Predator odor avoidance as a rodent model of anxiety: learning-mediated consequences beyond the initial exposure

Prey animals such as rats display innate defensive responses when exposed to the odor of a predator, providing a valuable means of studying the neurobiology of anxiety. While the unconditioned behavioral and neural responses to a single predator odor exposure have been well documented, the paradigm can also be used to study learning-dependent adaptations that occur following repeated exposure to a stressor or associated stimuli. In developing preclinical models for human anxiety disorders this is advantageous, as anxiety disorders seldom involve a single acute experience of anxiety, but rather are chronic and/or recurring illnesses. Part 1 of this review summarizes current research on the three most commonly used predator-related odors: cat odor, ferret odor, and trimethylthiazoline (a component of fox odor). Part 2 reviews the learning-based behavioral and neural adaptations that underlie predator odor-induced context conditioning, one-trial tolerance, sensitization, habituation and dishabituation.     

Distinct neural mechanisms mediate olfactory memory formation at different timescales

Habituation is one of the oldest forms of learning, broadly expressed across sensory systems and taxa. Here, we demonstrate that olfactory habituation induced at different timescales (comprising different odor exposure and intertrial interval durations) is mediated by different neural mechanisms. First, the persistence of habituation memory is greater when mice are habituated on longer timescales. Second, the specificity of the memory (degree of cross-habituation to similar stimuli) also depends on induction timescale. Third, we demonstrate a pharmacological double dissociation between the glutamatergic mechanisms underlying short- and long-timescale odor habituation. LY341495, a class II/III metabotropic glutamate receptor antagonist, blocked habituation only when the induction timescale was short. Conversely, MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, prevented habituation only when the timescale was long. Finally, whereas short-timescale odor habituation is mediated within the anterior piriform cortex, infusion of MK-801 into the olfactory bulbs prevented odor habituation only at longer timescales. Thus, we demonstrate two neural mechanisms underlying simple olfactory learning, distinguished by their persistence and specificity, mediated by different olfactory structures and pharmacological effectors, and differentially utilized based solely on the timescale of odor presentation.     

Sex-dependent habituation to novelty in rats

Male and female rats were familiarized with one-half of an exploration box for 30 or 60 minutes. When later tested, females showed higher preferences for the other novel half than males. Females also showed higher levels of rearing and ambulation. No sex differences were evident for grooming, freezing or defecation. The results were interpreted as females having habituated to the forced-exposure novelty of the familiar half of the apparatus more rapidly than males when the familiarization period was relatively short. With a longer period, males would have had more time to reach a similar level of habituation to that attained by females familiarized for 30 minutes. Relationships between novelty preferences and both rearing and ambulation as indices of exploration were also discussed in the light of significant correlations for males only.     

Sex-dependent habituation to novelty in rats

Male and female rats were familiarized with one-half of an exploration box for 30 or 60 minutes. When later tested, females showed higher preferences for the other novel half than males. Females also showed higher levels of rearing and ambulation. No sex differences were evident for grooming, freezing or defecation. The results were interpreted as females having habituated to the forced-exposure novelty of the familiar half of the apparatus more rapidly than males when the familiarization period was relatively short. With a longer period, males would have had more time to reach a similar level of habituation to that attained by females familiarized for 30 minutes. Relationships between novelty preferences and both rearing and ambulation as indices of exploration were also discussed in the light of significant correlations for males only.     

The effects of preexposure to a learning apparatus

ICR mice were given exposure to a one-way shuttle apparatus until locomotor activity had been habituated. When compared with nonexposed animals, the exposed animals displayed superior shuttle avoidance learning. Animals exposed to both the apparatus and the CS did not differ from the nonexposed group. It was concluded that preexposure to the learning apparatus facilitated acquisition, while preexposure to the apparatus in the presence of the CS did not. The relationship between this effect and habituation was considered.     

Influence of long-term sensitization on long-term habituation of the acoustic startle response in rats: central gray lesions, preexposure, and extinction

The relation between long-term decrements of the acoustic startle response in rats and the development of freezing behavior during habituation training was examined. Freezing behavior developed over the initial trials of habituation training, and the rate of long-term response decrements was found to be inversely related to the development of freezing. Manipulations (neurological or behavioral) that either reduced the level of freezing or retarded its development promoted startle response decrements. In Experiment 1, rats receiving electrolytic lesions of the ventrolateral periaqueductal gray demonstrated both accelerated long-term startle response decrements and retarded development of freezing behavior. In Experiment 2, preexposure to the startle apparatus (i.e., latent inhibition) accelerated long-term startle decrements and inhibited development of freezing. In Experiment 3, exposure to the startle apparatus following initial habituation training (i.e., extinction) reduced both freezing behavior and startle response amplitudes. The results are discussed in terms of the influence of Pavlovian fear conditioning on long-term habituation of the acoustic startle response.     

Amygdala and periaqueductal gray lesions only partially attenuate unconditional defensive responses in rats exposed to a cat

Defensive responses to a cat were observed in rats given excitotoxic lesions of the central nucleus of the amygdala (ACe), dorsolateral periaqueductal gray (dlPAG), ventral periaqueductal gray (vPAG), or sham lesions. Rats were placed adjacent to a compartment containing a cat. Sham-lesioned rats avoided the area nearest the cat and preferred the area furthest away from the cat. They also exhibited numerous defensive responses including, climbing, escape from the apparatus, and freezing. Rats with lesions of the ACe reacted like the sham lesioned rats by preferring the area of the apparatus furthest from the cat, however they climbed and escaped significantly less than sham lesioned rats. Avoidance of the area adjacent to the cat was attenuated in rats with lesions of the vPAG. Climbing along the walls of the apparatus was also attenuated in rats with lesions of the vPAG. Escapes from the apparatus were not significantly reduced by lesions of the vPAG and dlPAG. Thus, ACe lesions attenuated climbing and eliminated escapes, but did not impair locomotion of the rat away from the cat.     

Amygdala and Periaqueductal Gray Lesions Only Partially Attenuate Unconditional Defensive Responses in Rats Exposed to a Cat

Defensive responses to a cat were observed in rats given excitotoxic lesions of the central nucleus of the amygdala (ACe), dorsolateral periaqueductal gray (dlPAG), ventral periaqueductal gray (vPAG), or sham lesions. Rats were placed adjacent to a compartment containing a cat. Sham-lesioned rats avoided the area nearest the cat and preferred the area furthest away from the cat. They also exhibited numerous defensive responses including, climbing, escape from the apparatus, and freezing. Rats with lesions of the ACe reacted like the sham lesioned rats by preferring the area of the apparatus furthest from the cat, however they climbed and escaped significantly less than sham lesioned rats. Avoidance of the area adjacent to the cat was attenuated in rats with lesions of the vPAG. Climbing along the walls of the apparatus was also attenuated in rats with lesions of the vPAG. Escapes from the apparatus were not significantly reduced by lesions of the vPAG and dlPAG. Thus, ACe lesions attenuated climbing and eliminated escapes, but did not impair locomotion of the rat away from the cat.     

Acute exposure to a novel stressor further reduces the habituated corticosterone response to restraint in rats

The present study sought to identify dishabituation of the hypothalamic-pituitary-adrenal(HPA) axis response to different psychological stressors. Young adult male Sprague Dawley rats were exposed to five, 1 h sessions of restraint stress on five consecutive days. On the sixth day, and 2 h before additional exposure to restraint, animals were subjected to 30 min of a small (27cm square), elevated open field stressor (pedestal), which served as the dishabituating stimulus. We predicted HPA axis response dishabituation in chronically restrained rats exposed to the novel pedestal. Rats which underwent five days of restraint stress showed significantly blunted plasma corticosterone levels to restraint (habituation) as compared to restraint-nai've rats. However, rats which underwent five sessions of restraint responded with an enhanced habituation response when confronted with restraint shortly after exposure to the novel pedestal. Instead of HPA axis response dishabituation, we observed enhanced habituation. Subsequent experiments determined that a 1.25 mgkg corticosterone injection could substitute for pedestal exposure to produce enhanced restraint habituation.Combined treatment with both the glucocorticoid receptor antagonist RU40555 (30 mgkg)and the mineralocorticoid receptor antagonist RU283 18 (50 mgkg) blocked the expression of enhanced habituation after pedestal exposure. Thus, the delayed corticosterone negative feedback produced by novel stress led to enhanced expression of corticosterone response habituation.     

The "anxiety state" and its relation with rat models of memory and habituation.

Rats selected as "anxious", "nonanxious," or normal according to their behavior in an elevated plus maze were submitted to memory tasks and the densities of central benzodiazepine receptors in the amygdala and the hippocampus were studied. Anxious rats exibited better retention scores in the inhibitory avoidance task while nonanxious rats exibited worse retention scores in inhibitory and two-way active avoidance tasks compared to normal rats. No significant differences were detected in the retention scores for habituation to an open field. Nonanxious rats presented a lower benzodiazepine receptor density in the hippocampus but not in the amygdala compared to the other groups. These data suggest that the benzodiazepine receptors are involved in the effect of "anxiety" or emotional states on memory storage processes.     

The effect of temporal separation and prior habituation upon preconditioning

The effect of temporal separation and prior habituation was tested by measuring response transfer in a shuttle box following different initial treatments. Thirty hooded rats were split into three main groups and six sub-groups. One main group was preconditioned with concurrent light and sound stimuli, another with temporally spaced stimuli, and a control group had no preconditioning. Half of each main group was permitted to explore the apparatus before preconditioning on the supposition that this might reduce emotionality and hence reduce startle or attention responses.

It was found that concurrent stimulation produced significantly greater response transfer than in controls, whereas preconditioning with spaced stimuli did not. Prior habituation produced no significant effect on the amount of response transfer.     

Effects of excitotoxic brain lesions on taste-mediated odor learning in the rat

The association learning between taste and odor is important in ingestive behavior. For a better understanding of this learning, we have developed a convenient and useful paradigm to assess the taste-mediated odor learning. In the training session, Wistar male rats drank water from two bottles in their home cages and from eight small glass dishes. In the learning session they were exposed in their home cages and also in a circular open-field apparatus to 0.005 M Na-saccharin and 0.02 M quinine hydrochloride which contained either banana or almond odors. One learning trial consisted of this pair of exposures. The preceding behavioral experiment has shown that these two odors are not aversive and are differentially perceived by rats. In the test session, the animals were put in the open-field apparatus equipped with eight dishes: four contained water with banana, and another four, with almond. Normal control rats preferred to drink water with the odor previously associated with saccharin. Stronger and more persistent preference was attained after two or three learning trials. To elucidate the brain sites responsible for this taste-mediated odor learning, the same procedure was assessed on brain-lesioned rats. Rats with lesions in the amygdala showed rapid extinction of preference to the saccharin-associated odor, whereas control rats did not. However, rats with lesions in the insular cortex showed retention of learning similar to that of the control rats. Rats with lesions in the sulcal prefrontal or cingulate cortices showed moderate disruptive effects on preference to the saccharin-associated odor. In conclusion, the odor learning established in our experimental paradigm is based on the association between the quality of odor and hedonics of taste. The amygdala may play a role in the formation, at least in the retention process, of this taste-odor association learning.     

Potentiation of the acoustic startle response by a conditioned stimulus paired with acoustic startle stimulus in rats

The hypothesis that the standard acoustic startle habituation paradigm contains the elements of Pavlovian fear conditioning was tested. In a potentiated startle response paradigm, a startle stimulus and a light conditioned stimulus (CS) were paired. A startle stimulus then was tested alone or following the CS. Freezing behavior was measured to index conditioned fear. The startle response was potentiated on CS trials, and rats froze more in CS than in non-CS periods. In Experiment 1, response to a previously habituated, weak startle stimulus was potentiated. In Experiment 2, response to the same stimulus used as the unconditioned stimulus (US) in training was potentiated. This CS-potentiated response retarded the course of response decrements over training sessions as compared with an explictly unpaired control group. Conditioned fear is a standard feature of this habituation paradigm, serves to potentiate the startle response, and provides an associative dimension lacking in the habituation process per se.     

Extraversion and neuroticism in orienting reflex dishabituation

The present study examined the effects of extraversion (E) and neuroticism (N) as individual difference dimensions and of stimulus intensity on electrodermal orienting response habituation, test response, and dishabituation. Subjects were 162 college students selected from a screening sample of 860 as representing extreme groups of high, medium, and low E crossing with high, medium, and low N. Subjects in all nine resulting groups were habituated to criterion on auditory stimuli of 60, 80, and 100 db. A novel (test) stimulus and a dishabituating standard stimulus followed criteron habituation at each intensity. Results indicated that response amplitude for initial, test, and dishabituation responses was a direct function and that habituation rate (trials-to-criterion) was an inverse function of stimulus intensity. Extraverts habituated more rapidly than introverts at high intensity and gave smaller test and dishabituation responses. Some results differed when regression coefficients defined habituation rate and when first stimulus intensities alone were examined. Correlations among initial, test, and dishabituation responses and between each of these and the two measures of habituation rate are also reported. Results were interpreted as partially supportive of Eysenck's theory.     

Spatial memory for food hidden by rats (Rattus norvegicus) on the radial maze: studies of memory for where,what, and when

Rats (Rattus norvegicus) were allowed to hide food items on an 8-arm radial maze by carrying the items from the center to boxes at the end of each arm. Retrieval tests given after rats had hidden 4 items showed that they selectively returned to the maze arms where food had been hidden (Experiments 1 and 2). When rats were allowed to hide pieces of cheese (refed food) and pretzels (less preferred food) on different arms, they both hid and retrieved cheese before pretzels (Experiments 2-5). In Experiment 6, rats chose between arms where cheese and pretzels were hidden,with cheese degraded at one delay interval but not the other. Together, these experiments indicate memory for what and where but not when.     

Habituation of exploratory activity to new stimuli, to the absence of a previously presented stimulus and to new contexts, in rats

Rats trained to run a shuttle-alleyway for food exhibited exploratory behaviour to a novel visual/tactual stimulus presented in a specific place of the alley. Intermittent presentation of the same stimulus in the same place resulted in reduction of the exploratory activity (habituation), both within and between sessions. The animals also directed exploratory activity to the absence of the previously presented stimulus at the place where the stimulus had been presented before; this latter response also habituated. The presentation (1) of the same stimulus to which the animals had already habituated in another place in the alleyway or when the animals were running in an opposite direction to the previous trials of stimulus presentation, or (2) of another stimulus, resulted, again, in the appearance of exploratory activity. These results lead to the conclusion that the animals code external events, store this information, and use it to detect spatial- and directional-contextual changes and to generate predictions about that specific environment.     

How bumblebees first find flowers: Habituation of visual pattern preferences, spontaneous recovery, and dishabituation

Two experiments examined the exploratory behaviour of flower-naïve bumblebees. Bees were tested four times in a 12-arm radial arm maze in which they never received reward. Patterned and unpatterned stimuli were presented at the end of each corridor and the choices of the bees were recorded. We examined the effects of two variables, time and the presentation of a new stimulus, on habituation to a visual pattern. In Experiment 1, once bees had shown an unlearned preference for a pattern, and the preference had habituated, a response decrement was maintained after a confinement in the colony of two hours. After a 24 h confinement, however, the pattern preference was restored, i.e., spontaneous recovery was observed. In Experiment 2, the presentation of a new stimulus triggered renewed responding to the old pattern to which the bees had habituated, i.e., dishabituation was observed. Habituation to visual patterns is modulated by time and by exposure to new stimuli so that exploration in the face of failure to find food persists and unrewarding visual patterns are not eliminated for long as possible sources of food.     

Short- and long-term decrements in toxicosis-induced odor-aversion learning: the role of duration of exposure to an odor

Six experiments employed an odor-aversion paradigm to investigate the role of the duration of exposure to an odor in determining that odor's subsequent associability with illness. Rats were exposed to an odor at times T1 and T2, and the second of these exposures was followed by toxicosis. When the initial odor exposure was brief, the odor aversion was attenuated with a moderate T1-T2 interval of 3 hr (Experiment 1) but not with long intervals of 28 hr and 76 hr (Experiment 2). In contrast, when the initial odor exposure was long, the odor aversion was attenuated at a long T1-T2 interval (Experiment 3). With a T1-T2 interval of 24 hr, a brief initial exposure did not attenuate odor aversions when the context either remained the same or was changed from T1 to T2, whereas a long initial exposure attenuated such aversions when the context remained the same but not when the context was changed (Experiment 4). With a T1-T2 interval of 3 hr, a brief initial exposure attenuated odor aversions when the context remained the same or was changed from T1 to T2, whereas a long initial exposure attenuated such aversions when the context remained the same but not when the context was changed (Experiment 5). A brief exposure at T1, either with or without a subsequent context "extinction," attenuated odor aversions when the T1-T2 interval was 3 hr but not when this interval was 24 hr; a long initial exposure at T1, without a subsequent context "extinction," attenuated odor aversions when the T1-T2 interval was 4 hr and 24 hr but with a subsequent context "extinction" did not attenuate such aversions at either 4-hr or 24-hr T1-T2 intervals (Experiment 6). The results demonstrate that the duration of exposure to an odor determined whether that odor presentation caused short- or long-term decrements in odor conditionability and are discussed in terms of the relation between self- and retrieval-generated processes.     

An automated odor test for rats

The odor-testing apparatus described consists of a circular open-field area opening into four glass tubes, each with an odor source at the far end. When a rat is introduced, each tube is blocked by a grid; when the rat has touched each grid (sampled each odor), the tubes automatically open and recording of preference behavior starts. Two measures of preference are recorded by a photocell outside each tube, the number of visits and the time spent near the odor source. A uniformity test with food odor in all tubes showed that the apparatus did not promote position bias. In two separate sensitivity tests, one with food odor and one with estrous female urine odor, male rats significantly preferred an attractive odor in one tube to odorless controls. The apparatus yields relatively sensitive and reliable odor preference determinations by ensuring sampling of each odor before data are recorded and by eliminating visual, auditory and gustatory cues.     

Development of the perception of invariants: substance and shape

Three experiments investigated the perception of substance and shape as invariant properties of objects by three-month-old infants. In experiment 1, infants were habituated to two differently shaped objects undergoing a rigid motion. After habituation of the infants, the objects were presented undergoing a different rigid motion, or undergoing a deforming motion, or undergoing the same rigid motion. Habituation was maintained to the new rigid motion, indicating that the two rigid motions were perceived as sharing an invariant property. Dishabituation, on the other hand, occurred when a deforming motion followed a rigid one. In experiment 2, infants were habituated to one shape undergoing two different rigid motions. After habituation, the shape was changed but the same two motions continued. Dishabituation occurred, compared to a group with no shape change, indicating that shape is distinguished as an invariant property over two rigid motions. In experiment 3, habituation to a shape undergoing two rigid motions was followed by a new shape presented motionless, or the same shape presented motionless. Cessation of motion did not prevent recognition of shape as invariant. Two properties of an object, substance and shape, thus appear to be detectable as invariant in an event sequence, an instance of "phenomenal doubling" at an early age.