retention tests, most rats also showed excellent memory for odors used in prior problems. ... tests of memory, given after training, show that rats may have.
Physiology& Behavior, Vol. 50, pp. 555-561. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Olfactory Learning and Odor Memory in the Rat B U R T O N M. S L O T N I C K , A N G E L A K U F E R A A N D A L A N M. S I L B E R B E R G
Department o f Psychology, The American University, Washington, DC 20016 R e c e i v e d 19 February 1991 SLOTNICK, B. M., A. KUFERA AND A. M. SILBERBERG. Olfactory learning and odor memory in the rat. PHYSIOL BEHAV 50(3) 555-561, 1991.--Rats were trained on a series of 9 tasks, each of which required discrimination among 8 different and unique odors. Discrimination accuracy improved across successive problems and, by the end of training, most rats made few errors after their initial exposure to each new odor. Despite the number of stimuli to be discriminated, this acquisition of a learning set was not appreciably different from that demonstrated in an earlier study that used only 2 odors per task. In subsequent retention tests, most rats also showed excellent memory for odors used in prior problems. Odor discrimination
Odor learning
Odor memory
Olfactory learning set
RATS have an exceptional ability to master instrumental learning tasks when odors are used as discriminative stimuli. Learning abilities that had not been previously demonstrated in the rat, such as errorless acquisition of simple detection and 2-odor discriminations and the acquisition of a learning set, are readily obtained when rats are trained using odors. In particular, learning set performance is quite impressive. Thus in both successive odor reversal tasks and in multiple 2-odor discriminations rats generally show positive transfer on the second problem of the series and achieve near errorless performance after only 5-8 such problems. Indeed, in one recent study (12), rats demonstrated such rapid acquisition of each of a series of 13 novel 2-odor discrimination tasks that a measure of errors to criterion failed to reveal improvement over the series although this could be shown in an analysis of performance in the first 20 trials of each problem. Clearly, the demands of such tasks are not particularly challenging for the rat and probably do not reveal the limits of the animal's ability to discriminate among odors, form a learning set or adopt response strategies. To further explore such capacities, we trained rats on a series of multiple odor discriminations. In each task rats discriminated among 8 different and novel odors. Our results show that rats not only acquire such tasks rapidly, but also show improvement to a level of near-errorless learning over a series of these multiple stimulus discriminations. Further, tests of memory, given after training, show that rats may have considerable retention of odors learned in earlier problems of the series.
meter previously described by Lu and Slotnick (Fig. 1). The test chamber was a 15 cm x 20 cm x 20 cm Plexiglas box fitted with a stainless steel mesh floor. A 2.5 cm diameter vertical tube (stimulus delivery tube), attached to one end of the chamber was used to present odor stimuli. The animal could sample stimuli by inserting its snout through a 2 cm diameter hole (sampling port) in the stimulus delivery tube (Fig. 1). Insertion of the snout into the sampling port was detected by a photobeam. Water reinforcement was delivered through a 19 gauge stainless steel spout located within the sampling tube. The water delivery spout was connected to a water reservoir via a 2-way solenoid. Operation of the solenoid delivered 0.05 ml of water and produced an audible click. Licking responses on the spout were detected with a lickometer circuit (8). The olfactometer was washed with 95% ethanol and air dried for at least 8 hours before the introduction of new odors.
Odor Stimuli Odors for each 8-odor set were obtained by random selection from a stock of 100 pure chemicals, cosmetics, food flavorings and other miscellaneous odorants (Table 1). Concentrations for each odor were adjusted before use to be approximately at or just above the recognition threshold of a human observer.
Procedures Initial training. Animals were trained to detect and discriminate odors using a discrete trial go, no-go operant conditioning procedure (16) controlled by an Apple IIe computer and digital interface. Training was initiated after the animals had been on a 10 ml/day water deprivation schedule for at least 14 days. They received no water for 36--48 h before the first training session. Initially, rats were trained to detect 0.5% (of vapor saturation) amyl acetate vapor (S +, amyl acetate; S - , air) following the procedures described in detail by Lu and Slotnick (12).' All rats were given a single 200-trial session on the amyl acetate detection task and were then trained to discriminate between 0.5% butanol (S +) and 0.1% cineol ( S - ) in two 200-trial sessions.
METHOD
Subjects Ten adult male albino rats were housed in individual plastic cages in a temperature- and humidity-controlled vivarium. The animals were maintained on a 10 ml/day water deprivation schedule throughout the study.
Apparatus Odor stimuli were generated using an eight-channel olfacto-
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SLOTNICK. KUFERA AND SILBERBERG
RESUI .'fs
Acquisition of lndividual Problems
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FIG. 1. Top, single-dilution state olfactometer to control 8 stimuli. Air flows through the saturators could be varied from 0-100 cc/min. Main air flow was set at 2.7 liters/min. A photocell across the sampling tube (not shown in figure) was used to detect trial initiation responses. Upon breaking the photobeam after an intertrial interval the odor and final valve operate. During the 1-s final valve period the odor and carrier stream mix and are shunted to an exhaust path. When the final valve is deenergized the odor is presented to the animal for a maximum of 4 sec. Responses made during the first 2 s of the odor-on period were ignored. On an S ÷ trial the first response made in the remaining 2 s was reinforced with 0.05 ml of water and terminated the trial. See text for additional details.
Learning set task. Beginning on the next day each rat was tested on the first of a series of 9 consecutive novel 8-odor tasks. In each task 4 odors were arbitrarily designated as positive stimuli (S +) and the remaining 4 as negative stimuli ( S - ) . Within a session odors were presented randomly with the restriction that each odor was presented 5 times in each block of 40 trials. Each rat was given 2 test sessions on each set of odors. However, the number of trials per session, the time between sessions and the odors used in each session were varied. Three rats (R610, R611, R613) were given two 240-trial sessions on each odor set (30 presentations of each odor/session) and individual sessions were separated by 24 h (Group A). Three rats (R637, R638, R653) received only 160 trials in each session (20 presentations of each odor/session) and individual sessions were separated by 24 h (Group B). The remaining 4 rats (R710, R711, R712, R715) also received 160 trials per session but both sessions for each odor set were given on the same day (Group C). These sessions were separated by 2 - 4 hours. Odor sets (Table 1) for problems 1-9 were J-R, O-S, followed by J-M and A-I, for Groups A, B and C, respectively. Memory tests. Twenty-four h after completion of the nine 8-odor tasks the first of two memory tests was given. This consisted of a 160-trial test using odors from problem 3. In the second memory test, given approximately 24 h later, each rat was given a 160-trial test session using odors from problem 6. However, for this second memory test the significance of all odors was reversed. Thus the 4 odors that had served previously as S + stimuli were now S - stimuli and those that served previously as S - were now S ÷ stimuli. For purposes of analyses, performance accuracy was determined for blocks of 40 consecutive trials. Each such block contained 20 S + and 20 S - trials and, on average, each odor was presented 5 times. Repeated measures ANOVA and within group t-tests were used in statistical analyses.
Figure 2 illustrates the performance in blocks of 40 trials for each rat for each set of odors. As shown, 6 of the 10 rats achieved performance accuracy of 85% or better it1 the first problem; the remaining 4 rats performed at chance. Only 2 rats failed to show discrimination on the second problem, but all rats achieved high levels of performance in problems 3-9. In most cases, performance accuracy in the first 40-trial block of initial problems (5 presentations of each odor) was at or near chance levels for most rats, but their accuracy rapidly improved in subsequent blocks of trials.
Acquisition of a Learning Set Analysis of group means illustrated several interesting patterns. But, because the 3 rats in Group A received 240 trials/ session, it seemed inappropriate to combine their scores with the 7 that received only 160 trials per session and, therefore, graphs illustrating group means were based on the 7 rats that received 160 trials/session. Also, for these analyses, the response on the first presentation of each odor was considered an information trial (the first trial in which the animal encountered that odor) and was not used in scoring accuracy. Mean performance across problems showed a strong positive transfer-to-training effect (Figs. 3 and 4) and the improvement in accuracy in first 40 trials among odor sets was significant (all rats, F = 10.1, p < 0 . 0 0 1 ) . The rate of improvement was greatest in initial problems and asymptotic performance was achieved by problem 4 or 5 at which point, on average, rats performed at approximately 75% accuracy in the first block of trials and at 80% or better in the second block of trials on each odor set. The pattern of performance illustrated in Figs. 3 and 4 was not characteristic of all rats. Several (e.g., R711, R712, R611, R653, Fig. 2) consistently performed at or near chance in the initial block of trials on most or all problems while others, notably R613, R637 and R638 (Fig. 2), showed discriminative responding within the first 40 trials (approximately 5 presentations of each odor) of each problem. However, all rats demonstrated positive transfer of training across the 9 problems and even the rat that did most poorly (Fig. 2, R653) showed much more rapid acquisition of the last problem than it did in the first 3 problems of the series. There was a small but consistent decrease in performance in the beginning of the second session of each problem (Fig. 3, arrows). Thus, for every rat in virtually all problems, performance accuracy in the first block of trials in the second session of a problem was lower than the last block in the prior session. But, as described below, the magnitude of this effect varied somewhat depending upon specific training conditions.
Comparisons of Groups To determine the effects of presenting 240 vs. 160 trials/session and having all training on one problem completed in one day vs. having a 24 h break between sessions, groups were compared for their performance on the first and second session of each problem (Fig. 5). As shown in Fig. 5A and B, there was little effect of 160 vs. 240 trials/session for either rate of acquisition of each problem or initial performance in the first block of trials in the first or second session of each problem. However, rats given both sessions of a problem on the same day tended to perform somewhat less accurately in initial problems than those given only 1 session each day (Fig. 5C and D). Thus relative to rats that had a 24 h intersession-interval, those given
OLFACTORY LEARNING
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TABLE 1 ODORS USED IN EACH SET Set A
S+Odors S - Odors
Set B
S + Odors
Set C
S -Odors S + Odors S - Odors
Set D
S + Odors S - Odors
Set E
S ÷ Odors S - Odors
Set F
S ÷ Odors S - Odors
Set G
S ÷ Odors S - Odors
Set H
S ÷ Odors S - Odors
Set I Set J
Set K
S ÷ Odors S - Odors S ÷ Odors S - Odors S ÷ Odors S - Odors
Set L
S ÷ Odors S -Odors
Set M
S + Odors
Set N
S - Odors S ÷ Odors S - Odors
Set O
S ÷ Odors S - Odors
Set P Set Q
S ÷ Odors S - Odors S + Odors
Set R
S - Odors S+Odors
Set S
S - Odors S + Odors S-Odors
Beef Flavoring, Mimosa Perfume, Ethyl Acetoacetate, Green Soap Tincture Benzyl Acetate, Coconut Extract, Lavender Perfume, Violet Perfume Lysol Disinfectant, Pine Perfume, Narcissus Perfume, Lavoris Mouth Wash Cyclohexanone, Geranium Perfume, Fougere Perfume, Fufural Strawberry Extract, Maple Extract, Airwick Air Freshener, Butter Extract Anise Extract, Scope Mouth Wash, Vanilla Extract, Teriyaki Sauce Mennen Skin Bracer, Orange Perfume, Lemon Perfume, Acetone Apple Flavoring, Terpineol, Mint and Peppermint Extract, Soy Sauce Benzene, Trefle Perfume, Lemon Extract, Citral Vanilla Butter Extract, Lilac Perfume, Phenyl Propyl Acetate, Honeysuckle Perfume Rum Extract, Dichloroethane, Magnolia Perfume, Sea Breeze Antiseptic Black Cherry Juice, Sweet Pea Perfume, Chocolate Perfume, Geraniol Perfume Downy Soap, Methyl Eugenol, Root Beer Extract, Isopropyl Acetate Vitalis Hair Tonic, Trichloroethane, Banana Extract, Tuberose Perfume Terpinyl Acetate, Linalyl Perfume, Musk Keytone Crystals, Almond Extract Methyl Isoeugenol, Lotus Candle Odor, Gardenia Perfume, Imitation Brandy Extract Xylene, Sherry Extract, Orange Extract, Black Walnut Extract Acetic Acid, Tomato Flavoring, Violet Perfume, Caproic Acid Lavoris, Lemon Extract, Butter Extract, Soy Sauce Chypre Perfume, Black Cherry Juice, Sherry Extract, Tuberose Perfume Lysol, Benzyl Propionate, Rose Perfume, Vanilla- Butter- Nut Extract Apple Flavoring, Cinnamon Flavoring, Citronella, Trefle Perfume Downy Soap, Tincture Green Soap, Sea Breeze Cleaner, Tomato Flavoring Gardenia Perfume, Orange Extract, Teriyaki Sauce, Vitalis Hair Tonic Anise Extract, Pine, Strawberry Candle Odor, Cranberry Candle Odor Ethyl Acetate, Rum Extract, Sweet Pea Perfume, Violet Perfume Banana Extract, Linalyl Acetate, Noxema, Root Beer Extract Jasmine Perfume, Lotus Candle Odor, Orange Perfume, Strawberry Extract Chocolate Extract, Cyclohexanone, Lemon Perfume, Phenyl Propyl Acetate Coconut Extract, Eucalyptal Oil, Citral, T e n - O - Six Skin Antiseptic Beef Flavoring, Caproic Acid, Magnolia Perfume, Camphor Brandy Extract, Honeysuckle Perfume, Acetone, Moth Bails Airwick Air Freshener, Carnation Perfume, Fougere Perfume, Mimosa Perfume Maple Extract, Celery Juice, Methyl Salicylate, Musk Keytone Geranium Perfume, Lilac Perfume, Narcissus Perfume, Musk Candle Odor Isopropyl Acetate, Vanilla Extract, Skin Bracer, Cascara Sagrada Almond Extract, Geraniol Perfume, Lavender Perfume, Scope Mouth Wash Black Walnut, Benzyl Acetate Extra, Mint and Peppermint, Terpinyl Acetate
558
SLOTNICK, KUFERA AND SILBERBERG
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Problem FIG. 2, Percent correct responding for each of the 10 rats for the 9 novel 8-odor discrimination tasks and for the 2 memory tests (M1 and M2). Each data point represents the mean of 40 trials. Rats 610, 611 and 613 (Group A) were given two 240-trial sessions on each odor set and individual sessions were separated by 24 h. Only the first 320 trials on the novel discriminations are plotted for these rats. Rats 637, 638 and 653 were given two 160-trial sessions on each odor set and individual sessions were separated by 24 h. Rats 710, 711, 712 and 715 were given two 160-trial session on each odor set and individual sessions on each set were separated by 2--4 h. All rats received a single 160-trial session on each of the 2 memory tests. M1, retest on set 3; M2, retest on set 6 but with the significance of each odor reversed. See text for additional details.
2 sessions/day performed more poorly in the second session of the first problem, the first session of the second problem and in initial trials in the second session of problems 2, 3 and 4. But, there were no consistent differences between these groups in the last 6 problems.
Memory Test Both measures of memory (retraining on problem 3 and acquisition of reversal of problem 6) demonstrated retention for the sign (whether S + Odors or S - Odors) of previously learned odors.
Seven of the 10 rats performed better on the problem 3 retest than in the original learning of these odors (Fig. 2). However, this measure of memory is confounded because the rats had acquired a learning set and, thus, would be expected to do well on any subsequent problem. A better measure of retention is given by the initial and terminal performance on the reversal of problem 6. In this test, performance on the first block of 40 trials of the reversal was at or near chance levels for most rats and was significantly worse than performance in the first block of trials on the prior 6 odor sets (p
