A direct measure of short-term memory in mice utilizing a successive reversal learning set

BEHAVIORAL BIOLOGY, 7, 723-732 (1972), Abstract No. 1-120R

A Direct Measure of Short-Term Memory in Mice Utilizing a Successive Reversal Learning Set I

HERBERT P. ALPERN and JOHN G. MARRIOTT

Department o f Psychology and Institute for Behavioral Genetics University of Colorado BouMer, Colorado 80302

C57BL/6 mice were trained in a T-maze to develop a successive reversal learning set. After obtaining the set, animals were tested at several delay intervals after a signal to reverse. In Expt. I, peak performance occurred at 2-15 min and fell markedly at 60 min. Experiment II compared the performance of animals trained using a 2-rain ITI with those trained using a 60-rain ITI. Subjects trained with a 60-rain ITI did not develop the set. When the ITI was reduced to 15 rain, 50% of the subjects reached criterion. Significant differences in test performance between the groups differentially trained were not found; however, both groups confirmed the gradients found in Expt. I. Experiment III showed that a single daily test trial produced results similar to those from multiple daily test trials. The results indicate that delay-interval test performance indexes the activity of the STM trace.

Although the mouse has been used extensively in investigations concerned with memory processes (McGaugh, 1966; Deutsch, 1969; Borer et al., 1968; Glassman, 1969; McGaugh and Dawson, 1971), little is known about short-term memory (STM) in this organism. The major problem encountered in attempting to assess STM in rodents is that the various types of delayed-response procedures developed for primates are not amenable to rodent research. Consequently, investigators concerned with memory in rodents have been forced to speculate about STM in experiments which demonstrate an improvement of performance within a block of daily training trials but little retention across days (Bovet et al., 1968; Duncan et al., 1971). Although experiments like this do offer evidence for a STM system sustaining 1This investigation was supported by National Institute of Mental Health Grant MH 11167 and National Institute of General Medical Sciences Grant GM 14547. 723 Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

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daily performan.ce, they do not and cannot provide the evidence necessary to disclose the qualitative, much less the quantitative, nature of STM. The purpose of this investigation was to measure STM in mice with a degree of sensitivity fine enough to allow us to assess the quantitative aspects of this process. The strategy of Expt I was to train mice to develop a simple successive reversal learning set, to then give them a single piece of information (the signal to reverse), and to finally determine the length of time for which that bit of information (as indexed by reversal behavior) can be retained. Petrinovich and Bolles (1957) used a somewhat similar procedure to assess the temporal limit of delayed alteration in rats. To solve the successive reversal problem, a memory system capable of holding information from trial to trial is required. If our technique, indeed, allows us to isolate a short-term memory system-in other words, if the animal can utilize information only when presented within the span of short-term m e m o r y - t h e n the animal should not even be able to develop the successive reversal set (i.e., reverse on a single trial) when information is presented beyond the temporal boundary of STM as revealed in Expt I. This prediction was tested in Expt II. In Expts I and II, five test trials were used to assess retention of the signal to reverse. Since reinforcement was present on each of the five test trials, additive effects or long-term memory influences could not be entirely excluded. Experiment III, therefore, was designed to ascertain possible additive effects on retention of multiple test trials within a given daily session. EXPERIMENT I

Methods Subjects. Five 90-day-old naive female mice of the C57BL/6 strain bred in our laboratory served as subjects. Apparatus. A T-maze constructed from white Plexiglas was used. The start runway's dimensions were 29-112 × 4 × 9 cm and each arm measured 21 × 4 X 9 cm. Doors which could be raised from below were located at the entrance of the start runway and at the ends of each arm of the maze. The grid floor of the maze, constructed from approx 2-ram diam stainless-steel rods spaced 4.1 mm apart, was connected to a grid scrambler and an alternating current shock source which delivered 3 mA to a 10 K resistance. Procedure. The mice, individually housed during daily runs, were trained to develop a successive reversal set by the following procedure. On Day 1 and on each trial throughout the experiment, each animal was placed into the start runway and administered not more than 3 mA of footshock if it failed to make a choice within 2.5 sec or if it chose the incorrect alley (either right or left). If the animal avoided footshock by entering the correct alley, it was allowed to exit into its 23 × 10 X 10 cm stainless-steel holding cage 2.5 sec

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after the onset of the trial. If the animal failed to avoid and/or chose the incorrect arm, the correction procedure employed allowed it to escape into the correct alley and exit into its holding cage. An error was scored if the subjects did not make a choice within 2.5 sec and/or if it did not select the correct alley. When each subject avoided the footshock on five successive trials (reversal criterion) employing a 2-rain intertrial interval (ITI), training ceased for Day 1. On Day 2, the position discrimination was reversed and the previously correct position became the punished position. Since footshock was the cue signaling a reversal, each subject had to enter the incorrect alley at least once in attaining the 5/5 reversal criterion. Employing the aforementioned procedure, all animals were trained to two reversals with a 10-rain pause separating each reversal of the discrimination; on Day 3, three reversals; on Day 4, four reversals; on Day 5, five reversals; and on Day 6, three reversals. On each day, the subjects were reversed from the last position learned on the previous day. Although multiple reversals were given each day throughout training, the criterion for acquiring the learning set was less than one mean error for the group on the first reversal of each day for 3 consecutive days. On the test or delayed-response phase of the experiment, each subject was placed into the start runway and given a sign-trial footshock regardless of which alley it chose (subjects invariably chose the side trained to last on the previous day). After the sign trial, the subjects were given five test trials, all separated by the same ITI, with the shock still present in the alley in which the subject was footshocked on the sign trial. Errors were assessed as in the training phase. On the first day of testing, the ITI was zero rain (less than 5 sec). On subsequent test days, the subjects were tested at ITIs of 2, 15, 60, 240, and 480 min in first an ascending and then a descending fashion. For example, if the animals were tested at the 60-rain ITI, they were given a single sign tjcial followed by five test trials each hour for the next 5 hr. Each test day was followed by a control day: animals were trained to two reversals with a 2-min ITI separating all trials and each reversal separated by 10 rain. Results

By the sixth day (the fifteenth reversal), the subjects met the reversal set criterion. Figure 1 shows the development of the reversal set. No significant differences were found using t tests between the scores obtained on the ascending and descending test series, so the data for each ITI were pooled. The major finding was t h a t retention of the information acquired on the sign trial after reaching a peak at 2 rain and remaining stable for as long as 15 rain fell markedly at 60 min (see Fig. 2). An analysis of variance with repeated measures of the error scores revealed a highly significant intertrial interval effect [ F = ( 5 , 20) 5.15, P < 0 . 0 1 ] . ~ O f the planned orthogonal comparisons made, only the differences between the

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0-min and 2-rain 1TI groups I F = ( 1 , 20) 5.45, P < 0 . 0 5 ] and the 15-rain and 60-min ITI groups [F = (1, 20) 5.75, P < 0.05] were significant.

EXPERIMENT II

Methods Subjects. Twenty 90-day-old naive female mice of the C57BL/6 strain bred in our laboratory were subjects. Apparatus. The apparatus was the same as the one employed in Expt I. Procedure. The reversal-set training procedure used was similar to that used in Expt I, except: (1) the subjects were given only 10 trials/day; and (2) half of the animals were trained with a 2-min ITI, and the other half with a 60-min 1TI. After 250 trials, the 60-min ITI was changed to 15 min. The problem was reversed when each subject successfully avoided footshock on five successive trials. Since only 10 trials were given within a day, the five reversal criterion trials could occur within a day or across two successive days. Each reversal of the position discrimination was separated by the training ITI. The learning-set criterion for each subject was no more than one error on three consecutive reversal problems. The test or delayed-response phase of the experiment began for each subject the day following attainment of the set criterion. Each subject reaching the criterion was tested at ITls of 0 min (<5 sec), 2, 15, 60, and 240 min. Only one interval was presented on a given day. Each test day was followed by a control day: subjects were trained to two reversals with the same 2-min or 15-min ITI used in training, and each reversal was separated by the time equivalent to the ITI. Ascending and descending series with respect to ITI were run. To assess whether the footshock had some sort of nonspecific effect upon performance when the 0-min ITI was employed, each subject was tested after completion of the series, at its training ITI (either a sign trial followed by five test trials all separated by 2 min or a sign trial followed by five test trials all spaced 15 min apart) with a noncontingent footshock immediately preceding each test trial. The noncontingent footshock (3 mA delivered to a 10 K resistance) was administered through a grid floor in a white Plexiglas box measuring 15 × 15 × 7 cm. Results By the 250th trial, six subjects in the 2-min ITI group met the reversal set criterion; whereas, no subjects from the 60-min ITI group reached criterion. Fisher's exact test for a 2 × 2 contingency table revealed that this difference was highly significant (P <0.0l; Hays, 1963). After the ITI for the 60-min group was lowered to 15 min, five of the

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group reached the reversal-set criterion within 100 trials. By trial 350, a total of nine subjects in the 2-rain ITI group attained this criterion. Testing was performed on these samples. T tests revealed no significant differences between the scores obtained on the ascending and descending series, thus the data for each ITI were pooled. The results for both the 2- and 15-min-ITI-trained groups replicated the findings of Expt I: retention of the information acquired on the sign trial after reaching a peak at 2 rain and remaining somewhat stable to 15 rain fell dramatically at 60 min (see Fig. 3). 9O

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Fig. 3. The percentage of correct responses made by the animals at each ITI tested in Expt. II for: (1) the group initially trained with a 2-rain ITI and (2) the group initially trained with a 60-rain and then 15-min ITI. An analysis of variance with repeated measures of the error scores revealed a highly significant intertrial interval effect [F= (5, 60) 14.64, P < 0 . 0 0 1 ] ; a significant difference between the training procedures was not found (F = (1,12) 2.37). Orthogonal comparisons between consecutive intervals collapsed across training procedures revealed that, as in Expt I, only the differences between the 0-min and 2-min ITIs [ F = ( 1 , 60)12.38, P < 0 . 0 0 1 ] and the 15-min and 60-min ITIs I F = ( 1 , 60) 12.38, P < 0 . 0 0 1 ] were significant. No differences were found when the noncontingent footshock groups were compared with their respective control intervals (either the 2-min or the 15-min test interval). EXPERIMENT III Methods S u b j e c t s . Eight female mice of the C57BL/6J strain were acquired from The Jackson Laboratory, Bar Harbor, Me. The subjects were 90 days of age at the beginning of training.

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Apparatus. The apparatus was the same as that employed in Expts I and II. One addition, however, was made to the T-maze. A speaker which generated a 4000-Hz, 105-db tone was located directly above the choice point. This tone was sounded for 0.5 Sec and was terminated at the onset of shock in the start alley. Procedure. Training of the subjects was conducted in a similar manner to that described in Expt I, except: (1) a 5-min ITI separated all trials during set training; (2)5 min separated successive reversals given on the same day; (3) the position reversal criteria were correct responses on 5/6 trials with correct responses on the last two trials; (4) the training schedule was modified such that after the initial discrimination was learned on Day 1, two position reversals were given on Day 2, three position reversals on Days 3-7, two position reversals on Days 8 and 9, and thereafter only one position reversal until set criterion was attained; (5)the set criterion was less than 0.5 mean erxors for the group on 2/3 successive days. Delayed-response testing was conducted in a fashion similar to that described for Expt I, except: (1)the sign trial was followed, after a given delay interval, by just a single test trial; (2) thereafter, using a 5-rain ITI, the animals were trained to a 5/6 criterion with correct responses on the last two trials; (3)after the 5/6 criterion was met, no further position reversals were administered; (4)on the first 5 days of delayed-response testing, the delay interval between the sign trial and test trial was 0 rain (< 5 sec). Subsequently, delay intervals of 5, 15, 30, 45 and 60 min were each presented for 5 successive days in an ascending manner; (5) control days were not interspersed between days because all the animals were trained to criterion each day. Results By Day 21 (the thirty-third reversal), the subjects met the reversal set criterion. The major finding for the delayed-response phase was that retention of information acquired on the sign trial after reaching a peak at 5 rain decayed gradually to chance levels at 60 rain (see Fig. 4). An analysis of variance with repeated measures of the errors made on the daily test trial revealed a significant intertrial interval effect [F(5, 30)= 2.76, P < 0 . 0 5 ] . Of the planned comparisons made between the peak-performance group and all other groups, only the differences between the 5-min and the 0-min delay-interval groups [F (1,35)=4.28, P<0.05] and the 5-rain and the 60-rain delay-interval groups IF (1,35) = 7.12, P < 0.02] were significant. DISCUSSION The results provide a clear indication of the efficacy of measuring STM in mice with the delayed-response technique employed and indicates that the

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Fig. 4. The percentage of correct responses at each delay interval made by the animals on a single daily test trial in Expt III. STM trace, after reaching a peak within 2-5 min after an experience, decays within an hour. An alternative interpretation of the results, excluding a memorial hypothesis, is that the results represent temporal generalization around the ITI used during training. This explanation can be rejected since peak performance during the testing phase occurred at 2 rain regardless of whether the mice were trained initially with a 2- or 15-rain ITI. Further support for a STM interpretation is that the animals trained to acquire the reversal set with ITI of 60 rain (an interval found to be beyond the temporal limit for errorless reversal responding) failed to develop the set within 250 training trials, whereas, animals trained with an ITI of 2 min had little difficulty developing the set within the same number of trials. It might be argued that the training procedure for the 60-rain ITI was not pursued long enough. Nevertheless, within the 250 trials administered, no member of the group even approached the set criterion. If a STM system were sustaining performance, a breakdown of delayed-reversal behavior should have occurred when information was presented beyond the span of STM. The results support this supposition. Since each day the animals were given a sign trial followed by five test trials in the test phase of Expts I and II, it might be claimed that the five test trials were equivalent to repetitive sign trials; and, consequently a possible influence of long-term memory cannot be completely discounted. Experiment III was designed to clarify the possible effects of multiple trials. Here, a single test trial followed the sign trial. These results thus reflect retention of information processed after a single exposure. The significant finding was that the general shape of retention curves found in Expts I and II were replicated. If there was an effect of multiple trials, it was merely to increase by some

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constant amount each ordinate value of the curve. Moreover, numerous investigations concerned with long-term memory processes reveal that performance on maze problems correlates positively with temporal spacing (as great as 24hr) between trials (McGaugh et al., 1962; McGaugh and Cole, 1965). Performance should, thus, have been elevated at the longer delay intervals if long-term memory were, indeed, an influencing factor. No such observation was made (see Figs. 2 and 3). It should be noted that the learning-set criteria for Expts I and III were group criteria. Because of the type of question asked and the training procedure employed, an individual learning-set criterion was more appropriate for Expt II. Where a group criterion was used, it represented the performance of the majority of the animals within that group. That the effects of the different criteria were negligible is substantiated by the almost identical retention curves found in Expts I and II. Moreover, performance on control days was virtually the same in these two experiments. During the test phase of Expt I, performance at the 0-min ITI was as poor as performance at 60 rain. The noncontingent shock control groups employed in Expt II do not support the contention that some nonspecific or proactive effect of footshock produced the observed decrement in performance at the 0-min ITI. Furthermore, employing a similar procedure, Alpern and Marriott (1972) provided three inbred strains of mice a light and a shock cue on sign trials. Whether the animals were using the shock or the light could be determined. Although there were strain differences in the proportion of the time each cue was utilized, retention over the delay intervals within a strain was the same regardless of whicti cue was used. It appears, therefore, that this decrement is a real memorial effect and implies that the STM trace requires some modicum of time to attain maximal development. The fact that mice were able to develop the successive reversal learning set suggests that this species has the capacity to utilize simple conceptual processes in problem solving. Although previously reported for rats (Petrinovich and Bolles, 1957; Petrinovich et al., 1965), this represents the first clear demonstration of conceptual ability in mice.

REFERENCES Alpern, H.P. and Marriott, J.G. (1972). An analysis of short-term memory and conceptual behavior in three inbred strains of mice. Behav. Biol. in press. Bovet, D., Bovet-Nitti, F., and Oliverio, A. (1969). Genetic aspects of learning and memory in mice. Science 163, 139-149. Deutsch, J.A. (1969). The physiological basis of memory. Annu. Rev. Psychol. 20, 85-104.

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Duncan, N.C., Grossen, N.E., and Hunt, E.B. (1971). Apparent memory differences in inbred mice produced by differential reaction to stress. J. Physiol. Comp. Psychol. 74, 383-389. Glassman, E. (1969). The biochemistry of learning: An evaluation of the role of RNA and protein. Annu. Rev. Biochem. 38, 605-646. Hays, William L. (1963). "Statistics," p. 598. New York: Holt, Rinehart and Winston. McGaugb, J.L. (1966). Time-dependent processes in memory storage. Science 153, 135141358. McGaugh, J.L. and Cole, J.M. (1965). Age and strain differences in the effect of distribution of practice on maze learning. Psychon. Sci. 2, 253-254. McGaugh, J.L. and Dawson, R.G. (1971). Modification of memory storage processes. Behav. Sci. 16, 45-63. McGaugh, J.L., Jennings, R.D., and Thomson, C.W. (1962). Effect of distribution of practice on the maze learning of descendants of the Tryon maze bright and maze dull strains. Psychol. Rep. 10, 147-150. Petrinovich, L., Bradford, D., and McGaugh, ].L. (1965). Drug facilitation of memory in rats. Psychon. Sci. 2, 191-192. Petdnovich, L. and Bolles, R. (1957). Delayed alternation: Evidence for symbolic processes in the rat. J. Comp. Physiol. Psychol. 50, 363-365.