Effects of scopolamine on performance of rats in a delayed-response radial maze task

Effects of scopolamine on performance of rats in a delayed-response radial maze task

Physiology & Behavior, Vol. 43, pp. 403-409. Copyright©Pergamon Press plc, 1988. Printed in the U.S.A. 0031-9384/88 $3.00 + .00 Effects of Scopolami...

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Physiology & Behavior, Vol. 43, pp. 403-409. Copyright©Pergamon Press plc, 1988. Printed in the U.S.A.

0031-9384/88 $3.00 + .00

Effects of Scopolamine on Performance of Rats in a Delayed-Response Radial Maze Task J. J. B O L H U I S , A. M. S T R I J K S T R A A N D R. J. K. K R A M E R S University o f Groningen, Zoological Laboratory P. O. Box 14, 9750 A A Haren, The Netherlands R e c e i v e d 31 M a r c h 1987 BOLHUIS, J. J., A. M. STRIJKSTRA AND R. J. K. KRAMERS. Effects of scopolamine on performance of rats in a delayed-response radial maze task. PHYSIOL BEHAV 43(4) 403--409, 1988.--The effects of the cholinergic blocker scopolamine on performance of rats in an 8-arm radial maze were studied. In Experiment 1, rats received injections of scopolamine-HBr (0.2 mg/kg, IP) or saline, 20 min before a trial. The drug impaired performance only when midtrial delays were introduced (10 sec, 5, 20, 60 or 120 min), during which the animals were removed from the apparatus, and to a similar extent at all delays. Injection of scopolamine directly after choice 4 in a 20 rain delayed trial affected performance at a dose of 0.4 mg/kg, but not at 0.2 mg/kg. In Experiment 2, the rats were kept in the apparatus during the delay. There was a significant effect of scopolamine at 0.1 and at 0.2 mg/kg, initially irrespective of the length of the delay (10 sec, 2.5 or 5 min). After considerable training, administration of scopolamine (0.2 mg/kg) had no significant effect at the 10 sec delay but did impair performance after longer (5 or 10 min) midtrial intervals. These results suggest that scopolamine has differential effects, depending on dose and degree of training, and that an effect on memory storage may be one of them. Scopolamine Spatial memory Working memory Radial maze Cholinergic blockade Delayed response Memory storage

T H E role of central cholinergic mechanisms in learning and memory has been the subject of many recent studies (for reviews see [13,42]). This renewed interest is partly due to the predominance of the 'cholinergic hypothesis' in research into dementia of the Alzheimer type [1, 13-15, 37]. Postmortem investigation of the brains of patients with Alzheimer's disease has revealed substantial loss of cholinergic markers [15,38]. These cholinergic abnormalities have been shown to correlate with memory impairments in these patients [39]. There is increasing evidence that good performance of rats in spatial memory tasks is critically dependent upon normal functioning of the septo-hippocampal system ([29, 32, 35], but see also [26]). There is a prominent cholinergic projection from the medial septum and diagonal band to the hippocampus [18, 28, 30] and it has been argued that the effects of anticholinergic drugs on spatial memory are similar to those of hippocampal lesions in a number of tasks [32]. Olton and co-workers [33,35] have shown that lesions to the septo-hippocampal system impair performance of rats in a radial maze. According to these authors, hippocampal lesions selectively impair working memory (holding information useful for one trial), whilst reference memory (holding information for a series of trials) remains unimpaired. Several studies have found similar differential effects of the anticholinergic scopolamine on rats' behaviour in a radial maze [3, 27, 48]. In contrast, Okaichi and Jarrard [31] found that scopolamine impaired both working and reference memory.

Rat

Learning

This apparent discrepancy in the results may be due to the different doses of scopolamine that were used [3]. In all of these studies, at least spatial working memory was impaired by scopolamine (cf. [17,23]). Acquisition of the radial maze task is also disrupted by daily pretrial injections of the drug [44,47]. Godding et al. [19] did not find significant effects of scopolamine on radial maze performance of trained rats. This may have been due to the timing of the injections, 3 hr before the start of a trial [8], Buregovfi et al. [8] studied the effects of cholinergic blockade on spatial memory, using an aversively motivated 8-arm radial water maze task [6,11]. The effects of scopolamine (0.2 mg/kg) on performance of rats in this task were proportional to the length of the midtrial delay. Scopolamine did not impair performance when it was injected directly after choice 4 in a delayed trial, suggesting that the drug did not affect retrieval processes. An earlier study by Bure~ovfi and Bure~ [9] suggests that delaydependent effects of the drug may also be seen in a foodrewarded radial maze. The authors did not find significant effects of 0.1 mg/kg scopolamine on the performance of overtrained rats in a 12-arm radial maze. However, the drug did affect performance when a 5 rain delay was inserted between choices 6 and 7. Effects of scopolamine, dependent on the length of the retention interval, have been reported in experiments using a delayed response procedure in the rhesus monkey [2, 36, 40]. However, in several studies, using delayed response proce-

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BOLHUIS, STRIJKSTRA AND KRAMERS

dures in the rat it was found that the disruptive effect of scopolamine on performance was similar at different retention intervals [16, 21, 22, 24, 43]. It has been argued that effects of anticholinergics that are proportional to the length of the retention interval indicate that the drugs affect memory storage or retention [21, 22, 42]. Thus, it is important to establish whether delay-dependent effects as reported by Buregovfi et al. [8] also occur in a food rewarded radial maze task. In the present study, the effects of scopolamine on performance of rats in such a task were studied, both in uninterrupted trials as well as in trials with various midtrial delays. To assess the effects of the drug on acquisition and retrieval, it was injected both before and after the first half of a delayed trial. EXPERIMENT 1 In this experiment, the effects of scopolamine on performance of rats were studied in a food-rewarded radial maze. Various midtrial delays were introduced, during which the animals were removed from the apparatus, similar to the procedure that Buregovfi et al. [8] employed for the radial water maze.

2.c

NO HANDLING

HANDLING

~ SCOP

iJ ; .~ ~.c

j+,AL mm m SAL SCOP

SAL SCOP

no delay

,

5

20

60

120

min

delay (log)

FIG. 1. Mean number of errors (_+S.E.M.) in the second half of a trial after injection of scopolamine-HBr (scop., 0.2 mg/kg) or saline (sal.), 20 min before the start of a trial. The effects of handling on performance are shown on the left of the figure. In the 'handling' condition the rats were removed from the maze and placed in their home cage during a brief midtrial delay. In the 'no-handling' condition the rats were left in the apparatus during the delay. The right part of the figure shows the mean number of errors after various midtrial delays (n= 14).

METHOD Animals Fourteen male albino rats (Wistar), 9 months old at the beginning of the experiments, were used. They were housed in two cages, placed in the same room as the testing apparatus, under an L/D: 12/12 schedule (lights were on from 8.00 to 20.00 hr). Body weight was reduced to 85-9(~c of freefeeding weight by offering food for 20--40 min after a trial was completed. The rats had been used in previous radial maze experiments [5,45]. In the latter study, the rats were divided in two groups of 7 rats that received different training schedules. In the present experiments, performance in control trials did not differ significantly between these two groups. The results of all animals were pooled. Apparatus A radial maze was used similar to that described by Olton and Samuelson [34]. From a central platform (34 cm in diameter) 8 radial arms extended (86 cm long, 7 cm wide, with 3 cm high side walls). Five cm from the end of each arm was a food cup, 1.5 cm deep and 2 cm wide, in which black plastic cups were inserted. The central platform was enclosed by a 16 cm high wooden wall with 8 Plexiglas guillotine doors which could be raised and lowered by means of overhead strings. The apparatus was painted matt grey. Drugs Scopolamine-HBr was dissolved in saline in 0.14 mg/ml and 0.28 mg/ml concentrations. With 0.41-0.59 ml injection fluid, depending on the bodyweight of the animals, a dose of 0.2 mg/kg bodyweight or 0.4 mg/kg bodyweight was administered. The volume of saline injections was also adapted to bodyweight. Procedure One trial per rat per day was run. Testing took place in the first half of the light period. At the start of a trial a rat was placed on the central platform with all guillotine doors lowered, and allowed to explore for 10 seconds. Then all doors were raised simultaneously and the rat could enter an arm

and walk up to the food cup to get the reward, 3 drops of a 23% sucrose solution. When a rat had placed all four paws in an arm it was considered to have made a choice. When an arm had been entered, the doors of all other arms were lowered. After the rat had returned to the platform, the door of the visited arm was also lowered. Before the animal was allowed to make another choice it was confined on the central platform for 5 seconds, in order to prevent choice strategies [5]. An uninterrupted trial consisted of 8 or more consecutive choices, as many as the animal needed to enter all of the arms. In a delayed trial, the rat was allowed 4 choices, after which it was taken from the apparatus to its home cage. After the delay the rat was returned to the maze to complete the trial. When 8 choices took more than 8 rain the trial was stopped. An error was defined as a reentry in an arm already visited. The effects of scopolamine or saline on performance were tested by giving the animals injections of scopolamine-Hbr (0.2 mg/kg, IP) or a balanced amount of saline 20 min before the start of a trial. The effects were determined with uninterrupted and delayed trials. The delays used were 5, 20, 60 and 120 min, introduced in a quasi-random order. Scopolamine trials were run, with 20 min pretrial scopolamine and saline injections. In these trials the animal was taken out of the maze to its home cage after choice 4 and immediately taken back to the apparatus. This took approximately 10 seconds in a trial. In order to determine whether scopolamine affects acquistion (first half of a delayed trial) and/or retrieval (second half of a delayed trial), trials were run with a 20 min delay. In these trials saline or scopolamine (0.2 mg/kg, IP) was injected immediately after choice 4. Finally, effects of a higher dose of scopolamine (0.4 mg/kg) were determined in uninterrupted trials with injections 20 rain before the start of a trial, and in 20 min delayed trials with injections immediately after choice 4.

RESULTS AND DISCUSSION On a number of occasions the animals did not complete the trial within 8 min or they did not make any choice. The

SCOPOLAMINE AND SPATIAL MEMORY

405

2.0

2.0

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CO I

._t2

o

.0_

1.0

0 ~

SAL

SCOP 0.2

0

SCOP 0.4

SAL

~.OP 0.2

SCOP 0.4

FIG. 2. Mean number of errors (-S.E.M.) in the second half of an uninterrupted trial after injection of scopolamine (scop., 0.2 or 0.4 mg/kg) or saline (sal.), 20 min before the start of a trial (n=14).

FIG. 3. Mean number of errors (-S.E.M.) after injection of scopolamine (scop., 0.2 or 0.4 mg/kg) or saline (sal.), immediately after choice 4 of trials with a midtrial delay of 20 rain (n= 14).

percentage of such noncompleted trials was significantly higher in the scopolamine condition (14.3%) than after saline treatment, 0.9%, ×2(1)=13.0, p<0.001. Performance of the rats in uninterrupted and delayed trials after injection of scopolamine (0.2 mg/kg) or saline is illustrated in Fig. 1. A two-factor analysis of variance with repeated measures on both factors was performed on the number of errors in choices 5 to 8 in the trials in which the animals were removed from the maze during a delay ('handling'). The analysis revealed significant effects of Delay and Drug (scopolamine or saline) on the number of errors, F(4,52)=7.29, and F(1.13)= 126.6, respectively, p<0.001, but no significant interaction between these two factors. Newman-Keuls multiple comparison tests revealed that every mean value in the scopolamine trials was significantly different from the mean in the saline trial at the same delay (,o<0.01). The mean number of errors after the 120 min delay under scopolamine treatment was not significantly different from the random choice value (2.0 errors in choices 5-8). The results of the 'no delay' trials, in which the animals were either left in the maze (no handling) or removed for a few seconds (handling), are shown on the left of Fig. 1. A two-factor A N O V A with repeated measures on both factors revealed a significant interaction between the factors Treatment (handling or no handling) and Drug (scopolamine or saline) F(1,13)=5.44, p<0.05, indicating that the effect of scopolamine on performance was dependent on whether the animals were removed from the maze or not. Newman-Keuls tests showed that the mean number of errors in the scopolamine/handling condition differed significantly from all other means (p<0.01). Figure 2 shows the performance of the rats in uninterrupted trials after injection of saline or scopolamine at a dose of either 0.2 mg/kg or 0.4 mg/kg. A one-factor A N O V A with repeated measures revealed a significant effect of Drug on the number of errors in choices 5 to 8, F(2,26)=13.45, p<0.001. Newman-Keuls tests showed that only the mean number of errors at the 0.4 mg/kg dose was significantly different from the mean after saline injection (p <0.01). Performance in delayed trials with injection of saline or scopolamine immediately after choice 4 is shown in Fig. 3. A one-factor A N O V A with repeated measures performed on the number of errors revealed a significant effect of Drug,

F(2,26)=8.17, p<0.005. Newman-Keuls tests showed that only the mean number of errors after injection of 0.4 mg/kg differed significantly from the mean after saline injection. EXPERIMENT 2 The results of Experiment 1 indicated that scopolamine increased the effect of handling on performance of the rats in the radial maze. This handling effect may have obscured possible delay-dependent effects of the drug, that were expected on the basis of the results of Buregowi et al. [8]. To test this possibility, in Experiment 2 the rats received delayed trials in the radial maze, during which they remained on the central platform and were not handled. METHOD

Subjects Fourteen male albino rats (Wistar) were used, different from those in Experiment 1, and 12 months old at the beginning of the experiment. The animals were kept under the same conditions as in Experiment 1. Before the start of this experiment they had received a total of 50 trials in the radial maze with various delays ranging from 30 see to 16 min, using a free choice procedure to study serial position effects, as described by Bolhuis and Van Kampen [7]. The rats had not received trials (and were fed ad lib) for a period of 5 months before the start of this experiment.

Apparatus The same maze was used as in Experiment 1.

Drugs Scopolamine-HBr was administered IP, as in Experiment 1, but now doses of 0.1 and 0.2 mg/kg were used.

Procedure Pretraining (20 trials) consisted of 2 trials per day, using raisins as reward at the end of the arms. Two trials before the start of the drug treatment the reward was changed to 3 drops of a 23% sucrose solution for each arm and the animals were given only one trial per day, 5 days a week.

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B O L H U I S , STRIJKSTRA A N D K R A M E R S

2.0

2.5min

10 sec

TABLE 1

5min

MEAN N U M B E R OF ERRORS (-+ S.E.M.) IN T H E S E C O N D H A L F OF A T R I A L A F T E R INJECTION O F S C O P O L A M I N E (0.1 mg/kg) O R S A L I N E IN TRIALS WITH D E L A Y S O F 10 SEC, 5 OR 10 MIN, DURING W H I C H T H E A N I M A L WAS R E M O V E D FROM T H E M A Z E (n= 14)

tO

0

1.0 vIYY -/w

SAL

SCOP 0.1

SCOP 0.2

Fqililil SAL SCOP SCOP 0.1 0.2

10 Sec

5 Min

10 Min

Saline

0.29 (0.13)

0.29 (0.13)

0.36 (0.13)

Scopolamine

0.77 (0.21)

1.08 (0.22)

0.86 (0.25)

iilili

.....i

£

Delay

SAL

SCOP 0.1

SCOP 0,2

FIG. 4. Mean number of errors (_+S.E.M.) in the second half of a trial after injection of scopolamine (scop., 0.1 or 0.2 mg/kg) or saline (sal.), 20 min before the start of a trial with midtrial delays of 10 sec, 2.5 or 5 min (n=14). In the first part of the experiment the animals received trials with midtrial delays as in Experiment I, except that now the rats were not removed from the apparatus during the delays, but remained on the central platform and were not handled. Delays used were 10 sec, 2.5 and 5 min, introduced in a quasi-random order. A scopolamine trial with a particular delay was always preceded by a saline trial with the same delay. Scopolamine (0.1 or 0.2 mg/kg, IP) or saline was injected 20 min before the start of a trial. In the second part of the experiment the animals were removed from the maze during the midtrial delays and placed in their home cage as in Experiment I. Delays used were 10 sec, 5 and 10 min. Scopolamine was injected at a dose of 0.1 mg/kg only. In the last part of the experiment the animals were given a series of delayed trials again in which they remained on the central platform during the delay (10 sec, 5 or 10 min). Scopolamine was administered at a dose of 0.2 mg/kg only. There were two replications of each combination of delay and treatment, except for the 10 sec delay trials, of which there were three replications. Finally, the possible interaction between the effects of handling and scopolamine treatment were investigated by injecting 0.2 mg/kg scopolamine or saline, 20 min before the start of a 10 sec delay trial either with or without placing the animal in its home cage during the 10 sec interval. R E S U L T S A N D DISCUSSION

The percentage of noncompleted trials in Experiment 2, after injection of scopolamine was 8.0% at dose 0.1 and 8.8% at dose 0.2 mg/kg, compared to 1.4% after saline treatment. The results of the first part of Experiment 2 are shown in Fig. 4. A two-factor A N O V A with repeated measures on both factors, performed on the number of errors in the second half of the trials, revealed significant effects of Dose, F(2,26)<27.50, p<0.0001, and of Delay, F(2,26)=12.06, p<0.001, but no significant interaction between these two factors. Newman-Keuls tests showed that there was a significant difference between the means at both doses and the saline condition (p<0.05), but no difference between the means at the two doses. These results do not suggest that

there is a delay dependent effect of scopolamine, even though the rats were not handled during the midtrial delay. Table 1 shows the results of the second part of the experiment in which the rats were handled during the delays and placed in their home cage as in Experiment 1. Repeated measures A N O V A revealed a significant effect of Dose (saline or 0.1 mg/kg scopolamine) only, F(1,13)=14.59, p<0.005, and no significant interaction between Dose and Delay, F(2,26)=0.66. These results confirm those of Experiment 1 where the effects of the drug were also not dependent upon the length of the retention interval. The present experiment shows that this is also the case at shorter midtrial delays and with a lower dose of scopolamine. The effects of scopolamine in the second part of the experiment (with handling) are not more pronounced than those of the same dose in the first part (without handling). This is somewhat surprising in view of the results of experiment 1, where scopolamine appeared to increase the effects of handling on performance. It is possible that this is a result of the fact that the rats had received more training in the second part of the experiment and that consequently, working memory had become more resistant to disruption by scopolamine (cf. [45]). The finding that, in the first half of the experiment, scopolamine significantly impaired performance at the 10 sec delays, even at a dose of 0.1 mg/kg, appears to be inconsistent with the results of Experiment 1. The difference between the animals in Experiment 1 and those in the present experiment is that the former had received far more trials before drug treatment than the latter. To test the possible contribution of degree of training to the results, the animals received yet another series of trials without handling during delays (10 sec, 5 or 10 min). When this part of the experiment was started, the rats had received a total of 43 trials. The results are shown in Fig. 5. A N O V A revealed significant effects of Drug, F(1,13)=61.61, p<0.0001, and Delay, F(2,26)= 11.78, p<0.001, and a significant interaction between these two factors, F(2,26)=4.68, p<0.02. That is, the effect of the drug was dependent on the length of the midtrial delay. Newman-Keuls tests showed that the means at the 10 sec delay did not differ significantly. At the 5 and 10 min delays, the means after scopolamine treatment differed significantly from those in the saline condition, (p<0.01). The three means after saline treatment did not differ significantly, whereas in the scopolamine condition the means at the 5 and 10 min delays differed from the mean at 10 sec. Table 2 shows the results of the 10 sec delay trials with and without handling. A N O V A revealed a significant effect of Drug, F(1,13)=7.58, but not of Treatment (handling or no handling) on the number of errors. There was no significant interaction between these two factors, F(1,13)= 2.81, p =0.12.

S C O P O L A M I N E A N D S P A T I A L MEMORY

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lOsec

5min

407 TABLE 2

lOmin

MEAN NUMBER OF ERRORS (_+ S.E.M.) AFTER INJECTION OF SALINE OR SCOPOLAMINE (0.2 mg/kg), 20 MIN BEFORE TRIALS WITH A 10 SEC MID-TRIAL DELAY, DURING WHICH THE ANIMAL WAS EITHER LEFT IN THE MAZE ('NO HANDLING') OR BRIEFLY PLACED IN ITS HOME CAGE ('HANDLING') (n=14)

CO

Q~

No Handling

Handling

Saline

0.14 (0.10)

0.14 (0.10)

Scopolamine

0.21 (0. l 1)

0.54 (0.17)

tO

o to U

SAL

SCOP

SAL

SCOP

SAL

SCOP

FIG. 5. Mean number of errors (_S.E.M.) in the second half of a trial after injection of scopolamine (scop., 0.2 mg/kg) or saline (sal.) in trials with delays of 10 sec, 5 or 10 min during which the animal was left in the maze (n=14).

These results show that after considerable training scopolamine did not significantly affect performance at the 10 sec delay at a dose of 0.2 mg/kg. This is consistent with the results of Experiment 1. Overall, performance with and without scopolamine had improved, compared to that in the first part of Experiment 2. Furthermore, when the animals were not handled during the midtrial delay, the effects of scopolamine on performance were proportional to the length of the retention interval. In this experiment, unlike Experiment 1, there was no increased effect of handling on performance after injection of scopolamine, although Table 2 shows that there is a trend towards an interaction between handling and scopolamine treatment. GENERAL DISCUSSION These results show that scopolamine impairs performance of rats in a radial maze, depending on dose, degree of training, and on the conditions under which the animals were tested. After considerable training and when the rats were not handled during the midtrial delay, the effects of scopolamine on performance increased with the length of the retention interval. In Experiment 1, the effects of the drug at the lowest dose (0.2 mg/kg) suggested that the impairment of performance was dependent on whether or not the animal was removed from the maze and handled. These findings are concordant with those reported by Bure~ov~i and Bure~ [9]. These authors only used a midtrial delay of 5 min. The interaction between the effects of scopolamine at the 0.2 mg/kg dose and handling in Experiment 1 suggests an increased susceptibility of the memory to interference [9, 25, 47, 48]. The results of the last part of Experiment 2 (Table 2) show that the interaction between handling and scopolamine treatment may wane after the animals have received prolonged training. Bure~ov~i et al. [8] have argued that in their experiments with rats in a radial water maze and in a water tank navigation task, scopolamine affected acquisition rather than retrieval. The results of the present Experiment 1 provide support for this conclusion.-Scopolamine impaired performance at a dose of 0.2 mg/kg when it was injected before a 20 min delayed trial, but not when the same dose was injected im-

mediately after choice 4 in a trial with 20 min delay. Injection of scopolamine after choice 4 was also ineffective in experiments by Godding et al. [19] and Beatty and Bierley [4], where midtrial delays of 5 and 4 hours were used, respectively. The main result of Experiment 2 was that when the rats had received considerable training and were not handled during the midtrial delay, the effects of scopolamine on performance were proportional to the length of the retention interval. This is consistent with the findings of Bure~ov~i et al. [8] who reported similar effects using an aversively motivated spatial task, a radial water maze [6,11]. Delaydependent effects of scopolamine and atropine have also been found using delayed response tasks in rhesus monkeys [2, 13, 36, 40] and humans [13]. Pontecorvo and Evans [40] found a delay-dependent effect of scopolamine on delayedmatching-to-sample in monkeys, at a dose of 0.02 mg/kg, but not at 0.03 mg/kg. Recently, Viscardi and Heise [46] reported that the effects of scopolamine in a delayed response task in rats were proportional to the length of the retention interval at a dose of 0.5 mg/kg, not at lower doses of the drug. The delay-dependent effects in the present study suggest that under certain conditions, cholinergic blockade affects memory storage or retention. It appears that these effects may be masked b y i n c r e a s e d effects of handling (as in Experiment 1) or because performance in the maze is more vulnerable to anticholinergic treatment at low levels of training (as suggested by the results of Experiment 2). It has been suggested in recent studies by Bolhuis et al. [5] and Strijkstra and Bolhuis ([45], cf. [6]) that after extensive training of rats in a radial maze, different memory mechanisms become engaged. These authors reported exponential decay of performance with time in a radial maze. Performance could be improved considerably, however, when the rats received extensive training with delayed trials. If indeed different mechanisms are engaged, depending on the degree of training, they may have a different vulnerability to cholinergic blockade. In the study of Bure~ovfi et al. [8], the animals were not overtrained. It is obvious, however, that spatial working memory in the aversively motivated radial water maze task is much more robust than that in a food-rewarded radial maze [5,6]. On the basis of their results, Bure~ovfi et al. [8] have sugested that the memory trace formed under scopolamine treatment is weaker than that in untreated animals, leading to faster memory decay. In Experiment 1, the effects of scopolamine were less selective when a dose of 0.4 mg/kg was used. Performance was impaired in uninterrupted trials as well as when the drug was injected after choice 4 in a 20 min delayed trial, suggest-

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ing that at a higher dose, scopolamine affects retrieval processes as well as acquistition. This is in contrast with results of Godding et al. [19] who did not find significant effects of scopolamine (at doses of 1.0-5.0 mg/kg) injected either immediately or 2 hr after choice 4 in a 5 hr delayed trial. The rats in the study of Godding et al. [19] had received considerable training with long delays before the start of the experiment, resulting in very persistent working memory (cf. [45]). Also, injection timing (5 or 3 hr before the second half of the trial) may have been the reason for the absence of significant effects on performance in the second half of the trial (cf. [10]). The importance of the dose of scopolamine used is illustrated by the results of a number of authors. Hiraga and Iwasaki [23] found that a dose of 0.25 mg/kg did not have a consistent effect on performance in uninterrupted trials in a radial maze, whilst there was a significant impairment at a dose of 0.5 mg/kg. Eckermann et al. [17] found that a dose of 1.0 mg/kg scopolamine was needed to disrupt performance in uninterrupted trials in a radial maze. As noted before, the discrepancy between the results of Okaichi and Jarrard [31] and those of a number of other authors [3, 27, 48] may be due to the relatively large doses of scopolamine (0.5-1.5 mg/kg) used by the former. Beatty and Bierley [3] suggested that at a low dose the drug affects working memory only, and that both working memory and reference memory are affected at higher doses, such as those used by Okaichi and Jarrard [31]. Explanations of the effects of anticholinergics, other than on memory processes directly, have been put forward by several authors (e.g., [12, 19, 20, 24, 31]). Although in the present study the animals did not complete a considerable proportion of trials under scopolamine treatment (cf. [31],

other studies show that explanations of the results in terms of motivational changes or peripheral effects are unlikely. For instance, Bure~ov~i et al. [8] showed that scopolamine impaired performance of rats in an aversively motivated radial maze task [6,11]. Furthermore, several authors have demonstrated that peripherally acting anticholinergics did not have a significant effect on radial maze preformance of rats [8, 17, 23, 27]. Other interpretations of the effects of cholinergic blockade on performance have been that it affects discrimination, attention or arousal (e.g., [12,19, 31]). A recent study by Softie and Lamberty [41] showed that at a relatively high dose (0.5 mg/kg), scopolamine disrupted visual reversal in rats, without affecting the first discrimination. The difference in effect of injection of the drug before or after the first half of a trial in the present Experiment 1 and in other studies [4,8] does not support an explanation in terms of effects on either attention or arousal. The effects of 0.4 mg/kg scopolamine suggest that processes not directly related to memory may be affected at higher doses of the drug. The findings of the present study, that the impairment caused by scopolamine is dependent on the dose, on whether the rats are handled, and on the degree of training indicate that the drug may have differential effects. The delay-dependent impairment found in Experiment 2 suggests that an effect on memory storage may be one of them. ACKNOWLEDGEMENTS We are grateful to Jan Buret, Jerry Hogan, Jaap Kruijt and Mark Johnson for valuable comments.

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