Conditioning of an odor aversion in preweanlings with isolation from home nest as the unconditioned stimulus

Conditioning of an Odor Aversion in Preweanlings with Isolation from Home Nest as the Unconditioned Stimulus

GREGORY J. SMITH DAVID KUCHARSKI NORMAN E. SPEAR

Department of Psychology State University of New York at Binghamton

Binghamton, New York

Four experiments assessed the acquisition of aversions by preweanling rats to odors experienced during isolation from their home-nest environment. Conditioned aversion occurred for animals given a second novel odor paired with the home-nest environment, depending upon the duration of the odor-isolation pairing. With a brief duration of pairing (two pairings of 3 min each) novel odors became more preferred when paired with isolation or, to a lesser extent, when paired with home. With longer durations of pairing (two pairings of 4 hr each) the odor paired with isolation became absolutely aversive, although the odor paired with home did not become absolutely preferred. With intermediate durations neither aversion nor preference for the odors was substantial. Animals conditioned at ages 9, 16, or 23 days postnatal acquired aversions to odors paired with isolation, but 30-day-old rats did not.

Recent experiments have revealed that odorants with no inherent signal value to the organism can be rendered attractive and hence preferred over others if preweanling rodents have experienced continuous or daily exposure to the odorant from birth (Galef, 1982; Leon, Galef & Behse, 1977), or association of the odorant with home environmental stimuli (Brake, 1981; Galef, 1981; Wigal, Kucharski, & Spear, 1984). Two testable hypotheses have been suggested. First, simply familiarity with a given stimulus enhances the relative attractiveness of that stimulus to a young animal. This notion carries with it the suggestion that a simple, nonassociative mechanism could explain the development and the establishment of maternal and pheromonal bonds in immature mammals. An alternative explanation is, immature animals learn that stimuli signal the availability, or absence, or some desired event and that they come to prefer, or avoid, these stimuli based on their acquired signal value. While this latter interpretation works well to explain how stimuli associated with the home-nest environment come to be preferred, it has difficulty explaining why constant or daily exposure to an odorant outside the home

Reprint requests should be sent to Norman E. Spear, Department of Psychology, State University of New York, Binghamton, New York 13901, U.S.A.

Received for publication 16 Jul 1984 Revised for publication 23 Feb 1985 DevelopmenraZ?psychobiology, 18(5):421-434 (1985) 0 1985 by John Wiley & Sons, Inc. CCC 0012- 1630/85/05O421- 14$04.OO

422 SMITH, KUCHARSKI, AND SPEAR

should cause it to become preferred (see Leon et al., 1977). The most likely explanation is that both processes, nonassociative and associative learning, play a part in the development of early bonding. We found in the present experiment that short familiarity with an odorant can render it more preferable than a novel odor, but longer familiarity with an odorant, without imposing a form of extinction as Leon et al. (1977), can render that odor less preferred than a novel odor.

To put these experiments in the context that followed our reasoning, we were initially less interested in how home preferences are formed and more interested in observing the stress associated with separation from the nest and how this stress could contribute to the learning of an aversion to some nonnatural odorant associated with separation. Our reasoning was that since young animals undergo intense isolation stress upon removal from the home environment (see Bell & Smotherman, 1980; Campbell & Randall, 1975; Elliott & Scott, 1961), we expected that stimuli anticipatory to isolation from the home nest would acquire aversive or nonpreferred properties. A preliminary study performed in this laboratory confirmed our expectation. What follows below is our initial analysis of this effect. Experiment I is a more systematic study of our preliminary result. Exper- iment I1 is designed to assess whether rats acquire an aversion for the odorant associated with isolation or a preference for the odor associated with the home cage. Experiment 111 examines how length of familiarity in isolation can change the value of an odorant from preferred to nonpreferred. Experiment IV investigates how the aversive qualities of isolation changes as the rodent approaches the age of weaning.

Experiment I This experiment examined the effectiveness of two 4-hour sessions of isolation from

the home nest as an unconditioned stimulus (US) in an odor-aversion paradigm with 1 I-day-old rats.

Methods

Subjects The subjects were 16, 11-day-old rats selected from two litters, culled at birth to

eight pups per litter. These rats were derived from the Sprague-Dawley strain born and reared in the State University of New York at Binghamton colony. All rats were housed in standard polypropylene nest cages with both parents and littermates, except during explicit periods of training and testing. Animals were maintained in a vivarium operating on a 16:8 light/dark cycle with lights on at 0700 hours. Training began at 0800 hours and tests were administered between 1400-1800 hours.

Apparatus Standard laboratory wire mesh cages (24.5 X 17.5 X 17.5 cm) lined with 5-gallon

plastic bags were used to train isoluted subjects. Each bag was filled with 500 ml of clean wood shavings. Commercially available liquid odorants (McCormack brand) were presented in a saturation cotton ball suspended from a clear acrylic plastic lid which covered the entire apparatus.

Standard laboratory polypropylene nest cages (47.5 X 26 x 15 cm) were used to train litter control subjects. Each was filled with clean wood shavings. Commercially

CONDITIONING OF AN ODOR AVERSION IN PREWEANLINGS 423

available liquid odorants were presented in a saturated cotton ball suspended from a clear acrylic plastic lid which covered the entire apparatus.

Test apparatus was a rectangular container (29.5 x 19 x 9.7 cm) made from, and enclosed within, clear acrylic plastic. The apparatus floor was divided into two equal halves (29.5 x 9.5 X 9.7 cm), each half held 500 ml of the scented wood shavings. Resting 3 mm above the scented shavings was a wire mesh floor which covered the entire apparatus.

Procedure Training required 18 hr, during which time each rat was exposed intermittently to

one of two odorants that served as conditioned stimuli (CS-1 and CS-2). The CS-1 odorant was experienced during the first 4 hr of training. Rats assigned

to the isolation treatment experienced the CS-1 in isolation from littermates and parents. Rats assigned to the litter control treatment experienced the CS-1 in the presence of littermates and parents.

Following CS-1 exposure, rats were exposed to the CS-2 for the next 4 hr. In each treatment condition, rats experienced the CS-2 in the presence of littermates and parents.

Following CS-2 exposure, rats were reexposed to the CS-1 for the next 4 hr. Rats in the isolation treatment experienced the CS-1, as before, in the isolation apparatus. Littermates assigned to the litter control treatment reexperienced the CS- 1 in the presence of parents and siblings.

After completion of the second CS-1 exposure, all rats were reexposed to the CS-2 for 6 hr in the presence of parents and littermates.

Tests were administered 36 hr after the completion of training. Each test consisted of two, 2-min preference tests. In each case, rats were placed in the center of the apparatus, straddling the midline between the CS-1 and CS-2 scented compartments. The response measure was time spent over the CS-2 odor compartment. For a rat to be judged over a compartment, three paws and the snout had to be extended over that compartment. These data were subjected to tests of analysis of variance with p < .05 set as the criterion for rejecting the null hypothesis.

Design The between-group factor was training condition, isolation (I) and litter control (LC).

There were eight rats assigned to each treatment. The odorants, liquid lemon and liquid orange, were presented in counterbalanced fashion across litters such that one full litter experienced orange odor as the CS-1 and the lemon odor as the CS-2, and the next litter experienced the opposite, lemon as CS-1 and orange as CS-2. Note that only half of the animals in each litter (the isolated rats) were exposed to the CS-1 odor while isolated. The response measure was transformed into percentage time over the CS-1 odorant and analyzed using analysis of variance tests.

Results and Discussion The results indicated that isolation-treated rats spent 35.5% (S.E. = 8.2) of the test

session over the CS-1, the odor previo.usly associated with isolation, whereas rats assigned to the litter control treatment spent 55% (S.E. = 5.1) over that odorant.

A 2 x 2 analysis of these data indicated that the isolation group spent reliably less time over the CS-1 odorant than the litter control group, F (1,12) = 3.86, p < .05.


Neither the order of odorant presentation, F < 1, nor the interaction, F (1,12) = 1.17, proved statistically significant.

The suggestion of these data is that immature rats acquire an aversion for odors associated with isolation from the home-nest environment. On the surface, these results make intuitive sense. There may very well be an advantage bestowed upon the nursing pup that learns to avoid those areas of its environment which restricts it from the lactating dam. The significance of these observations is that separation from the mother signals “danger” to the infant (e.g., loss of warmth, protection, nutritional needs). However, based solely on these data, we cannot unequivocally infer that our pups acquired an aversion for the CS-1 without first eliminating the alternate explanation that our rats had instead acquired a preference for the CS-2 odor through its previous association with the home nest. Said another way, our animals may have learned a conditioned aversion to the CS + (e.g., the odor paired with isolation) or a conditioned preference to the CS - (e.g., the odor explicitly unpaired with isolation). Such appetitive conditioning has been reported in a number of previous experiments (cf. Bronstein & Crockett, 1976; Johanson & Hall, 1982). Experiment I1 sought to distinguish between these alternative explana- tions.

Experiment I1 To decide whether our pups were actually acquiring an aversion to the CS-1 odor

associated with isolation or learning a preference for the CS-2 odor associated with the home nest environment, we conditioned preweanling rats as in Experiment I but applied a new testing procedure. In this testing procedure, we arranged three odorants juxtapo- sitioned in the same test apparatus, the CS-1 odor, a neutral, never-before-experienced odor, and the CS-2 odor. During testing, we recorded the amount of time spent over each odorant and then compared these scores across treatment conditions. Using this procedure, we are able to compare both preferences for the CS-2 and aversions for cs-1.

Methods

Subjects

The subjects were 26, 9-day-old rats selected from our colony litters.

Apparatus and Procedures

The training apparatus and procedures were identical to those used in Experiment I and are described there.

The test apparatus (37.5 X 20.5 X 10 cm) was constructed from clear acrylic plastic. The bottom of the apparatus was divided widthwise into three equal compartments (each 20 x 12 x 4.5 cm). Each compartment held approximately 500 ml of clean wood shavings. For all tests, the middle compartment was scented with 2.5 ml of a previously unexperienced liquid garlic odor. The compartment at one end of the apparatus was scented with 2.5 ml of liquid orange odor; the compartment at the other end of the apparatus was scented with 2.5 ml of liquid lemon odor. One-piece stainless steel wire mesh screen covered the entire floor.


During test, each rat was given two, 2-min preference tests. Each test began with placement over the novel, garlic odor. Care was taken not to face rats toward either of the other compartments. For each test trial, time spent over each odorant was recorded and later transformed into percentages.

Design The between-group factor was training treatment, isolation, or litter control. The

within-group factor was percentage time over each of three odorants, CS-I, novel garlic, or CS-2. For each training treatment, a counterbalanced procedure using liquid lemon and orange odorants assured that both odors were used as the CS-1 and CS-2.

Results and Discussion The results of this experiment are presented in Figure 1. Note that the left panel

reports the preference scores for the isolation treatment. The right panel contains those for the litter control treatment.

In general terms, rats exposed to CS-1 odor in isolation (Fig. 1, left panel) spent less time over the CS-1 odor than litter controls (right panel). Rats assigned to the litter control treatment spent approximately an equivalent amount of time over CS- 1, CS-2, and the novel odor. Rats assigned to the isolation treatment spent the vast majority of the test session (63%) over the neutral odor, however, and time over CS-2 was about equal for rats in the isolation and home control treatments. Indications are that CS-2 was not rendered preferred as might have been expected through association with the home- nest environment. If anything, the conditioned aversion may have generalized from CS- 1 to CS-2. Generalization learning of this type has been reported in other conditioned aversion experiments (Rudy & Cheatle, 1977).

Analysis of these data supported our interpretations. A statistical interaction involving training treatment and odorant, F (2,48) = 3.74, p < .05, was, in a large part, because isolated-treated rats spent less time over CS-1 than litter controls and more time

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Fig. 1. Mean percentage time spent over each of the test odors (S + refers to the CS-I odor, neutral refers to novel garlic, and S- refers to the CS-2 odor).


over the neutral odor than their respective controls. In contrast, there were no differences among odorant scores for rats assigned to the litter control treatment. Finally, while the isolated rats spent less time over CS-2 than litter controls, this difference did not reach statistical significance. It should be pointed out, however, that several studies from this laboratory (e.g., Kucharski & Spear, 1984) have found that odors frequently associated with the absence of an aversive event do not become preferred and often are responded to as if they are aversive, when tested against a novel odor. Yet when given a test between the odor that had signaled the aversive event (CS-1) and the odor that had signaled the absence of the aversive event (CS-2), animals choose to spend most of their time over the CS-2. This result was found in Experiment I.

The present experiment employed a procedure to determine whether isolated rats were forming an aversion to the CS-1 odor (previously paired with isolation) or a preference for the CS-2 odor (previously paired with the home nest). The results suggested that the isolated rats had developed an aversion to CS-1, rather than a preference for CS- 2. Although we found no indication that familiarity generally led to a greater preference, as previously reported by Leon, Galef, and Behse (1977), the experiment was not designed for that purpose. This led us to perform Experiment 111. In this experiment, we sought to determine under what circumstances an odorant would become preferred or nonpreferred following familiarity with it.

Experiment I11 Experiments I and I1 indicated that immature rats can acquire and express an aversion

to an odor associated with isolation. However, previous experiments conducted in this laboratory and others have demonstrated a seemingly contradictory result. Preweanlings exposed to an odor outside their nest have revealed an increased preference for that odor at a later time (Caza & Spear, 1984; Leon, Galef, & Behse, 1977; Wigal, Kucharski, & Spear, 1984; also see Hill, 1978, for a review of similar effects). Under closer ex- amination, two subtle features of the “conditioning” procedure may account for whether an aversion or preference is conditioned. First, our preliminary, unsuccessful attempts to condition an odor aversion in the present circumstances included presentation of only an odor in isolation, without an alternative odor of the same type present in the home environment. There is evidence that aversion to an odor paired with footshock may develop for infant rats only if an alternative odor signals the absence of footshock (Ku- charski & Spear, 1984). This evidence was heeded in the procedures of the present experiment. Second, Caza and Spear (1984) reported that whereas short exposures to an odor resulted in increased preference for that odor, this effect tended to be reduced with longer exposure intervals. Perhaps young rats acquire a conditioned aversion to an odor that signals isolation from the home nest environment only if this isolation period is of sufficiently long duration. Thus, in the present experiment, we varied the duration of the isolation exposure and examined changes in preference for the associated odor.

Another purpose of the present experiment was to resolve some procedural problems associated with our previous experiments. In those tests, “isolated” subjects were exposed to the CS-1 odor in isolation, whereas “litter control” subjects were exposed to the CS-1 odor in the context of the home-nest environment. The decreased preference for the CS-1 odor by the isolated subjects was judged to be the result of a conditioned aversion to that odor. Alternatively, it is possible that the differences obtained were not due to the isolated subjects learning a conditioned aversion to the odor paired with isolation, but instead due to nonassociative effects of isolation itself altering the baseline


preference for these odors. In the present experiment, two additional control groups were added to eliminate this interpretative problem. For the first group, rats received the same treatment as the isolated subjects except that neither the CS-1 nor CS-2 were presented during the conditioning phase of the experiment. For the second group, rats received the same treatment as the litter control rats except that neither the CS-1 nor the CS-2 were presented during the conditioning phase of the experiment. If isolation paired with the odor led to the conditioned aversion for CS- 1, then our experimental isolated rats should express less of a preference for the odor than the control isolated groups. Alternately, if the experimental litter-control rats were learning a conditioned preference, they should express more of a preference for that odor than control rats not exposed to it in the home- nest environment.

Additionally, animals in the present experiment were given two separate tests, one that pitted the CS-1 against the novel odor and a second that pitted the CS-2 against the novel odor. Our test procedure in Experiment I1 had pitted all three odors against one another simultaneously. The result of that test indicated that isolated rats had acquired an aversion to the odor associated with isolation but no preference for the odor associated with the home nest. The present test procedure was designed to reassess the conclusions drawn from Experiment 11 with the more common testing procedure of testing preference for two odors at a time.

Methods

Subjects and Apparatus The subjects were 135 9-day-old rats of the Sprague-Dawley strain born and reared

in our colony. They were treated identically to those in Experiment I. The training and testing apparatuses were those described in Experiment I. For the test preference between the CS-1 odor and the novel odor, one container was scented with 2.5 ml of liquid lemon odor and the other with 2.5 ml of liquid orange odor. For the apparatus that served to test the preference between CS-2 odor and the novel odor, one container was scented with 2.5 ml of liquid peppermint odor and the other with 2.5 ml of liquid orange odor.

Design and Procedure

The design of this experiment was a 3 X 2 x 2 factorial, involving three conditioning durations (3 min, 1 hr, or 4 hr) X two training treatments (isolation vs litter control) x two odor conditions (odor present or odor absent during training). Rats in the isolation treatment spent either 3 min, 1 hr, or 4 hr (depending on the conditioning duration assigned them) separated from their home-nest litters followed by another 3 min, 1 hr, or 4 hr in the home-nest environment. Once the sequence was repeated all the animals were returned to the home cage and left undisturbed for 36 hr. Animals in the odor present conditions experienced the CS-1 and the CS-2 odors. For the isolated condition, the CS-1 odor was present while the rat was in isolation; for the litter control condition the CS- 1 odor was presented in the presence of littermates and parents. Animals in the no odor present condition were not exposed to either CS-1 or CS-2 until testing.

Two preference tests, counterbalanced for order, were administered 36 hr after completion of training. At the beginning of each test trial, rats were placed in the center of the apparatus, straddling the midline that divided the odor compartments. For the CS-1 versus novel odor test, the response measure was time spent over the lemon (CS-1) odor relative to the time spent over the orange (novel) odor. Other procedural details were as described in Experiment I.


Results

Preference between the CS-1 and Novel Odor

The test results are presented in Figure 2. These scores reflect the mean percentage of time spent over the CS-1 odor. Separate planned comparisons were carried out for the three different conditioning durations. For each of the conditioning durations, no reliable differences were found between the two no-odors-present control groups ( p > .25). Therefore, for subsequent analyses data were pooled across these two control groups.

For the 3-min conditioning duration, a planned comparison between the isolated- odors present group and the no-odors-present controls revealed that the former group expressed a significantly enhanced preference for the CS-1 (lemon), F (1,123) = 5.96, p < .05. No comparable differences occurred between the litter-control odor-present group and the no-odors-present controls, F (1,123) = 2.02, p > . lo.

For the I -hr conditioning duration, no reliable differences were obtained between the isolated-odors present and the no odors present control groups ( p > .25). Further, a comparison between litter-control odors-present and no-odors-present controls indicated no reliable differences ( p > .25).

For the 4-hr conditioning duration, the isolated odors-present group demonstrated reliably lower preferences for the CS- 1 (lemon) than the no-odor-present control groups, F (1,123) = 6.11, p < .05. This indicates a conditioned aversion to lemon due to its pairing with isolation. No reliable differences were obtained between the litter-control odor-present and the no-odor-present controls ( p > .25).

These results indicate that a short isolated exposure to an odor increases preference for the odor, and as the duration of this exposure increases, the preference shifts to an aversion.

EXPOSURE DURATION 3 MIN.

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Fig. 2 . Mean percentage of time spent over the CS-I (S+) odorant during the test as a function of group (isolated or litter controls), conditioning duration (3 min, 1 hr, 4 hr) and odor exposure (odors present or no-odors present) during conditioning.


Preference between the CS-2 and Novel Odors

The test results are presented in Figure 3. These scores reflect the mean percentage of time spent over the CS-2 odor. Separate analyses were performed for each of the three different exposure durations. No differences were obtained between the two no-odor- present control groups, so these groups were collapsed into one group for further analyses.

For the 3-min conditioning duration, the isolated odors-present group expressed greater preference for the CS-2 (peppermint) than the no-odors-present control groups, F(1,123) = 4.99, p < .05. The comparison between the litter-control odors-present group and the collapsed control groups failed to reveal any significant differences.

For the 1-hr conditioning duration, no significant differences were obtained between either the isolated odors-present and no-odors-present controls or the litter-control odors- present and no-odors-present controls.

For the 4-hr conditioning duration, no significant differences were obtained in the comparison between the isolated odors-present and collapsed controls or between the litter-control odors-present and no-odor-present controls.

Discussion This experiment revealed that infant rats isolated with an odor for 4 hr expressed

lower preferences for that odor than animals not similarly exposed. This indicates a conditioned aversion in which isolation from home had served as the unconditioned stimulus.

The present experiment revealed that if infant rats were exposed to an odor for a very short duration, 3 min, while isolated from littermates and parents, their preference for that odor was increased. Animals exposed to the same odor in the presence of

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Fig. 3. Mean percentage of time spent over the CS-2 (S-) odorant during the test as a function of group (isolated or litter controls), conditioning duration (3 min, 1 hr or 4 hr) and odor exposure (odors present or no odors present) during conditioning.


littermates and parents also had an increase in preference for that odor, although not as robust as that seen in the previous condition. It is somewhat paradoxical that animals given the 3-min exposure to the odor paired with isolation expressed an increased preference for the odor presented in the context of their home. The paradox is that the isolated odors-present group expressed an enhanced preference for odors presented both outside and inside the home, whereas the litter-control odors-present group did not express a similar enhanced preference to either of the odors presented in the context of the home. This paradox may be resolved by considering that the isolated animals are probably more aroused than the litter controls due to separation from the nest. This enhanced arousal may promote processing of the olfactory stimuli and carryover when the animals are returned to the nest after 3 min of isolation. This explanation may account for the finding of enhanced preference for both CS-1 and CS-2 odors among the isolated-odor present animals.

The absence of either conditioned preference or aversion to an odor that had been paired with isolation for 1 hr suggests that the two exposure phenomena (i.e., the preference acquired after short duration exposures and the aversion which develops after longer exposures) may cancel each other out with an intermediate duration of odor- exposure-in-isolation.

Experiment IV The previous experiments revealed that for 9-day-old and 1 I-day-old pups, stimuli

associated with isolation from the home nest may acquire aversive properties if the exposure duration is sufficiently long. In the present experiment, we examine this relation as a function of age. If, for instance, pups acquire the aversion because of the absence of nutrients, prey protection, thermal protection, etc., we should find that the effects of isolation will dissipate with age. As the animal ages and becomes less dependent on the dam for nutrients, protection, and maintenance of bodily functioning, the consequences of isolation should become less aversive. In Experiment IV, the effects of isolation were examined across ages ranging from 9-30 days postpartum.

Methods

Subjects and Apparatus

The subjects were 160 male and female Sprague-Dawley rats obtained from 16 litters born and reared in our colony. These animals were maintained identically to those in the previous experiments.

The training and testing apparatus were described in Experiment I.

Procedure and Design From each of the litters, five pups were assigned to the isolation treatment and five

pups to the litter control treatment. Within the isolated treatment, pups were divided in counterbalanced fashion, between an isolation treatment where pups were maintained at room temperature (22-26°C) and an isolation treatment where pups were maintained at nest temperature (33-37°C). This manipulation was included to examine the possible hypothermic effect accompanying isolation on the conditioned aversion. Conceivably, pups isolated at room temperature could be learning an odor aversion where hypothermia represents the unconditioned stimulus (Hinderliter, Misanin, Baker & Topper, 1978).


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The design of the present experiment was a 2 X 4 factorial with two training treatments (isolation vs odor control) and four age groups (9-, 16-, 23- and 30-day-old rats). Within each of the eight orthogonal treatment conditions, there were assigned 20 rats. Test scores were transformed into percentage time spent over the odor associated with isolation (lemon) for analysis.

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Results and Discussion The results are presented in Figure 4. These results reflect the mean percentage of

time spent over the odor associated with isolation by rats in the isolation and litter control treatments. While apparatus temperature may have contributed to the present results, the severity of isolation by itself apparently overshadowed the effect of ambient temperature and led to no statistically significant differences between isolation treatments. Analysis of isolation versus isolation +plus +warm treatments indicated no significant differences between these conditions across any of the four ages: 9 days, F (1,18) = 1.63; 16 days, F (1,18) = <1; 23 days, F (1,18) = 2.93; 30 days, F (1,18) = 3.77. Henceforth, within each age, scores were pooled across isolation treatments for the purpose of further analysis and graphic representation.

As seen in Figure 4, the effects of isolation on the acquired odor aversion were diminished with increasing age of the subject. At 9 days and 16 days postpartum, a sizable odor aversion resulted from the 4-hour odor-isolation experience. The effect was somewhat weaker at 23 days postpartum and was completely eliminated by 30 days postpartum.

A 2 X 4 analysis of these data supported these interpretations by indicating a reliable training treatment by age interaction, F (3,152) = 2.39, p < .035) along with main effects for age, F (3,152) = 2 . 3 4 , ~ < .035, and training treatment, F (1,152) = 13.4, P < .001. Planned comparisons between isolation and litter control treatments within each of the four age groups indicated a lessening of the aversive effects of isolation as the rats approached the age of natural weaning, approximately 27 days postpartum (see Ostadalova, Bibr, Babicky , Parizek, & Kolar, 1971). Comparisons revealed that 9-day-

RETENTION TEST

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Fig. 4. Percentage time during the test session that animals spent over the CS-1 ( S + ) odorant as a function of age at training (9, 16, 23, or 30 days of age) and conditioning group (isolated or litter control).


old, 16-day-old, and 23-day-old members of the isolation treatments spent reliably less time over the odor associated with isolation than litter control subjects, F (1,152) = 13.41, p < .001; F (1,152) = 4 . 5 , ~ < .025; F (1,152) = 2 . 7 1 , ~ < .05, respectively. At 30 days postpartum, however, group differences failed to achieve statistical significance, F < 1.

The results of the present experiment indicate that as the developing rat approaches the age of weaning, isolation from the home nest becomes less and less aversive. We observed in Experiments I, 11, and 111, as well as the present one, that 9-1 1-day-old rats will acquire an aversion to odors associated with 4 hr of isolation from littermates and parents. Add to this finding that 16-23-day-old rats also react toward isolation-associated odors by developing a conditioned aversion for the odor. But there was no evidence that such an aversion is acquired by 30-day-old rats. Apparently, near the age of natural weaning, the conditioned aversion fails to develop.

General Discussion The results of the present studies demonstrate: (1) Reweanling rats will acquire a

conditioned aversion for stimuli that signal an extended period of isolation from the home nest environment and (2) the magnitude of the aversion is lessened as the rat’s age approaches that of natural weaning. The significance of these observations is that separation from mother signals “danger” to the infant (e.g., loss of warmth, protection, nutritional needs). As long as the infant rat needs to rely on the caregiving behaviors provided in the home nest, isolation from this environment seems to be aversive and conditionable to stimuli that predict the occurrence of isolation. Yet, on the other hand, as the infant becomes more autonomous from the home-nest environment, the aversion subsides.

Two recent developments in this line of research should be pointed out. First, research subsequent to these experiments suggests that it is critical for the development of the aversion that we present both an odor associated with isolation as well as an odor associated with the home environment. Our attempts to condition an odor aversion by pairing only odor and isolation, without a discriminative stimulus of odor and home-nest environment, have failed to produce conditioning. Interestingly, we have found also that odor-footshock conditioning is sensitive to similar manipulations (Kucharski & Spear, 1984). Second, we have collected data to indicate that the conditioned aversion with isolation as the unconditioned stimulus may be attenuated with subcutaneous injections of morphine sulfate (.3 and 1.0 mg/kg) immediately prior to isolation (Smith, Spear, & Horowitz, 1979).

A general implication is that more than one process controls odor preferences for the preweanling rat. The present experiments, and others, suggest that enhancement of preference for an odor by short-term mere exposure to that odor is one important process. (Caza & Spear, 1984). This short-duration enhancement of preferences appears to increase if the animal is aroused to some extent (see Experiment 111; Johanson & Hall, 1982; Pedersen, Williams, & Blass, 1982; R. G. Bryan, personal communication; J. W. Whitlow, personal communication). This enhancement in odor preference decreases and the odor becomes aversive as the duration of isolation exposure increases, provided that an alternative novel odor has been experienced in nonisolation circumstances. In support of the notion of arousal, it is interesting to note that isolation from the home nest produces a marked elevation of plasma corticosteroids in rats (Smotherman, Wiener, Mendoza, & Levine, 1976) and plasma cortisol in squirrel monkeys (Coe, Mendoza, Smotherman, & Levine, 1978) and rhesus macaques (Smotherman, Hunt, McGinnis, & Levine, 1979).


The synthesis and release of the adrenal corticoids is elicited by the pituitary hormone, adrenocorticotropic hormone (ACTH) and the release of ACTH is highly correlated with stress in the organism, hence adrenocorticoid levels in plasma are often used for an indirect measurement of ACTH activity and inferred “stress.” In regard to the present discussion concerning the effects of isolation stress on learning and conditioning, short exposure to isolation may evoke internal hormonal consequences that are conducive for learning a conditioned preference (Pedersen et al., 1982) while longer exposures are accompanied by need states that should be avoided by preweanling rats.

A second process that may be involved is based on the finding that young animals increase their preference to an odor if it has been experienced in the context of the home environment (i.e., in the presence of heat, littermates, and/or parents; Alberts, 1981). In this case of exposure for long periods of time, the odors take on properties charac- teristic of home litter shavings (Wigal, Kucharski, & Spear, 1984). Galef (1982) has observed that this attraction increases as a function of pretest exposure.

Although the young rat increases its preferences for an odor after very short durations of exposure to it, with long durations of exposure the rat pup is capable of learning not only to approach and prefer odors associated with the home nest environment but also to avoid stimuli that signal the absence of home environmental stimuli. This learning wanes at the time of weaning. These results suggest that under certain conditions the younger, more immature organism will acquire information that older animals will not. Our theoretical bias leads us to point to these data as another example of age-specific processes which uniquely determine the information processing behavior of the organism. It is less likely that learning is impossible at certain ages, but rather, learning or its expression may be constrained by the adaptive specializations for that stage of development.

Notes This research was supported by a grant from the National Institute of Mental Health (1 RO1 MH35219)

to Norman E. Spear. The authors are grateful for the assistance of Norman Richter in conducting the research and to Teri Tanenhaus in preparing this report.

‘Now at the Department of Psychology, Kent State University, Kent, Ohio 44242.

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Conditioning of an odor aversion in preweanlings with isolation from home nest as the unconditioned stimulus