BEHAVIORALAND NEURALBIOLOGY58, 144--151 (1992)
Those Cheating Rats Male and Female Rats Use Odor Trails in a Water-Escape "Working Memory" Task LARRY W . MEANS, STEVEN R. ALEXANDER, AND MARGARET F. O ' N E A L 1
Department of Psychology, East Carolina University, Greenville, North Carolina 27858
tial delayed matching-to-sample). The optimal retention interval for acquisition appears to be 5 min (Means & Dent, 1991). To save time, rats are run in squads with all animals in a squad receiving their information run before any rat in the squad is given a test run. The task is sensitive to age (Means & Kennard, 1991), estrogen therapy in ovariectomized females (O'Neal, Poole, Hamm, & Means, 1990), and gender (Means & Dent, 1991). An earlier version of the task is sensitive to nootropics (Means, Comer, & Moore, 1991) and a three-choice version is sensitive to tranquilizers (Bass, Means, & McMillen, 1992). When we first used female rather than male rats in the task (O'Neal et al., 1990), we were surprised to notice behaviors we had not seen with males. We observed that as well as sniffing the water, females often swam next to and sniffed the bar of the Tbarrier opposite the start position (Fig. 1) before making a choice. Sometimes they swam to the opening of a choice section, clung to the divider, and seemed to be sniffing the air inside the section. We questioned whether in a water maze, rats might be able to use odor cues to locate the escape platform. It is well documented that in dry mazes rats use their own odor trails or those of previously run rats to solve tasks in which working memory is pertinent. In the T-maze, rats use odor trails in spontaneous alternation (Douglas, 1966; F r a n k e n & Baker, 1969) and in single (Amsel, Hug, & Surridge, 1969) and double (Ludvigson & Sytsma, 1967; Prytula & Colbert, 1975) alternation acquisition. Although rats learned to follow the trail of a previously reinforced rat in a T-maze (Means, Hardy, Gabriel, & Uphold, 1971), they were only able to do so when a homogeneous running system was employed. A homogeneous running system involves rewarding all subjects for going to the same choice section of the apparatus. In a heterogeneous system
Three-month-old Sprague-Dawley rats were trained on a working memory win-stay (spatial delayed matchingto-sample) water-escape task with the escape platform location the same for all subjects on a given trial, a procedure that maximizes the buildup of an odor trail to the escape platform. In subsequent tests during which the location of the escape platform varied randomly between subjects, the rats, especially the females, while continuing to perform above chance level, made increased errors. Varying the platform location between subjects eliminated odor trail as a nonambiguous cue for locating the escape platform. In a second experiment females performed better than males on a reference memory odor trail discrimination task which involved following the path of like-gender "pathmaker" rats to the escape platform. The relatively poor use of odor trails by the males was associated with a high frequency of choosing a preferred choice section or returning to the choice section selected first on the immediately preceding trial (perseveration). Collectively, the two experiments demonstrate that rats can use either working memory or odor trails to locate an escape platform in a water maze, and that they, especially females, will use odor trails in a working memory task if odor trails are available. Clearly, the location of the escape platform should be varied randomly between subjects in tests of working memory. © 1 9 9 2 Academic Press,
Inc.
In our laboratory we have used a two-choice water-escape task (apparatus is shown in Fig. 1) to assess working memory (Comer & Means, 1989; Means, 1988). The task, as currently used, involves giving subjects trials consisting of a forced-choice information run (pathway to incorrect choice section is blocked) and a free-choice test run following a retention interval. Typically, on the test run the escape platform is placed in the same choice section as on the information run (called w i n - s t a y or spai Please address correspondence and reprint requests to Larry W. Means. 144 0163-1047/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
USE OF ODOR TRAILS BY RATS IN MEMORY TASKS odor trails are confounded by rats going to different choice sections to receive reward. Use of a heterogeneous procedure in the double-alternation dry maze task also reduced the effects of odor cues to the point that they were no longer influential (Ludvigson & Sytsma, 1967). However, for several reasons we thought it unlikely that rats used odor cues in our water-escape working memory task. Many researchers have demonstrated in dry maze situations that experimental contamination by odor cues is rather easily avoided. Researchers have concluded that odor cues are airborne and wire mesh tops on dry mazes (Pitt, Davis, & Brown, 1973) or exhaust fans (Phillips & Bloom, 1971) are probably sufficient to prevent use of trails. Even without using special procedures to control for odor cues, rats reportedly do not rely on odor to avoid previously entered alleys in the radial arm maze (Olton & Samuelson, 1976; Zoladek & Roberts, 1978). In several of our earlier water maze studies, however (Comer & Means, 1989; Means, 1988; Means & Bass, 1990; Means & Kennard, 1991), we used a homogeneous testing procedure with male rats, and thus, we wondered if use of odor trails might have contaminated the experiments. Alternatively, since we had never observed extensive sniffing in males, we suspected that females might be more inclined than males to use odor trails. The present study, therefore, was conducted to determine whether male and/or female rats use odor trails to locate the escape platform while performing a working memory task in the water maze. We also attempted to evaluate the importance of response biases in influencing choices made by male and female rats in water-escape tasks.
~OAPE PLATFORM LOCATIONS
DIVIDERS
_IDINEI PANEL
qG POSITION
F I G . 1.
All subjects were naive S p r a g u e - D a w l e y rats (Charles River), 7 5 - 9 0 days old at the beginning of the experiments. The male and female p a t h m a k e r rats used in Experiment 2 were 90 and 150 days of ,age, respectively. Animals were housed individually in 32 z 27 x 20 cm plastic cages and had free access to food and water. The housing and testing rooms were maintained at 22 _+ 2°C. A 16:8 h light:dark cycle was in effect in which the lights came on at 700 h.
T h e c i r c u l a r two-choice w a t e r m a z e is i l l u s t r a t e d .
water at room temperature to a depth of approximately 34 cm. The water was changed every 5 days, and a tablespoon of Clorox bleach was added each morning. The water was clouded by the addition of approximately 150 ml of nontoxic white paint. A Tshaped stainless steel divider (51 cm high, stem 105 cm long, bar 72 cm long) divided the circular tank into a start section and two choice sections. Adjacent to the bar was a sliding panel used to close either choice section off from the start section. The start section was 35 cm long at its longest point, and the width of the entrance to either choice section was approximately 21 cm. A circular platform, 29 cm in diameter, was placed in the center of the correct choice section. The top of the platform was submerged approximately 1 cm below the surface of the water. The maze was located in a rectangular room with two solid walls, a wall with a row of windows covered by Venetian blinds and a wall with a distinct door; prominent extramaze cues were available.
GENERAL M E T H O D
Subjects
145
EXPERIMENT 1 The first experiment was conducted to determine if while being trained on a working memory task, either male or female rats would use the odor trails of rats that preceded them to locate the escape platform on test trials. Thus, we trained rats under homogeneous conditions and subsequently tested them under both homogeneous and heterogeneous conditions. We assumed that if the animals used odor trails to locate the escape platform, their performance should drop significantly when tested under heterogeneous conditions which eliminates unambiguous odor trails.
Apparatus
Procedure
The maze (see Fig. 1) was a circular metal tank, 140 cm in diameter and 60 cm in height, filled with
Ten male and ten female rats were handled individually for 4 min once a day for 2 days and then
146
MEANS, ALEXANDER, AND O'NEAL
given two sessions of platform training per day for 2 days. Each session of platform training consisted of two trials, one in each choice section of the maze. For each trial, each subject was placed just inside the entrance of a choice section in which the platform was located. The sliding panel was closed and the animal was allowed 2 min to find and climb onto the platform; an animal that did not find the platform in the allocated time was placed on it. The subject remained on the platform for approximately 10 s while the escape latency was recorded. Maze training began the day after platform training was completed. Each rat was given a pair of trials during each of two daily training sessions which were at 0700 and at 1300 h Monday through Friday. A pair of trials consisted of a forced choice information trial (the incorrect choice section was closed) followed 5 min later by a free choice test trial (neither choice section was closed). Subjects were randomly assigned to a running order, and that order was followed each day. Subjects were run in squads of five or six animals. All subjects within a squad were given information trials; then all were given test trials. The platform was in the same position for all animals within a session (homogeneous condition); the position of the platform varied randomly between the AM and PM sessions. On all trials, the subject was placed in the start section against the perimeter of the tank, facing the center of the bar. The rat was released, and a stopwatch was activated to begin the latency measure; timing was terminated when the subject's paw touched the platform. If the subject entered the incorrect choice section, the panel was closed, confining the animal to the incorrect choice section for 30 s. The panel was then opened allowing the rat to swim back into the start section and then into the correct choice section. Thus, a correction procedure was used. Subjects were not confined to the incorrect choice section more than once on a given trial, and the 30-s confinement times were subtracted from the total latency. For test trials, a rat was considered to have entered a choice section when its entire body was within a choice section. If the choice section entered first contained the escape platform the choice was scored as correct. Once animals had reached a criterion of 9 correct choices on 10 consecutive test trials, they were given 48 pairs of trials over 24 days. The procedure was identical to training except that for a randomly determined 25% of the trials, the location of the escape platform varied randomly between subjects (heterogeneous condition) within a session rather than being the same for all animals. For the purpose of
analysis, the 48 pairs of trials were divided into three blocks of 16 pairs of trials to determine if animals response pattern changed across trials.
Results and Discussion Three female subjects and one male subject failed to reach criterion and were dropped from the study. None of the subjects dropped was the first of its gender to be run in the established order. Of the 16 remaining subjects, there was not a significant difference between males (M +_ SEM = 17 + 1.24) and females (M -+ SEM = 17 _+ 1.66) on number of trials needed to reach the maze acquisition criterion. Group means of individual rat's median latencies for the first five and the last five acquisition trials were also compared; a 2 x 2 (Gender x Block) ANOVA revealed that latency did not change across blocks but females (M = 15.1 s) were slower to escape than were males (M = 7.5 s): F(1, 14) = 11.75, p < .05. For the testing period the percentage of correct choices made was above chance level for both genders in both the heterogeneous and homogeneous conditions across all three blocks of trials (one-sample t tests, p < .05 in all cases). A 2 x 2 x 3 mixed factors ANOVA (Gender x Condition x B l o c k ) o f the percentage of correct choices produced significant main effects for Condition, i.e., whether the correct choice section was homogeneous or heterogeneous: F(1, 14) = 6.40, p < .05; and Block: F(2, 28) = 3.46, p < .05. The rats made a higher percentage of correct choices on homogeneous test trials (mean = 84.21) than on heterogeneous test trials (mean = 76.23). Also, the mean percentage of correct choices was numerically lower for the first block (mean = 74.25) than for the second (mean = 82.22) or third (mean = 84.19) blocks. However, N e w m a n Keuls tests did not reveal the differences to be significant. The Condition x Block effect approached significance: F(2, 28) = 3.11, p = .059; Fig. 2 reveals that the subjects, especially the females, made fewer correct choices during the first block of trials under the heterogeneous condition. Because the Block effect was significant and correct choice data for the first block appeared strikingly different, data for the first heterogeneous block were submitted to a 2 x 2 x 2 ANOVA (Gender x Condition x Gender of Preceder). As in the overall data, subjects made more correct choices when the correct choice section was homogeneous (see Fig. 3): F(1, 11) = 7.21, p = .021. There was also a significant interaction between Condition and Gender of Preceder: F(1, 11) = 9.11, p < .05. Percentage of correct choices was significantly lower (p
147
USE OF ODOR TRAILS BY RATS IN MEMORY TASKS
HOMOGENOUS
HETEROGENOUS
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I I I Females 90
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FIG. 2. Experiment 1: Correct choices across three blocks of trials are shown.
< .01, Newman-Keuls) when a subject was immediately preceded in the maze by a subject of the other gender in the heterogeneous condition. The three-way latency ANOVA revealed that as was the case during acquisition, during testing females (mean = 21.69 s) were slower to escape than males were (mean = 5.62 s): F(1, 14) = 12.03, p < .01), but there was no significant main effect for Condition or Block. Experiment 1 demonstrated that both male and female subjects used the trails of rats previously run in the maze to locate the escape platform, but not exclusively. Despite the use of trails, throughout the testing period subjects were able to make the correct choice based on the use of a working memory (win-stay) strategy at above chance level. They made fewer correct choices, however, when they ran in the heterogeneous condition, and trails of preceding rats did not predict the escape location. The tendency to use trails appeared strongest in the first block of the heterogeneous condition. Subjects apparently learned to use trails during training when the homogeneous condition was in effect, but abandoned the use of trails following experience with the heterogeneous condition. The fact that the first subject in the running order (a male) acquired the task further suggests that trails were not essential for learning water escape. Both genders were more likely to use trails if the immediately preceding rat was of the other sex. Females were slower than males to escape, perhaps due to the time spent sniffing and clinging to the barrier. In following the trail of previously run rats, it is possible that rats could see a turbulence in the cloudiness of the water. While we cannot rule out the use of visual cues, we did not observe the presence of any turbulence or any other visual cues at the
time that trials were initiated. Also, we observed considerable sniffing behavior, suggesting that the rats were using odor cues. Finally, the fact that both genders were more likely to use trails if the immediately preceding rat was of the other sex further suggests that odor was the cue being used. EXPERIMENT 2 Because the first experiment strongly suggested that the rats, especially the females, used odor trails to improve their performance on the "working memory" task, we reasoned that rats should be capable of learning to locate the escape platform when the odor trail of other rats is the only relevant cue. Thus, we sought to determine if rats could acquire an odor trail discrimination in the water maze. For this task working memory was irrelevant. We also did a post hoc analysis to examine unlearned response strategies that influenced the rats' choices in the water maze.
Procedure To avoid confounding effects of scents of both genders in the water maze simultaneously, Experiment 2 was conducted in two stages; the data were collected for male subjects first and then for females. For the males, 4 rats, previously trained on a winstay water-escape task, were used as "pathmakers," and 10 naive rats served as subjects or "followers." Each of the 14 animals was handled for 3 min twice per day for 3 days and once on the fourth day. The followers were then given two platform training sessions as described above. The procedure was the same for females, but there were 4 pathmakers and 11 followers. Subsequent procedures involving measurement of escape latencies and correct choices,
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GENDEROFPRECEDER FIG. 3. Experiment 1: Correct choices for the first block of trials are shown.
148
MEANS, ALEXANDER, AND O'NEAL
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FIG. 4. Experiment 2: Correct choices across six blocks of trials. (*p < .05; **p < .01).
release of subjects, failure to locate the platform, etc. were also the same as in Experiment 1. Three testing sessions (0800, 1300, and 1600 h) per day were given Monday through Friday for 25 days, with platform position random between trials and homogeneous between subjects on any given trial. During a session each p a t h m a k e r consecutively was forced to the choice section containing the escape platform. The followers were t h e n tested to determine if they could use odor cues left by the animals preceding them. Each follower was given one free choice trial. If the first follower made a correct choice, then the next follower was immediately tested, and so on for the remaining followers. If a follower made an incorrect choice, the front of the barrier, the sliding panel, the incorrect choice section and the start section walls were wiped with a cloth, and the water was stirred to diffuse possible odor cues. The four pathmakers were then run again, and the testing procedure continued with the next follower. Thus, at least four rats consecutively had swum to the escape platform before any follower was given a trial. All detectable ripples resulting from rats swimming in the water dissipated before
l
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FIG. 5. Experiment 2: Escape latencies across six blocks of trials are shown.
follower rats were placed into the maze, eliminating the possibility of follower rats using visual or tactile cues to find the platform. On Days 26-30, the subjects were tested to determine if they could find the escape platform when odor trails did not predict the location of the escape platform. Thus, the pathmakers were eliminated, and free choice trials were given to each follower on the same schedule described above. The platform position was heterogeneous between subjects. Maze walls and barriers were not wiped, and the water was stirred only after every fifth rat completed a trial. The 90 trials were divided into 6 blocks of 15 trials each. The m e a n n u m b e r of correct choices was determined for each rat for each block. Also, each rat's median escape latency was calculated based on the five trials given at 1300 h. Results and Discussion A 2 x 6 (Gender × Block) ANOVA (see Fig. 4) revealed that females (M = 9.87) made significantly more correct choices t h a n males (M = 8.48) did: F(1, 18) = 5.53, p < .05. The effect for Block was significant (F[5, 90] = 10.173, p < .001) as was the Gender x Block interaction (F[5, 90] = 2.800, p < .05). N e w m a n - K e u l s post hoc tests showed that during Blocks 2 and 5 females made significantly (p < .05) more correct choices t h a n did males. Figure 4 further reveals that with the exception of the first block, females made more correct choices t h a n would be expected by chance (one-sample t test, p < .05) on all blocks of trials in which pathmakers were included. Males were above chance level only on the last two acquisition blocks. Neither gender performed above chance with pathmakers absent (Block 6). Since the platform was present on all trials during Block 6, the chance level performance of both groups demonstrates that the rats were not using any cue (visual, tactile, odor) originating from the platform itself. Thus, the subjects were able to learn to use an odor trail to locate the escape platform. The females were much better at using odor trails t h a n were the males. The Gender x Block ANOVA for latency revealed that females were slower to escape t h a n were males: F(1, 18) = 6.07, p < .05 (see Fig. 5). The significant main effect for Block (F[5, 90] -14.63, p < .001) reflects a decrease in latency over acquisition trials. Because the males performed so poorly when an odor trail was the only relevant cue, a post hoc analysis was conducted. We reasoned that if males
USE OF ODORTRAILS BY RATS IN MEMORYTASKS
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.
MALES FEMALES
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.
.
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4o I ODOR PERSEV.WIN-mAYPREFER. RICHT CRITERION FIG. 6. Experiment 2: Escape strategies are shown. Asterisks above the bars represent performance that is significantly above chance level. Asterisks below the bar denote significant differences in performance between males and females. (*p < .05; **p < .01).
149
responses in accord with Perseveration (one-sample t test; p < .01), Position Preference (p < .01), and Win-Stay (p < .05) strategies, whereas females responded in accord with Position Preference (p < .01) and Perseveration (p < .05). In addition, males and females differed (two-sample t test) on the percentage of trials in which their responses were consistent with the various strategies. Females responded in accord with Odor Trials more frequently than did males (p < .05). Males choices were consistent with Perseveration and Win-Stay strategies more frequently than were females (p < .05 in each case). Because the data in Fig. 6 represent the same responses scored according to different criteria, statistical comparisons across criteria are not legitimate. GENERAL DISCUSSION
were having a difficult time using an odor trail to fiad the escape platform, unlearned response biases might have directed their choices. Thus, the data for all subjects were scored for number of choices made according to several potential strategies: (1) Perseveration, the percentage of first choices that were the same as the first choice on the immediately preceding trial; (2) Win-Stay, the percentage of first choices that were a return to the choice section where escape was made on the immediately preceding trial; (3) Position Preference, the percentage of choices made by each rat to its preferred choice section, the preferred choice section being the choice section entered first most frequently over all trials; (4) Right choice section Bias, percentage of first choices that were made to the right choice section. Figure 6 shows the percentage of first choices that involved correctly following the odor trail and that were made in accord with the above described alternative unlearned strategies. Every single response was scored according to all criteria. Thus, a given response could have been consistent with several of the strategies. For example, if a rat correctly followed an odor trail to the right choice section, which happened to have been the choice section it selected most frequently on all trials and the first choice section entered on the preceding trial, he was scored as having made a choice in accord with the Odor Trail, Right Bias, Position Preference, and Perseveration strategies. Even though in the present experiment all strategies other than following the odor trail would have resulted in only 50% correct choices, the rats choices were made in accord with other strategies significantly more often than would have been expected by chance (one-sample t tests). Thus, besides using odor trails, males made
As in the dry maze (Ludvigson, 1969), subjects in Experiment 1 were more successful with the task when run under homogeneous conditions, demonstrating that rats are able to use odor cues in the water maze. The experiment further showed that although rats, especially females, can employ odor cues in the water, they are able to use a working memory strategy (i.e., win-stay) to make correct choices at above chance level when olfactory cues provide ambiguous information. The finding reassures us in regard to conclusions made in previous studies run with males in homogeneous conditions (Means, 1988; Comer & Means, 1989; Means & Bass, 1990; Means & Kennard, 1991), but makes evident the fact that a heterogeneous running procedure should be used in water-maze working memory studies with either gender. The females' stronger tendency to use olfactory cues resulted in more correct choices for females than for males when the escape contingency was odor based. Females performed significantly above chance level by the second block of trials in the odor trail discrimination, whereas males performed above chance level only on the fourth and fifth blocks of trials. Also, the females attained and maintained a higher level of performance. It seems paradoxical that females appear more innately prepared than males to utilize odor cues, given the fact that during three of her four estrous cycle stages, the female rat is less sensitive to olfactory cues than is the male (Pietras & Moulton, 1974). Escape latencies for the females were longer than those for males in both experiments, probably due to time spent in olfactory investigation. Archer (1975) found that in the open field females spend
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MEANS, ALEXANDER, AND O'NEAL
more time sniffing than males do. In our study latencies for the females might be longer than for males because trying to discern odor cues in a water maze probably requires more time than making a choice based on the strategies that males prefer. The fact that odor cues might be more difficult to detect in a water maze than in a dry maze may explain the fact that we found gender differences in latencies, and Eslinger and Ludvigson (1980) did not; in the water maze females' use of their preferred odor trail strategy would presumably be a much more difficult and time consuming task. We assume that the females were actively searching for odor cues when they engaged in behaviors such as sniffing and clinging to the barriers, although the present study did not provide information about the locus of the odor trails. Our observations lead us to question whether our conclusions apply to water mazes that do not contain barriers. It may very well be that if the escape location is the same across trials and between subjects and the water is not stirred between subjects, rats are capable of using odor trails to locate the escape platform. The odor trial would not necessarily have to be a "path" to the platform to be valuable. If the procedure is such that the over the course of the experiment the platform is placed in only a few locations, as is commonly the case, the rats use reference memory to avoid those locations where the platform is never placed. Rats then would only need a detectable difference in the strength of odor cues near the relevant escape locations in order to use odor cues to make correct or efficient escape responses. A testing procedure in which the escape location is the same over trials within a session and/or between subjects tested within a session and the water is not stirred between trials would seem to maximize the potential for the buildup of a detectable odor difference. Also, many investigators use milk to cloud the water instead of nontoxic paint as we use. Milk may be a better medium for odor than is water-base paint. Clearly, whenever design restrictions permit, it would seem prudent, especially in working memory and "cognitive map" studies, to randomly vary the escape location between trials and/or subjects. If the experimental design precludes varying the escape location, portions of the apparatus touched by a subject should be wiped and the water should be stirred between trials. REFERENCES Amsel, A., Hug, J. J., & Surridge, C. T. (1969). Subject-to-subject trial sequence, odor trials, and patterning at 24-h ITI. Psychonomic Science, 15, 119-120.
Archer, J. (1975). Rodent sex differences in emotional and related behavior. Behavioral Biology, 14, 451-479. Bass, E. W., Means, L. W., & McMillen, B. A. (1992). Buspirone impairs performance of a three-choice working memory water escape task in rats. Brain Research Bulletin, 28, 455461. Comer, T. R., & Means, L. W. (1989). Overcoming unlearned response biases: Delayed escape following errors facilitates acquisition of win-stay and win-shift working memory waterescape tasks in rats. Behavioral and NeuralBiology, 52, 239250. Douglas, R. J. (1966). Cues for spontaneous alternation. Journal of Comparative and Physiological Psychology, 62, 171-183. Eslinger, P. J., & Ludvigson, H. (1980). Commonality among rats in production of reward and nonreward odor. Bulletin of the Psychonomic Society, 16, 191-193. Franken, R. E., & Baker, J. G. (1967). Drive level and cue utilization in spontaneous alternation. Psychonomic Science, 8, 89-90. Franken, R. E., & Baker, J. G. (1969). The effects of drive level on cues utilized in spontaneous alternation. Psychonomic Science, 16, 239-240. Ludvigson, H. W. (1969). Runway behavior of the rat as a function of intersubject reward contingencies and constancy of daily reward schedule. Psychonomic Science, 15, 41-43. Ludvigson, H. W., & Sytsma, D. (1967). The sweet smell of success: Apparent double alternation in the rat. Psychonomic Science, 67, 283-284. Means, L. W. (1988). Rats acquire win-stay more readily than win-shift in a water escape situation. Animal Learning and Behavior, 16, 303-311. Means, L. W., & Bass, E. W. (1990). Four-choice win-stay water escape acquisition by rats. Psychological Reports, 66, 67-70. Means, L. W., Comer, T. R., & Moore, R. (1991). Bmy 21502 and piracetam facilitate performance of two-choice win-stay water-escape in normal rats. Journal of Neural Transmission, 85, 109-116. Means, L.W., & Dent, M.F. (1991). The effects of number of trials per day, retention interval, gender and time of day on acquisition of a two-choice win-stay water-escape working memory task in the rat. Journal of Neuroscience Methods, 39, 77-87. Means, L. W., Hardy, W. T., Gabriel, M., & Uphold, J. D. (1971). Utilization of odor trails by rats in maze learning. Journal of Comparative and Physiological Psychology, 76, 160-164. Means, L. W., & Keunard, K. J. P. (1991). Working memory and the aged rat: Deficient two-choice win-stay water-escape acquisition and retention. Physiology and Behavior, 49, 301307. Olton, D. S., & Samuelson, R. J. (1976). Remembrance of places passed: Spatial memory in rats. Journal of Experimental Psychology: Animal Behavior Processes, 2, 97-116. O'Neal, M. F., Poole, M. C., Hamm, R. J., & Means, L. W. (1990). Estrogen protects performance on a working memory task in ovariectomized rats [Abstract]. Second Annual Convention of the American Psychological Society, 49. Phillips, J. M., & Bloom, J. M. (1971). Control of conspecific odors in the runway. Psychological Reports, 29, 838. Pietras, R.J., & Moulton, D. G. (1974). Hormonal influences on odor detection in rats: Changes associated with the estrous cycle, pseudopregnancy, ovariectomy, and administration of
USE OF ODOR TRAILS BY RATS IN MEMORY TASKS testosterone propionate. Physiology and Behavior, 12, 475491. Pitt, S., Davis, S.F., & Brown, B.R. (1973). Apparent double alternation in the rat: A failure to replicate. Bulletin of the Psychonomic Society, 2, 359-361. Prytula, R. E., & Colbert, J. C. (1975). Administration of trials
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within-Ss and double-alternation responding: A demonstration of a multiple-baseline reversal. Psychological Reports, 36, 131-137. Zoladek, L., & Roberts, W. A. (1978). The sensory basis of spatial memory in the rat. Animal Learning and Behavior, 6, 7781.
Those Cheating Rats Male and Female Rats Use Odor Trails in a Water-Escape "Working Memory" Task LARRY W . MEANS, STEVEN R. ALEXANDER, AND MARGARET F. O ' N E A L 1
Department of Psychology, East Carolina University, Greenville, North Carolina 27858
tial delayed matching-to-sample). The optimal retention interval for acquisition appears to be 5 min (Means & Dent, 1991). To save time, rats are run in squads with all animals in a squad receiving their information run before any rat in the squad is given a test run. The task is sensitive to age (Means & Kennard, 1991), estrogen therapy in ovariectomized females (O'Neal, Poole, Hamm, & Means, 1990), and gender (Means & Dent, 1991). An earlier version of the task is sensitive to nootropics (Means, Comer, & Moore, 1991) and a three-choice version is sensitive to tranquilizers (Bass, Means, & McMillen, 1992). When we first used female rather than male rats in the task (O'Neal et al., 1990), we were surprised to notice behaviors we had not seen with males. We observed that as well as sniffing the water, females often swam next to and sniffed the bar of the Tbarrier opposite the start position (Fig. 1) before making a choice. Sometimes they swam to the opening of a choice section, clung to the divider, and seemed to be sniffing the air inside the section. We questioned whether in a water maze, rats might be able to use odor cues to locate the escape platform. It is well documented that in dry mazes rats use their own odor trails or those of previously run rats to solve tasks in which working memory is pertinent. In the T-maze, rats use odor trails in spontaneous alternation (Douglas, 1966; F r a n k e n & Baker, 1969) and in single (Amsel, Hug, & Surridge, 1969) and double (Ludvigson & Sytsma, 1967; Prytula & Colbert, 1975) alternation acquisition. Although rats learned to follow the trail of a previously reinforced rat in a T-maze (Means, Hardy, Gabriel, & Uphold, 1971), they were only able to do so when a homogeneous running system was employed. A homogeneous running system involves rewarding all subjects for going to the same choice section of the apparatus. In a heterogeneous system
Three-month-old Sprague-Dawley rats were trained on a working memory win-stay (spatial delayed matchingto-sample) water-escape task with the escape platform location the same for all subjects on a given trial, a procedure that maximizes the buildup of an odor trail to the escape platform. In subsequent tests during which the location of the escape platform varied randomly between subjects, the rats, especially the females, while continuing to perform above chance level, made increased errors. Varying the platform location between subjects eliminated odor trail as a nonambiguous cue for locating the escape platform. In a second experiment females performed better than males on a reference memory odor trail discrimination task which involved following the path of like-gender "pathmaker" rats to the escape platform. The relatively poor use of odor trails by the males was associated with a high frequency of choosing a preferred choice section or returning to the choice section selected first on the immediately preceding trial (perseveration). Collectively, the two experiments demonstrate that rats can use either working memory or odor trails to locate an escape platform in a water maze, and that they, especially females, will use odor trails in a working memory task if odor trails are available. Clearly, the location of the escape platform should be varied randomly between subjects in tests of working memory. © 1 9 9 2 Academic Press,
Inc.
In our laboratory we have used a two-choice water-escape task (apparatus is shown in Fig. 1) to assess working memory (Comer & Means, 1989; Means, 1988). The task, as currently used, involves giving subjects trials consisting of a forced-choice information run (pathway to incorrect choice section is blocked) and a free-choice test run following a retention interval. Typically, on the test run the escape platform is placed in the same choice section as on the information run (called w i n - s t a y or spai Please address correspondence and reprint requests to Larry W. Means. 144 0163-1047/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
USE OF ODOR TRAILS BY RATS IN MEMORY TASKS odor trails are confounded by rats going to different choice sections to receive reward. Use of a heterogeneous procedure in the double-alternation dry maze task also reduced the effects of odor cues to the point that they were no longer influential (Ludvigson & Sytsma, 1967). However, for several reasons we thought it unlikely that rats used odor cues in our water-escape working memory task. Many researchers have demonstrated in dry maze situations that experimental contamination by odor cues is rather easily avoided. Researchers have concluded that odor cues are airborne and wire mesh tops on dry mazes (Pitt, Davis, & Brown, 1973) or exhaust fans (Phillips & Bloom, 1971) are probably sufficient to prevent use of trails. Even without using special procedures to control for odor cues, rats reportedly do not rely on odor to avoid previously entered alleys in the radial arm maze (Olton & Samuelson, 1976; Zoladek & Roberts, 1978). In several of our earlier water maze studies, however (Comer & Means, 1989; Means, 1988; Means & Bass, 1990; Means & Kennard, 1991), we used a homogeneous testing procedure with male rats, and thus, we wondered if use of odor trails might have contaminated the experiments. Alternatively, since we had never observed extensive sniffing in males, we suspected that females might be more inclined than males to use odor trails. The present study, therefore, was conducted to determine whether male and/or female rats use odor trails to locate the escape platform while performing a working memory task in the water maze. We also attempted to evaluate the importance of response biases in influencing choices made by male and female rats in water-escape tasks.
~OAPE PLATFORM LOCATIONS
DIVIDERS
_IDINEI PANEL
qG POSITION
F I G . 1.
All subjects were naive S p r a g u e - D a w l e y rats (Charles River), 7 5 - 9 0 days old at the beginning of the experiments. The male and female p a t h m a k e r rats used in Experiment 2 were 90 and 150 days of ,age, respectively. Animals were housed individually in 32 z 27 x 20 cm plastic cages and had free access to food and water. The housing and testing rooms were maintained at 22 _+ 2°C. A 16:8 h light:dark cycle was in effect in which the lights came on at 700 h.
T h e c i r c u l a r two-choice w a t e r m a z e is i l l u s t r a t e d .
water at room temperature to a depth of approximately 34 cm. The water was changed every 5 days, and a tablespoon of Clorox bleach was added each morning. The water was clouded by the addition of approximately 150 ml of nontoxic white paint. A Tshaped stainless steel divider (51 cm high, stem 105 cm long, bar 72 cm long) divided the circular tank into a start section and two choice sections. Adjacent to the bar was a sliding panel used to close either choice section off from the start section. The start section was 35 cm long at its longest point, and the width of the entrance to either choice section was approximately 21 cm. A circular platform, 29 cm in diameter, was placed in the center of the correct choice section. The top of the platform was submerged approximately 1 cm below the surface of the water. The maze was located in a rectangular room with two solid walls, a wall with a row of windows covered by Venetian blinds and a wall with a distinct door; prominent extramaze cues were available.
GENERAL M E T H O D
Subjects
145
EXPERIMENT 1 The first experiment was conducted to determine if while being trained on a working memory task, either male or female rats would use the odor trails of rats that preceded them to locate the escape platform on test trials. Thus, we trained rats under homogeneous conditions and subsequently tested them under both homogeneous and heterogeneous conditions. We assumed that if the animals used odor trails to locate the escape platform, their performance should drop significantly when tested under heterogeneous conditions which eliminates unambiguous odor trails.
Apparatus
Procedure
The maze (see Fig. 1) was a circular metal tank, 140 cm in diameter and 60 cm in height, filled with
Ten male and ten female rats were handled individually for 4 min once a day for 2 days and then
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MEANS, ALEXANDER, AND O'NEAL
given two sessions of platform training per day for 2 days. Each session of platform training consisted of two trials, one in each choice section of the maze. For each trial, each subject was placed just inside the entrance of a choice section in which the platform was located. The sliding panel was closed and the animal was allowed 2 min to find and climb onto the platform; an animal that did not find the platform in the allocated time was placed on it. The subject remained on the platform for approximately 10 s while the escape latency was recorded. Maze training began the day after platform training was completed. Each rat was given a pair of trials during each of two daily training sessions which were at 0700 and at 1300 h Monday through Friday. A pair of trials consisted of a forced choice information trial (the incorrect choice section was closed) followed 5 min later by a free choice test trial (neither choice section was closed). Subjects were randomly assigned to a running order, and that order was followed each day. Subjects were run in squads of five or six animals. All subjects within a squad were given information trials; then all were given test trials. The platform was in the same position for all animals within a session (homogeneous condition); the position of the platform varied randomly between the AM and PM sessions. On all trials, the subject was placed in the start section against the perimeter of the tank, facing the center of the bar. The rat was released, and a stopwatch was activated to begin the latency measure; timing was terminated when the subject's paw touched the platform. If the subject entered the incorrect choice section, the panel was closed, confining the animal to the incorrect choice section for 30 s. The panel was then opened allowing the rat to swim back into the start section and then into the correct choice section. Thus, a correction procedure was used. Subjects were not confined to the incorrect choice section more than once on a given trial, and the 30-s confinement times were subtracted from the total latency. For test trials, a rat was considered to have entered a choice section when its entire body was within a choice section. If the choice section entered first contained the escape platform the choice was scored as correct. Once animals had reached a criterion of 9 correct choices on 10 consecutive test trials, they were given 48 pairs of trials over 24 days. The procedure was identical to training except that for a randomly determined 25% of the trials, the location of the escape platform varied randomly between subjects (heterogeneous condition) within a session rather than being the same for all animals. For the purpose of
analysis, the 48 pairs of trials were divided into three blocks of 16 pairs of trials to determine if animals response pattern changed across trials.
Results and Discussion Three female subjects and one male subject failed to reach criterion and were dropped from the study. None of the subjects dropped was the first of its gender to be run in the established order. Of the 16 remaining subjects, there was not a significant difference between males (M +_ SEM = 17 + 1.24) and females (M -+ SEM = 17 _+ 1.66) on number of trials needed to reach the maze acquisition criterion. Group means of individual rat's median latencies for the first five and the last five acquisition trials were also compared; a 2 x 2 (Gender x Block) ANOVA revealed that latency did not change across blocks but females (M = 15.1 s) were slower to escape than were males (M = 7.5 s): F(1, 14) = 11.75, p < .05. For the testing period the percentage of correct choices made was above chance level for both genders in both the heterogeneous and homogeneous conditions across all three blocks of trials (one-sample t tests, p < .05 in all cases). A 2 x 2 x 3 mixed factors ANOVA (Gender x Condition x B l o c k ) o f the percentage of correct choices produced significant main effects for Condition, i.e., whether the correct choice section was homogeneous or heterogeneous: F(1, 14) = 6.40, p < .05; and Block: F(2, 28) = 3.46, p < .05. The rats made a higher percentage of correct choices on homogeneous test trials (mean = 84.21) than on heterogeneous test trials (mean = 76.23). Also, the mean percentage of correct choices was numerically lower for the first block (mean = 74.25) than for the second (mean = 82.22) or third (mean = 84.19) blocks. However, N e w m a n Keuls tests did not reveal the differences to be significant. The Condition x Block effect approached significance: F(2, 28) = 3.11, p = .059; Fig. 2 reveals that the subjects, especially the females, made fewer correct choices during the first block of trials under the heterogeneous condition. Because the Block effect was significant and correct choice data for the first block appeared strikingly different, data for the first heterogeneous block were submitted to a 2 x 2 x 2 ANOVA (Gender x Condition x Gender of Preceder). As in the overall data, subjects made more correct choices when the correct choice section was homogeneous (see Fig. 3): F(1, 11) = 7.21, p = .021. There was also a significant interaction between Condition and Gender of Preceder: F(1, 11) = 9.11, p < .05. Percentage of correct choices was significantly lower (p
147
USE OF ODOR TRAILS BY RATS IN MEMORY TASKS
HOMOGENOUS
HETEROGENOUS
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FIG. 2. Experiment 1: Correct choices across three blocks of trials are shown.
< .01, Newman-Keuls) when a subject was immediately preceded in the maze by a subject of the other gender in the heterogeneous condition. The three-way latency ANOVA revealed that as was the case during acquisition, during testing females (mean = 21.69 s) were slower to escape than males were (mean = 5.62 s): F(1, 14) = 12.03, p < .01), but there was no significant main effect for Condition or Block. Experiment 1 demonstrated that both male and female subjects used the trails of rats previously run in the maze to locate the escape platform, but not exclusively. Despite the use of trails, throughout the testing period subjects were able to make the correct choice based on the use of a working memory (win-stay) strategy at above chance level. They made fewer correct choices, however, when they ran in the heterogeneous condition, and trails of preceding rats did not predict the escape location. The tendency to use trails appeared strongest in the first block of the heterogeneous condition. Subjects apparently learned to use trails during training when the homogeneous condition was in effect, but abandoned the use of trails following experience with the heterogeneous condition. The fact that the first subject in the running order (a male) acquired the task further suggests that trails were not essential for learning water escape. Both genders were more likely to use trails if the immediately preceding rat was of the other sex. Females were slower than males to escape, perhaps due to the time spent sniffing and clinging to the barrier. In following the trail of previously run rats, it is possible that rats could see a turbulence in the cloudiness of the water. While we cannot rule out the use of visual cues, we did not observe the presence of any turbulence or any other visual cues at the
time that trials were initiated. Also, we observed considerable sniffing behavior, suggesting that the rats were using odor cues. Finally, the fact that both genders were more likely to use trails if the immediately preceding rat was of the other sex further suggests that odor was the cue being used. EXPERIMENT 2 Because the first experiment strongly suggested that the rats, especially the females, used odor trails to improve their performance on the "working memory" task, we reasoned that rats should be capable of learning to locate the escape platform when the odor trail of other rats is the only relevant cue. Thus, we sought to determine if rats could acquire an odor trail discrimination in the water maze. For this task working memory was irrelevant. We also did a post hoc analysis to examine unlearned response strategies that influenced the rats' choices in the water maze.
Procedure To avoid confounding effects of scents of both genders in the water maze simultaneously, Experiment 2 was conducted in two stages; the data were collected for male subjects first and then for females. For the males, 4 rats, previously trained on a winstay water-escape task, were used as "pathmakers," and 10 naive rats served as subjects or "followers." Each of the 14 animals was handled for 3 min twice per day for 3 days and once on the fourth day. The followers were then given two platform training sessions as described above. The procedure was the same for females, but there were 4 pathmakers and 11 followers. Subsequent procedures involving measurement of escape latencies and correct choices,
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GENDEROFPRECEDER FIG. 3. Experiment 1: Correct choices for the first block of trials are shown.
148
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FIG. 4. Experiment 2: Correct choices across six blocks of trials. (*p < .05; **p < .01).
release of subjects, failure to locate the platform, etc. were also the same as in Experiment 1. Three testing sessions (0800, 1300, and 1600 h) per day were given Monday through Friday for 25 days, with platform position random between trials and homogeneous between subjects on any given trial. During a session each p a t h m a k e r consecutively was forced to the choice section containing the escape platform. The followers were t h e n tested to determine if they could use odor cues left by the animals preceding them. Each follower was given one free choice trial. If the first follower made a correct choice, then the next follower was immediately tested, and so on for the remaining followers. If a follower made an incorrect choice, the front of the barrier, the sliding panel, the incorrect choice section and the start section walls were wiped with a cloth, and the water was stirred to diffuse possible odor cues. The four pathmakers were then run again, and the testing procedure continued with the next follower. Thus, at least four rats consecutively had swum to the escape platform before any follower was given a trial. All detectable ripples resulting from rats swimming in the water dissipated before
l
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FIG. 5. Experiment 2: Escape latencies across six blocks of trials are shown.
follower rats were placed into the maze, eliminating the possibility of follower rats using visual or tactile cues to find the platform. On Days 26-30, the subjects were tested to determine if they could find the escape platform when odor trails did not predict the location of the escape platform. Thus, the pathmakers were eliminated, and free choice trials were given to each follower on the same schedule described above. The platform position was heterogeneous between subjects. Maze walls and barriers were not wiped, and the water was stirred only after every fifth rat completed a trial. The 90 trials were divided into 6 blocks of 15 trials each. The m e a n n u m b e r of correct choices was determined for each rat for each block. Also, each rat's median escape latency was calculated based on the five trials given at 1300 h. Results and Discussion A 2 x 6 (Gender × Block) ANOVA (see Fig. 4) revealed that females (M = 9.87) made significantly more correct choices t h a n males (M = 8.48) did: F(1, 18) = 5.53, p < .05. The effect for Block was significant (F[5, 90] = 10.173, p < .001) as was the Gender x Block interaction (F[5, 90] = 2.800, p < .05). N e w m a n - K e u l s post hoc tests showed that during Blocks 2 and 5 females made significantly (p < .05) more correct choices t h a n did males. Figure 4 further reveals that with the exception of the first block, females made more correct choices t h a n would be expected by chance (one-sample t test, p < .05) on all blocks of trials in which pathmakers were included. Males were above chance level only on the last two acquisition blocks. Neither gender performed above chance with pathmakers absent (Block 6). Since the platform was present on all trials during Block 6, the chance level performance of both groups demonstrates that the rats were not using any cue (visual, tactile, odor) originating from the platform itself. Thus, the subjects were able to learn to use an odor trail to locate the escape platform. The females were much better at using odor trails t h a n were the males. The Gender x Block ANOVA for latency revealed that females were slower to escape t h a n were males: F(1, 18) = 6.07, p < .05 (see Fig. 5). The significant main effect for Block (F[5, 90] -14.63, p < .001) reflects a decrease in latency over acquisition trials. Because the males performed so poorly when an odor trail was the only relevant cue, a post hoc analysis was conducted. We reasoned that if males
USE OF ODORTRAILS BY RATS IN MEMORYTASKS
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4o I ODOR PERSEV.WIN-mAYPREFER. RICHT CRITERION FIG. 6. Experiment 2: Escape strategies are shown. Asterisks above the bars represent performance that is significantly above chance level. Asterisks below the bar denote significant differences in performance between males and females. (*p < .05; **p < .01).
149
responses in accord with Perseveration (one-sample t test; p < .01), Position Preference (p < .01), and Win-Stay (p < .05) strategies, whereas females responded in accord with Position Preference (p < .01) and Perseveration (p < .05). In addition, males and females differed (two-sample t test) on the percentage of trials in which their responses were consistent with the various strategies. Females responded in accord with Odor Trials more frequently than did males (p < .05). Males choices were consistent with Perseveration and Win-Stay strategies more frequently than were females (p < .05 in each case). Because the data in Fig. 6 represent the same responses scored according to different criteria, statistical comparisons across criteria are not legitimate. GENERAL DISCUSSION
were having a difficult time using an odor trail to fiad the escape platform, unlearned response biases might have directed their choices. Thus, the data for all subjects were scored for number of choices made according to several potential strategies: (1) Perseveration, the percentage of first choices that were the same as the first choice on the immediately preceding trial; (2) Win-Stay, the percentage of first choices that were a return to the choice section where escape was made on the immediately preceding trial; (3) Position Preference, the percentage of choices made by each rat to its preferred choice section, the preferred choice section being the choice section entered first most frequently over all trials; (4) Right choice section Bias, percentage of first choices that were made to the right choice section. Figure 6 shows the percentage of first choices that involved correctly following the odor trail and that were made in accord with the above described alternative unlearned strategies. Every single response was scored according to all criteria. Thus, a given response could have been consistent with several of the strategies. For example, if a rat correctly followed an odor trail to the right choice section, which happened to have been the choice section it selected most frequently on all trials and the first choice section entered on the preceding trial, he was scored as having made a choice in accord with the Odor Trail, Right Bias, Position Preference, and Perseveration strategies. Even though in the present experiment all strategies other than following the odor trail would have resulted in only 50% correct choices, the rats choices were made in accord with other strategies significantly more often than would have been expected by chance (one-sample t tests). Thus, besides using odor trails, males made
As in the dry maze (Ludvigson, 1969), subjects in Experiment 1 were more successful with the task when run under homogeneous conditions, demonstrating that rats are able to use odor cues in the water maze. The experiment further showed that although rats, especially females, can employ odor cues in the water, they are able to use a working memory strategy (i.e., win-stay) to make correct choices at above chance level when olfactory cues provide ambiguous information. The finding reassures us in regard to conclusions made in previous studies run with males in homogeneous conditions (Means, 1988; Comer & Means, 1989; Means & Bass, 1990; Means & Kennard, 1991), but makes evident the fact that a heterogeneous running procedure should be used in water-maze working memory studies with either gender. The females' stronger tendency to use olfactory cues resulted in more correct choices for females than for males when the escape contingency was odor based. Females performed significantly above chance level by the second block of trials in the odor trail discrimination, whereas males performed above chance level only on the fourth and fifth blocks of trials. Also, the females attained and maintained a higher level of performance. It seems paradoxical that females appear more innately prepared than males to utilize odor cues, given the fact that during three of her four estrous cycle stages, the female rat is less sensitive to olfactory cues than is the male (Pietras & Moulton, 1974). Escape latencies for the females were longer than those for males in both experiments, probably due to time spent in olfactory investigation. Archer (1975) found that in the open field females spend
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MEANS, ALEXANDER, AND O'NEAL
more time sniffing than males do. In our study latencies for the females might be longer than for males because trying to discern odor cues in a water maze probably requires more time than making a choice based on the strategies that males prefer. The fact that odor cues might be more difficult to detect in a water maze than in a dry maze may explain the fact that we found gender differences in latencies, and Eslinger and Ludvigson (1980) did not; in the water maze females' use of their preferred odor trail strategy would presumably be a much more difficult and time consuming task. We assume that the females were actively searching for odor cues when they engaged in behaviors such as sniffing and clinging to the barriers, although the present study did not provide information about the locus of the odor trails. Our observations lead us to question whether our conclusions apply to water mazes that do not contain barriers. It may very well be that if the escape location is the same across trials and between subjects and the water is not stirred between subjects, rats are capable of using odor trails to locate the escape platform. The odor trial would not necessarily have to be a "path" to the platform to be valuable. If the procedure is such that the over the course of the experiment the platform is placed in only a few locations, as is commonly the case, the rats use reference memory to avoid those locations where the platform is never placed. Rats then would only need a detectable difference in the strength of odor cues near the relevant escape locations in order to use odor cues to make correct or efficient escape responses. A testing procedure in which the escape location is the same over trials within a session and/or between subjects tested within a session and the water is not stirred between trials would seem to maximize the potential for the buildup of a detectable odor difference. Also, many investigators use milk to cloud the water instead of nontoxic paint as we use. Milk may be a better medium for odor than is water-base paint. Clearly, whenever design restrictions permit, it would seem prudent, especially in working memory and "cognitive map" studies, to randomly vary the escape location between trials and/or subjects. If the experimental design precludes varying the escape location, portions of the apparatus touched by a subject should be wiped and the water should be stirred between trials. REFERENCES Amsel, A., Hug, J. J., & Surridge, C. T. (1969). Subject-to-subject trial sequence, odor trials, and patterning at 24-h ITI. Psychonomic Science, 15, 119-120.
Archer, J. (1975). Rodent sex differences in emotional and related behavior. Behavioral Biology, 14, 451-479. Bass, E. W., Means, L. W., & McMillen, B. A. (1992). Buspirone impairs performance of a three-choice working memory water escape task in rats. Brain Research Bulletin, 28, 455461. Comer, T. R., & Means, L. W. (1989). Overcoming unlearned response biases: Delayed escape following errors facilitates acquisition of win-stay and win-shift working memory waterescape tasks in rats. Behavioral and NeuralBiology, 52, 239250. Douglas, R. J. (1966). Cues for spontaneous alternation. Journal of Comparative and Physiological Psychology, 62, 171-183. Eslinger, P. J., & Ludvigson, H. (1980). Commonality among rats in production of reward and nonreward odor. Bulletin of the Psychonomic Society, 16, 191-193. Franken, R. E., & Baker, J. G. (1967). Drive level and cue utilization in spontaneous alternation. Psychonomic Science, 8, 89-90. Franken, R. E., & Baker, J. G. (1969). The effects of drive level on cues utilized in spontaneous alternation. Psychonomic Science, 16, 239-240. Ludvigson, H. W. (1969). Runway behavior of the rat as a function of intersubject reward contingencies and constancy of daily reward schedule. Psychonomic Science, 15, 41-43. Ludvigson, H. W., & Sytsma, D. (1967). The sweet smell of success: Apparent double alternation in the rat. Psychonomic Science, 67, 283-284. Means, L. W. (1988). Rats acquire win-stay more readily than win-shift in a water escape situation. Animal Learning and Behavior, 16, 303-311. Means, L. W., & Bass, E. W. (1990). Four-choice win-stay water escape acquisition by rats. Psychological Reports, 66, 67-70. Means, L. W., Comer, T. R., & Moore, R. (1991). Bmy 21502 and piracetam facilitate performance of two-choice win-stay water-escape in normal rats. Journal of Neural Transmission, 85, 109-116. Means, L.W., & Dent, M.F. (1991). The effects of number of trials per day, retention interval, gender and time of day on acquisition of a two-choice win-stay water-escape working memory task in the rat. Journal of Neuroscience Methods, 39, 77-87. Means, L. W., Hardy, W. T., Gabriel, M., & Uphold, J. D. (1971). Utilization of odor trails by rats in maze learning. Journal of Comparative and Physiological Psychology, 76, 160-164. Means, L. W., & Keunard, K. J. P. (1991). Working memory and the aged rat: Deficient two-choice win-stay water-escape acquisition and retention. Physiology and Behavior, 49, 301307. Olton, D. S., & Samuelson, R. J. (1976). Remembrance of places passed: Spatial memory in rats. Journal of Experimental Psychology: Animal Behavior Processes, 2, 97-116. O'Neal, M. F., Poole, M. C., Hamm, R. J., & Means, L. W. (1990). Estrogen protects performance on a working memory task in ovariectomized rats [Abstract]. Second Annual Convention of the American Psychological Society, 49. Phillips, J. M., & Bloom, J. M. (1971). Control of conspecific odors in the runway. Psychological Reports, 29, 838. Pietras, R.J., & Moulton, D. G. (1974). Hormonal influences on odor detection in rats: Changes associated with the estrous cycle, pseudopregnancy, ovariectomy, and administration of
USE OF ODOR TRAILS BY RATS IN MEMORY TASKS testosterone propionate. Physiology and Behavior, 12, 475491. Pitt, S., Davis, S.F., & Brown, B.R. (1973). Apparent double alternation in the rat: A failure to replicate. Bulletin of the Psychonomic Society, 2, 359-361. Prytula, R. E., & Colbert, J. C. (1975). Administration of trials
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within-Ss and double-alternation responding: A demonstration of a multiple-baseline reversal. Psychological Reports, 36, 131-137. Zoladek, L., & Roberts, W. A. (1978). The sensory basis of spatial memory in the rat. Animal Learning and Behavior, 6, 7781.
