dawley albino rats, males weighing approximately 200 g at the beginning of the experiment, were obtained from Simonsen Laboratories. The animals were housed in individual cages and were fed approximately 20 g of lab chow each day and were given ad lib water.

Apparatus The apparatus was a conventional step-down platform [8]. Grason-Stadler programming equipment delivered the footshock (FS) for a 5 sec duration from a setting of 1.6 ma. A Beckman brief-stimulus generator was used to administer ECS of 40 ma for 0.5 see.

Procedure Twenty-four hr after arrival in the laboratory animals were adapted to ear clips (modified alligator clips) for three successive days. Hence animals could wear earclips with little irritation during the training trial, thus permitting ECS to be given immediately following training. On Day 4 each animal was placed on the platform for three successive pre-training trials in the step-down apparatus with ear clips attached. He was removed from the compartment 5 see after

1This research was supported in part by Research Grant No. MH 15861 from the National Institute of Mental Health. 2National Institute of Mental Health Predoctoral Fellow supported by Research Grant No. MH 10554 to the Department of Psychology, University of Southern California. 511

512

LEWIS AND BREGMAN TABLE 1 EXPERIMENTAL DESIGN AND DATA FOR EXPERIMENT I

Group

1 II II I IV V

FS

ECS

Yes Yes Yes Yes No

Yes No Yes No No

Injection

Prostigmine Prostigmine Saline Saline Prostigmine

stepping down. Upon stepping off the platform the fourth time, four groups received FS (refer to Table 1). Two of these four groups were convulsed immediately after the FS. The fifth group which did not receive FS was removed 5 sec after stepping down. Immediately after ECS or Sham ECS animals were returned to their living cages. Twenty-four hr later they were given an intraperitoneal (IP) injection of 0.04mg/kg prostigmine or 0.04mg/kg saline and tested 24 hr later.

Testing On Day 6, 24 hr after injection, all animals were placed back in the step-down apparatus for a single test trial. Step-down latencies were recorded and a trial was terminated if an animal failed to step down within 300 sec. RESULTS AND DISCUSSION

A Mann-Whitney analysis comparing ECS to Sham ECS yielded a U ~ 20, p < 0.002; analysis of the prostigmine-ECS versus saline-ECS revealed no differences (U ~- 13,p=0.242). Thus, reliable amnesia resulted but prostigmine did not attenuate the amnesia produced by ECS. It would appear that peripheral effects are not the cause of the retention deficits that have been reported. A similar result has been reported by Squire, Glick and Goldfarb [11]. EXPERIMENT 2 A more extensive experiment testing the effects on memory of physostigmine in combination with ECS was undertaken. The rationale for the experiment follows from the changes in the cholinergic system produced by both ECS and physostigmine. Richter and Crossland [10] demonstrated that ECS elicits the release of bound ACh, and thus, following ECS, both ACh and AChE concentrations should be above their usual levels since an increase in AChE follows an increase in ACh due to an enzyme induction mechanism. Adams, et aL [1] have shown an increase in AChE in rat brains following ECS with a return to normal 96 hr later. It seems reasonably certain, therefore, that ECS increases the amount of ACh. Carlton [2, 3] has argued that the cholinergic system mediates the effects of nonreinforcement (extinction), and Lewis and Maher [9] hypothesized that ECS is a massive inhibitor acting similarly to Pavlovian extinction. This could mean that ECS works its inhibitory-extinctive effect through increased ACh activity. If, then, one adds still further inhibition by the injection of physostigmine (which increases effective ACh by blocking its hydrolyzer), then the amnesia following ECS may be still more profound.

n

Mean Step-down Latency

6 6 6 7 7

25.99 sec 149.59 sec 64.24 sec 228.35 sec 7.00 sec

MET1 IOD

Anhnals The animals were 72 Sprague-Dawley albino rats, male, weighing approximately 200 g at the beginning of the experiment, obtained from Simonsen Laboratories. Apparatus The apparatus used was the same as described in Experiment I. Procedure The animals were pretrained as in Experiment 1 and then assigned to one of twelve groups in a 2 x 2 x 3 factorial design (See Table 2). The procedures used were the same as those in Experiment 1 except that after treatment (ECS or Sham ECS) the animal was injected at different times with 0.5 mg/kg of physostigmine [2, 12] or saline. Testing The testing day varied, but all animals were tested 24 hr after their respective injections. The response measure was the same as Experiment 1. RESULTS AND DISCUSSION

A Mann-Whitney analysis, comparing all of the ECS groups to the Sham ECS groups yielded a significant difference (Z = 6.83, p <0.00003); however, the only other significant difference was between physostigmine injected 24 hr after Sham ECS and its saline control (U = 5, p -----0.041). As in Experiment 1 amnesia was reliably produced when ECS was administered shortly after FS in the step-down apparatus. Physostigmine did not deepen the amnesia when given following ECS, but it did have an amnestic effect when administered alone 24 hr following learning. This effect was not observed at 1 hr or 7 days learning-injection interval. EXPERIMENT 3 In the next experiment the cholinergic system was manipulated through injections of scopolamine which decreases the amount of ACh because of competition at the receptor sites. Presumably this would also decrease the amount of amnesia. In this experiment the scopolamine was administered before the amnestic treatment. METHOD

Animals Thirty-six Sprague-Dawley albino rats, males, approximately 200 g were obtained from Simonsen Laboratories.

CHOLINERGIC SYSTEM, AMNESIA AND MEMORY

513 that injection of an anticholinergic, e.g. scopolamine, will attenuate or abolish the amnesia. No evidence for this hypothesis was found here.

Apparatus The apparatus was the same as in Experiments 1 and 2.

Procedure The animals were pretrained as in Experiment l and then assigned to one of four groups in a 2 × 2 factorial design (See Table 3). Animals were either injected with 1.0 mg/kg of scopola-

G E N E R A L DISCUSSION Considerable previous research [1, 4, 5, 6, 7, 12 and 13] implicates the cholinergic system in memory deficits. Adams and his colleagues [1,4] have combined cholinergiccompounds with ECS to enhance or attenuate the amount of amnesia

TABLE 2 EXPERIMENTAL DESIGN AND DATA FOR EXPERIMENT 2

Group I II I 11 IV V VI VII VIII IX X XI XII

FS

ECS

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes No No No No No No

Time of injection after ECS or NECS 1 hr 24 hr 7 days 1 hr 24 hr 7 days 1 hr 24 hr 7 days 1 hr 24 hr 7 days

Injection

n

Physostigmine Physostigmine Physostigmine Saline Saline Saline Physostigmine Physostigrnine Physostigmine Saline Saline Saline

6 6 6 6 6 6 6 6 6 6 6 6

Mean Step-down Latency 10.91 sec 7.86 sec 14.49 sec 6.34 sec 7.20 see 11.57 sec 135.86 sec 59.96 sec 158.49 sec 129.95 sec 192.99 sec 100.92 sec

TABLE 3 EXPERIMENTAL DESIGN AND DATA FOR EXPERIMENT 3

Group

1

11 111 IV

Injection

FS

ECS

n

Mean Step-down Latency

Scopolamine Saline Scopolamine Saline

Yes Yes Yes Yes

Yes Yes No No

9 9 9 9

12.91 sec 18.49 see 111.26 sec 144.68 sec

mine [2, 4, and 12] or an equivalent amount of saline one half hour prior to training followed immediately by treatment.

Testing Twenty four hr after treatment (ECS or Sham ECS) all animals were placed back in the step-down apparatus and tested, instead of being tested 24 hr after injection as in the two previous experiments. RESULTS AND DISCUSSION

A Mann-Whitney analysis comparing ECS to NECS groups yielded a U = 79, df-~ 18/18, p <0.01. Further analysis showed no significant differences between drug and saline (U = 148, d r = 18/18, p >0.05). Although a reliable ECS-produced amnesia was obtained, scopolamine made no difference. Our working hypothesis was that ECS causes amnesia due to an excess of ACh and

produced by ECS. Their successes give impressive support to the notion that ECS works through the cholinergic system. If Carlton [2, 3] is correct, this system mediates the effects of nonreinforcement and is inhibitory in nature. Lewis and Maher [9] have hypothesized that ECS acts as an inhibitor and therefore it is reasonable to assume that this inhibition is mediated through the cholinergic system. Unfortunately the data presented here is not very positive for these notions. We were not able to deepen amnesia by combining physostigmine with ECS nor to attenuate amnesia by using scopolamine even though both of these affects have been reported to some extent by Adams, et aL [1]; Weiner [12]; Davis, Thomas and Adams [4]. One reason for our failure was that we invariably tested 24 hr following injection, whereas the injection test time in the previous studies has varied from 30 rain to four and one-half hours at the most. In the previous studies, then, animals could have been still under the contemporary effects of the compounds; in our

514

LEWIS AND BREGMAN

studies the contemporary effects had probably dissipated. But this is the reason we chose the relatively long interval that we did. Since learning is defined to be relatively permanent, a 24 hr interval should not be unreasonably long to look for the effects on memory of these compounds. It should be

noted that Weiner and Deutsch [13] injected an anticholinesterase (DFP) intracranially 24 hr after learning on one occasion and tested 24 hr later and found no effect although they did find amnesia if D F P was injected 7 days after learning, contrary to our negative results with physostigmine.

REFERENCES 1. Adams, H. E., P. R. Hoblit and P. B. Sutker. Electroconvulsive shock, brain acetylcholinesterase activity and memory. Physiol. Behav. 4: 113-116, 1969. 2. Carlton, P. L. Cholinergic mechanisms in the control of behavior by the brain. Psychol. Rev. 70: 19-29, 1963. 3. Carlton, P. L. Brain acetylcholine and inhibition. In: Reinforcement and Behavior, edited by J. T. Tapp. New York: Academic Press, 1969, pp. 286-327. 4. Davis, J. W., R. K. Thomas, Jr. and H. E. Adams. Interactions of scopolamine and physostigmine with ECS and one trial learning. Physiol. Behav. 6: 219-222, 1971. 5. Deutsch, J. A., M. D. Hamburg and H. Dahl. Anticholinesterase-induced amnesia and its temporal aspects. Science 151: 221-223, 1966. 6. Deutsch, J. A. and S. F. Leibowitz. Amnesia or reversal of forgetting by anticholinesterase, depending simply on time of injection. Science 153: 1017-1018, 1966. 7. Hamburg, M. D. Retrograde amnesia produced by intraperitoneal injection of physostigmine. Science 156: 973-974, 1967.

8. Jarvik, M. E. and W. B. Essman. A simple'one-trial learning situation for mice. Psychol. Rep. 6: 290, 1960. 9. Lewis, D. J. and B. A. Maher. Electroconvulsive shock and inhibition: Some problems reconsidered. Psychol. Rev. 73: 388-392, 1966. 10. Richter, D. and J. Crossland. Variation in acetylcholine content of the brain with physiological state. Am. J. Physiol. 159: 247-255, 1949. 11. Squire, L. R., S. D. Glick and J. Goldfarb. Relearning at different times after training as affected by centrally and peripherally acting cholinergic drugs in the mouse. J. comp. physiol. Psychol. 74: 41--45, 1971. 12. Weiner, N. I. Electroconvulsive shock induced impairment and enhancement of a learned escape response. Physiol. Behav. 5: 971-974, 1970. 13. Weiner, N. and J. A. Deutsch. Temporal aspects of anticholinergic and anticholinesterase-induced amnesia for an appetitive habit. J. comp. physiol. Psychol. 66: 613-617, 1968.