Predator Odor as an Unconditioned Fear Stimulus in Rats: Elicitation of Freezing by Trimethylthiazoline, a Component of Fox Feces

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1 Behavioral Neuroscience Copyright 2000 by the American Psychological Association, Inc. 2000, Vol. 114, No. 5, /00/$5,00 DOI: // Predator Odor as an Unconditioned Fear Stimulus in Rats: Elicitation of Freezing by Trimethylthiazoline, a Component of Fox Feces Karin J. Wallace and Jeffrey B. Rosen University of Delaware Four experiments tested whether an odor from a rat predator can unconditionally elicit a fear response in rats. In a large chamber, rats displayed fear-related behaviors to trimethylthiazoline (TMT, a volatile compound isolated from fox feces), including avoidance and immobility, while showing less exploratory behavior. In a smaller chamber, TMT induced a species-typical fear response, freezing, whereas other odors did not. In addition, TMT systematically elicited more freezing as the amount of TMT increased. Moreover, there was no within-sessions or between-sessions habituation of freezing to TMT, nor did TMT promote contextual conditioning. The results indicate that the predator odor, TMT, can induce a fear-related behavioral response in rats that is controllable and quantifiable, suggesting that TMT-induced freezing may be a useful paradigm for a neurobehavioral system analysis of ecologically relevant, unconditioned fear. Fear is a functional behavioral response to a dangerous event (Rosen & Schulkin, 1998). Research on learned fear in rats, typically produced by Pavlovian conditioning (e.g., pairing neutral stimuli with aversive footshock), has been instrumental in advancing understanding of the neurobiology of fear (Davis, 1992; Le- Doux, 1996). In contrast, unconditioned fear, such as fear of a predator, has not been studied in as much depth. Antipredator defense models provide considerable analytical advantages over footshock-based models of fear and defense for studying the determinants of defensive behavior and the neural and neurochemical systems that control them (D. C. Blanchard, 1997). Footshocks are not typically encountered by rats in natural environments but may represent a class of rare stimuli that are painful. When pain does occur, the painful stimuli may be associated with predator attack rather than with other more common features of the environment that signal a predator (D. C. Blanchard, 1997). In the wild, when a rat is attacked by a predator, it is usually fatal and too late for defensive maneuvers (Bolles, 1970). On the other hand, presentation of predators and stimuli associated with predators are "natural" threats and can serve as painless, controllable stimuli to study the behavior and biology of fear and defense (D. C. Blanchard, 1997). The odor from the fur of cats has been used as an unconditioned, ecologically relevant olfactory fear stimulus for rats (e.g., R. J. Blanchard & Blanchard, 1989; Zangrossi & File, 1992). However, when rats are exposed to this odor (a cloth rubbed on a cat's fur), typical fear behaviors (i.e., freezing) are displayed only briefly or Karin J. Wallace and Jeffrey B. Rosen, Department of Psychology, University of Delaware. This study was supported by National Science Foundation Grant IBN Correspondence concerning this article should be addressed to Jeffrey B. Rosen, Depat~nent of Psychology, 220 Wolf Hall, University of Delaware, Newark, Delaware Electronic mail may be sent to jrosen@udel.edu. do not emerge at all (R. J. Blanchard, Blanchard, & Hori, 1989). Instead, rats display behaviors involved in risk assessment or approach-avoidance conflict, such as orientation to the threatening object with a flattened back and stretched attention postures, sniffing from a distance, approaching the odor source for only short periods of time (R. J. Blanchard et al., 1989; Kaesermann, 1986), and spending more time in a sheltered environment (Zangrossi & File, 1992). These milder forms of defensive behavior may be due to the intensity of the stimulus, in which cat odor is less intense than a whole cat, and have been hypothesized to model human anxiety as opposed to fear (D. C. Blanchard & Blanchard, 1988; R. J. Blanchard et al., 1998). However, complicating this analysis is that the amount of odor is unknown and not controlled and that the specific compounds in the fur that elicit these behaviors are also unknown. The latter problem can confound interpretation, as seen in a comparison of mice responding to odor of cats on a vegetarian versus a carnivorous diet, in which mice displayed more avoidance of feces from meat-eating cats (Berton, Vogel, & Belzung, 1998). There are other predator odors, including specific components from fox feces and weasel secretions, that elicit avoidance responses similar to those seen with presentation of a cat or cat odors. One predator odor that has received attention is trimethylthiazoline (TMT), a component of fox feces. TMT is one of several sulfur-containing odors isolated from fox feces but is unique in its ability to induce stress and emotional responses from wild and laboratory-bred rats (Vernet-Maury, Polak, & Demael, 1984). Wild rats (rattus rattus) placed in a terrarium will avoid TMT (Vernet-Maury, Constant, & Chanel, 1992), and wild rats naive to fox avoid food in the presence of TMT as they do when in the presence of the scent from a known predator (mongoose; Burwash, Tobin, Woolhouse, & Sullivan, 1998). Laboratory rats are more vigilant in the presence of TMT than in the presence of nonpredator animal odors (e.g., from lion; Cattarelli & Chanel, 1979). Furthermore, rats exhibit analgesia in the presence of TMT (Hotsenpiller & Williams, 1997) similar to fear-associated analgesia induced by stressed rats or footshock (Fanselow, 1985; Fanselow 912

2 UNCONDITIONED FREEZING TO A FOX ODOR 913 & Tighe, 1988). These data imply that TMT may be an effective natural fear stimulus for rats. However, as with cat odors (R. J. Blanchard et al., 1989), fear responses typically measured in tests of conditioned fear (e.g., freezing) are not easily evoked by TMT in the contexts in which animals have been tested (Vernet-Maury et al., 1984). It is possible that because the odor is only a signal for the presence of a predator, it does not elicit sufficient fear to induce freezing. On the other hand, the testing conditions used to date may not have been optimal for inducing freezing with predator odors. In long runways or large areas in which TMT has been tested, animals were able to stay away from the odor source and effectively create sufficient distance from the odor to reduce its intensity to nonthreatening levels. In the current series of experiments, the hypothesis that TMT is an unconditioned fear stimulus for rats was examined. Freezing was the prototypical fear response measured in most of the experiments. Several aspects of TMT-induced fear were examined. We tested whether TMT elicited freezing and other behavioral responses in a medium-sized chamber where the rat could move away from the source of TMT and in a confined space where the rat could not escape the odor source. In addition, to test whether the intensity of TMT affected the amount of freezing, we generated a response curve for increasing amounts of TMT. Furthermore, whether the unconditioned freezing response habituated to TMT after repeated exposure and whether it could be used as an unconditioned stimulus to support contextual fear conditioning were also tested. Experiment 1 In the first study we observed behavior in response to TMT and other odors in a typical laboratory shoebox cage where the rat was able to roam. We observed and measured avoidance of the odor and odor-induced immobility as well as nondefensive behaviors such as rearing and grooming. In addition, the risk-assessment behaviors of exploring and stretch-sniff at the odor source were measured. Me~od Subjects. Nine male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing g at the beginning of the experiment were used. The subjects were housed individually in hanging wire cages. They were maintained on a 12-hour light-dark cycle, and food and water were continuously available. All experimental procedures were approved by the University of Delaware Institutional Animal Care and Use Committee. Apparatus. Testing was conducted in a standard, clear Plexiglas shoebox cage measuring cm with woodchip bedding on the floor of the cage. A Plexiglas lid with holes to allow air circulation and tape marking three equally sized areas of cm was placed on top of the cage during the experiment (Area 1 contained odor, Area 2 was in the middle, and Area 3 was farthest from the odor). The odor was presented by pipetting it onto a 6 6 cm piece of Kimwipe taped to a cm piece of Plexiglas that was propped up against the endwall. The cage was placed in a fumehood during experimentation so the volatile odors would not spread through the experimental room. A new cage was used for each rat so no odor except for the odor of the woodchips would be present in the chamber. Odor remaining in the fumehood was allowed to dissipate before the next subject was run. The experiment was videotaped for later analysis. The camera was located directly above the test chamber. Procedure. All rats were handled for about 5 min a day in the testing room for approximately 1 week before testing. Subjects were transported for handling in the same type of cages as they were tested in, so the testing cage was not novel. On the day of testing each rat was placed in the chamber with a piece of Plexiglas containing no odor propped up against the endwall. The rat was videotaped for 5 min in this condition. The odorless Plexiglas was removed following the 5-min period and replaced by a piece of Plexiglas containing an odor for 20 min. Odors tested were TMT, a component of fox feces (molecular weight: 129.2; Phero Tech, Delta, British Columbia, Canada); butyric acid, an ester in butter with a rancid odor unpleasant to humans (molecular weight: 88.1; ICN Biomedicals, Aurora, Ohio); isoamyl acetate, odor of bananas and pears (molecular weight: 130.2; ICN Biomedicals); and the neutral odor of the test cage. Odors were not diluted and were used neat. Each subject was presented with 300 nmol of each odor in the same order: TMT (19.4 ~1), neutral odor, butyric acid (13.2 p~l), and isoamyl acetate (19.6 p,1). Avoidance, immobility, rearing, grooming, and stretch-sniff were measured for 5 min before the odor presentation and during the 20 min of odor presentation. An additional measure of exploring the odor source within 30 s of its being placed in the chamber was also scored. The behaviors were measured later by viewing the videotapes. The experimenter (Karin J, Wallace) rating the behaviors was aware of the odor presented in this and all subsequent experiments. The amount of time spent in each of the three areas was used as a measure of avoidance. More time spent in Area 3 was considered more avoidance. Immobility was considered a lack of movement and measured as amount of time spent immobile. Rearing was counted when the rat stood on its hind limbs and was not directly sniffing the odor source. Grooming was recorded as the amount of time spent cleaning. Stretch-sniff was counted when a rat had a stretched back with hind limbs splayed while sniffing in the direction of the odor; this was measured as number of times the animal stretch-sniffed. An additional measure of initial exploration of the odor source was scored if the rat entered Area 1 and sniffed at the Plexiglas within 30 s of its being placed into the chamber. Statistical analysis of avoidance involved a 4 (type of odor) 3 (area of box) 4 (5-min time bin) three-way analysis of variance (ANOVA). Significant effects were followed by individual contrasts. Analysis of immobility, grooming, and rearing involved 4 (type of odor) 4 (5-min time bin) two-way within-subjects ANOVAs. Rats did not perform measurable levels of stretch-sniff after the first 5 min of odor presentation. Therefore, stretch-sniff in only the 5 min before the odor and in the first 5 min of odor presentation was analyzed. A one-way (type of odor) withinsubjects ANOVA was performed on these data. For all the above behaviors, a one-way (type of odor) ANOVA was performed to determine whether the preodor exposure was equivalent for each odor. As appropriate, significant results were followed by mean comparison contrasts. Exploration of the odor was analyzed by a Cochran Q test for related samples, followed by binomial tests. Statistical significance was set at p <.05 for all tests. Results In the 5 min before odor presentation, there was a difference in which area the rats spent time, F(2, 16) = 6.8, p <.007. Post hoc comparisons showed that rats spent more time in Area 1 (area containing the odor source) and Area 3 (area farthest from the odor source) than in Area 2 (middle area). There was a tendency for subjects to spend more time in Area 1 than Area 3, but this was not significant (p =.07). This preference was likely due to the novel Plexiglas object in that area. In the analysis with odor present, there was no main effect of time, F(18, 126) = 1.55, ns, indicating that the amount of time spent in each area did not change throughout the 20-rain session. There was, however, a main effect of the

3 .. [~ 914 WALLACE AND ROSEN amount of time spent in the three areas of the chamber, F(2, 14) = 22.76, p <.0001, such that, overall, rats spent more time in Area 3. More importantly, there was a significant interaction effect of Odor Area, F(6, 42) = 6.74, p < This is shown in Figure 1A. Time spent in Area 1 was significantly greater with the neutral odor than the other odors (p <.001), whereas time spent in Area 3 was greater with the other odors than with the neutral odor (p <.0001). While in Area 1, rats sniffed the odor source, and the subjects with the neutral odor manipulated the object containing the odor. We conducted an ANOVA without the neutral odor to compare differences between the three odor groups. There were no differences between TMT, butyric acid, and isoamyl acetate in the amount of time spent in the three areas, F(4, 20) < 1, ns. With all three odors the majority of time was spent in Area 3. Measuring immobility, we found no differences in the 5 min before presentation of each odor, F(3, 24) = 1.6, ns. However, during the odor presentation there was more immobility to TMT than to the other odors (see Figure 1B). There was a main effect of odor, F(3, 21) = 5.6, p <.006, that was due to less movement to TMT than to neutral odor, butyric acid, or isoamyl acetate. There was also a main effect of time, F(3, 21) = 2.5, p <.01, that was due to significantly more immobility min after the presentation of the odors than within the first 5 min of odor presentation. An Odor Time interaction, F(9, 63) = 2.5, p <.01, and post hoc A 2so T [] Neu~ [] Butyric Acid I I~ Isoamyl Acetate ] TMT ] ~-~ iiii:!.'xx -.~.... r -k.r I B ~ ~ 120 -~ ~ ~ 100 ~ [] Neutral [] Butyric Acid 0 j... i Odor Middle Far Frm Area 3 ~ 4114 o [i 20 4 < 0 j Time in 5 min intervals C 70 T 60 D 20-1 r~ 40 ~ 30.~ "-" Neutral Butyric Acid Isoamyl TMT Acetate Odor ~ 12 ~'.~ 10 ~m ~g s ~ 6 g4 2 0 Neutral 1 II Butyric Acid Isoamyl TMT Acetate Odor Figure 1. A: Mean (+_SEM) seconds spent in each of the three areas of the large testing chamber: the area containing the odor source (Area 1), the middle transition area (Area 2), and the area far from the odor source (Area 3). *p <.0001; denotes that significantly more time was spent in the area with the odorless Plexiglas during the neutral condition and less time was spent in the area far from the Plexiglas compared with the other odors. B: Mean (±SEM) seconds spent immobile during the 20 min in the chamber divided into 5-min intervals. * denotes that rats were more immobile in response to trimethylthiazoline (TMT) than to the other odors (p <.001) and more immobile to TMT in the last three 5-min time intervals compared with the first 5 min of TMT exposure (p <.0001). + p <.0001; denotes that immobility to isoamyl acetate was greater during the last two 5-min time intervals than during the first two 5-min intervals. Even though immobility increased across time to isoamyl acetate, it never reached the levels induced by TMT. C: Mean (±SEM) seconds spent grooming during odor presentation. *p <.001; rats spent more time grooming with a neutral odor than with the other odors. D: Mean (+--SEM) number of times rearing. *p <.01; rats reared less in the presence of TMT than the other odors and neutral condition.

4 UNCONDITIONED FREEZING TO A FOX ODOR 915 comparisons revealed that during the first 5 min immobility was at low levels and similar for all odors but that during TMT presentation immobility significantly increased after 5 min and remained greater than the other odors for the remainder of the observation period. Immobility to isoamyl acetate was also increased after 10 min of exposure. However, the increase in immobility to isoamyl acetate never reached the levels induced by TMT. Prior to the odor presentation, there were no differences in stretch-sniffing behavior between the odors, F(3, 21) = 1.1, ns. During the first 5 min of odor presentation, there was a difference in the number of stretch-sniff bouts, F(3, 21) = 3.12, p <.05. Post hoc comparisons further showed that rats had more mean (+-SEM) stretch-sniff bouts to butyric acid (2.9 _+ 0.9) than to isoamyl acetate ( ), TMT ( ), or neutral odor ( ). Exploration and sniffing of the odor source in the first 30 s of exposure to the odors was significantly different, Cochran Q(3) = 11.07, p <.02. No exploration occurred with TMT, whereas there was significantly more exploration with neutral odor (p <.05), butyric acid (p <.02), and isoamyl acetate (p <.05; see Table 1 ). With each of the odors, 4 of the 9 rats tested were in Area 1 when the odor was placed in the chamber and quickly moved out of the area. All of the subjects with butyric acid, 3 of the subjects with isoamyl acetate, and 2 of the subjects with the neutral odor moved back into Area 1 to sniff the odor source. In contrast, none of the rats with TMT returned to Area 1 or approached the odor source. The nondefensive behaviors, grooming and rearing, were also measured. There was no difference in grooming before the odors were presented, F(3, 24) = 0.70, ns. During the odor presentations, there was more grooming with the neutral odor than with butyric acid, isoamyl acetate, or TMT, F(3, 24) = 7.6, p <.001 (see Figure 1C). There were no differences between butyric acid, isoamyl acetate, and TMT. Analysis of rearing found a difference between the odor groups before the odor presentations, F(3, 24) = 4.3, p <.01. Post hoc analysis showed that the number of rearings was less before TMT than before neutral odor or butyric acid. This was probably because TMT was tested first. During odor presentation there was a significant main effect of odor on rearing, F(3, 24) = 11.9, p <.0001, in that there was significantly less rearing to TMT than the other odors (p <.001; see Figure 1D). Rearing was marginally less to isoamyl acetate than to butyric acid and the neutral odor (p <.06). There was also a main effect of time, F(3, 24) , p <.0001, in that there was less rearing as the observation period progressed. The Odor Time interaction was not significant, F(9, 45) = 0.53, ns. Table 1 Number of Rats Exploring the Odor Source During the First 30 s of Odor Presentation Exploration TMT* Neutral odor Butyric acid Isoamyl acetate Yes No Note. TMT = trimethylthiazoline. *p <.05 (rats' behavior in response to TMT was significantly different from other odors). Discussion The rats avoided TMT. Avoidance has been found by others in laboratory and wild rats in response to TMT (Vernet-Maury et al., 1984) and cat odors (R. J. Blanchard et al., 1989; Zangrossi & File, 1992). However, in our experiment, rats also avoided butyric acid and isoamyl acetate, suggesting that rats avoid not only predator odors but also other odors that are novel. Thus, avoidance behavior did not distinguish between TMT and the other odors. Stretchsniff behavior, which occurs during risk assessment of danger (R. J. Blanchard et al., 1989), also was not displayed more to TMT than to the other odors. Butyric acid was the only odor to induce significant amounts of stretch-sniff behavior. The amount of the nondefensive behavior, grooming, also did not separate TMT from the other two odors, although grooming occurred more in the neutral odor condition than with the three odors. In contrast to avoidance, stretch-sniff, and grooming, immobility did discriminate between TMT and the other odors in that rats spent more time immobile to TMT. Immobility increased after 5 rain of exposure and remained high for the remaining 15 rain of the session, suggesting that it took several minutes for TMT to disperse through the chamber in sufficient levels to induce immobility. However, immobility to isoamyl acetate also increased after 10 min of exposure, but not to the levels of TMT-induced immobility. Although we believe that our measure of immobility is the same as fear-related freezing typically seen in response to novel or threatening stimuli, we are not completely confident that all of the immobility was fear-related freezing. Behavior was viewed and scored on videotape captured from a camera placed above the chamber. Because a videotaped image of behavior is not as clear as behavior viewed live and we could see only the backs of the subjects, it was difficult to be certain that immobility was truly freezing and not some other immobile behavior. Thus, although immobility to TMT was likely fear-related freezing, increased immobility to isoamyl acetate may also have been fear-related freezing. Nondefensive grooming behavior also decreased to TMT, butyric acid, and isoamyl acetate, as would be expected if fear were increased. However, in argument against the interpretation that all three odors induced fear, rearing--a nondefensive exploratory behavior--significantly decreased with exposure to TMT compared with the other odors. In addition, rats did not explore the TMT odor source when it was initially introduced into the chamber as they did with the other odors. A decrease in exploratory behavior would likely occur during danger and would support the notion that immobility to TMT is fear-related freezing. Experiment 2 Although the shoebox chamber in Experiment 1 allowed us to measure a variety of behaviors, it made it difficult to accurately measure the commonly evaluated fear behavior, freezing. In addition, it took time for the odor to disperse through the chamber and allowed the rat to move away from the odor, thus impeding experimental control of the situation. To better control the stimulus and response, we decided to measure freezing behavior in a smaller chamber where the source of the odor was inescapable and optimal for elicitation of freezing. Different configurations of testing chambers have been shown to generate diverse defensive behaviors in that avoidance and escape are more likely elicited in

5 916 WALLACE AND ROSEN large chambers and freezing is more probable in small environments (R. J. Blanchard et al., 1989). In Experiment 2 of the present study, freezing behavior to TMT and other odors was analyzed in a small, confining chamber. Each rat was able to move and turn around but was always facing an odor source and unable to escape the odor. Freezing to TMT was compared with butyric acid as before and to caproic acid (a goat-like odor found in milk), which rats have been shown to avoid when it was presented on a cotton swab in front of their nose (Heale, Vanderwolf, & Kavaliers, 1994). It was hypothesized that the small, confined area and the inability to escape would select freezing as the predominant species-specific defense reaction. Method Subjects and apparatus. Twenty naive male Sprague-Dawley rats (Harlan) weighing approximately g were used in this experiment. In addition to receiving the same housing conditions and pretesting handling treatment as described in Experiment 1, rats were also preexposed to the testing chamber as described below. Testing was conducted in a clear Plexiglas cylindrical chamber (8.6 cm diameter, 20.0 cm length, SR-Lab animal enclosure, San Diego Instruments, San Diego, CA). Plexiglas doors (the same pieces of Plexiglas placed in the chamber of Experiment 1) dropped into slots at each end of the cylinder kept the rat in the chamber. The rat was confined but not restrained and could move and turn around in the chamber. During testing, the odors were pipetted onto a piece of a Kimwipe that was taped to the inside of each door as in Experiment 1. The chamber was placed in a fumehood during the experiment so the volatile odors would not spread through the experimental room. The chamber was cleaned with 5% (vol/vol) ammonium hydroxide following each rat's test. Residual odor was allowed to dissipate before each subject was placed in the chamber. Procedure. In addition to being handled as described in Experiment 1, in the 3 to 4 days before testing rats were acclimated to the chamber. Acclimation involved placing a subject in the testing chamber for 4-10 min a day until it spent less than 20% of the time freezing during a 4-min observation period. During the acclimation period all conditions were the same as during testing, except the odors were not presented. On testing day, rats were placed in the chamber with odorless drop doors (as in the acclimation period) for 3 min to establish a base level of freezing for that day. If a rat froze less than 20% during the baseline period, then the odorless doors were replaced with doors that contained the odor. If freezing was greater than 20% of the time, then the rat was removed and tested again the next day or when freezing fell below 20% during the baseline period. Rats were exposed to 100 nmol TMT (6.5 p.1 per door), butyric acid (13.2/~l per door), caproic acid (6.0/xl per door, molecular weight: 116.2; ICN Biomedicals), or the neutral odor of the testing chamber. The 100 nmol refers to the combined amount applied to the two doors (50 nmol was applied to each door). A smaller amount of odor was presented in this experiment than in Experiment l because of the smaller size of the chamber. In the neutral-odor condition no odor was placed on the doors, but water was pipetted on the tissues to make them wet as in the other odor conditions. Each rat (n = 5 per odor) was exposed only once to a single odor. Freezing was observed for 9 min of odor exposure. Freezing was used as a measure of fear and is characterized by crouching with cessation of movement except for that associated with breathing (R. J. Blanchard & Blanchard, 1969). The observer (Karin J. Wallace) viewed the animal from the horizontal perspective looking at the full length of the body. The behavior was not videotaped, and the observer was aware of the experimental groupings. Freezing was measured as a sample of time spent freezing over a given length of time in the chamber. Behavior was assessed every 5 s as either freezing or not freezing. A percentage of time spent freezing was calculated for each rat ([number of observations freezing/total observations] 100). The time spent freezing to TMT and the neutral odor was first analyzed over the 9-min testing period with a two-way repeated-measures ANOVA. Subsequently, the percentage of time spent freezing during the whole observation period was used as a freezing score to compare freezing during TMT, butryic acid, caproic acid, and the neutral odor. We performed a one-way ANOVA on this data, followed by ScheffCs S post hoc analysis to determine differences in percentage of time spent freezing between various odors. Results When TMT was initially presented, rats sniffed a door and quickly turned around and continued sniffing for a few seconds, with intermittent bouts of freezing. Rats turned and sniffed a few times and then predominantly froze the rest of the time in the chamber. Freezing was analyzed in l-rain intervals for the entire 9 rain of exposure to TMT and the neutral odor. The mean (+-_SEM) percentage of time spent freezing in the l-rain intervals to TMT and neutral odor is shown in Figure 2. The range of the mean percentage of time spent freezing to TMT for each minute of the test session was 26%-57%. The range of the neutral odor group was 5%-25%. Freezing was significantly greater during TMT exposure than during the neutral odor, F(1, 8) = 15.75, p <.004. Freezing was consistent across the entire time in the chamber for rats exposed to either TMT or the neutral odor, F(8, 64) = 1.2, ns, and there were no differences in freezing rates between each minute the rat was in the chamber in the presence of each odor, F(8, 64) < 1, ns. Figure 3 presents the percentage of time spent freezing to each odor averaged across the entire test session. A one-factor ANOVA found a significant group difference, F(3, 16) = 8.1, p <,002. A ScheffCs S post hoc analysis (p <.01) revealed that the mean (r+_sem) percentage of time spent freezing to 100 nmol TMT ( ) was significantly greater than the percentage of time spent freezing to 100 nmol butyric acid (16.6 _-_ 3.7), caproic acid (16.4 +_ 2.7), or neutral odor ( ). The percentage of time 1oo 9o ~ 80 ~. 7o ~ ~0 N 4o ~ 3o ~ 2o to ~Neutral TMT Pre l Time of Odor Exposure (Min) Figure 2. Mean (~-SEM) percentage of time spent freezing to trimethylthiazoline (TMT) in the small testing chamber. Rats froze significantly more to TMT than to the neutral odor throughout the 9-rain testing session. There was no within-sessions habituation of freezing to TMT or the neutral odor.

6 UNCONDITIONED FREEZING TO A FOX ODOR 917 1oo r~ 50.e 40 ~ 30 ~ 20 ~, 10 0 il Neutral Caproic Acid Butyric Acid TMT Odor (100 amol) Figure 3. Mean (+_SEM) percentage of time spent freezing to trimethylthiazoline (TMT), caproic acid, butyric acid, and the neutral odor presented in the small testing chamber. * p <.002; freezing to TMT was significantly greater than freezing to the other odors. spent freezing to butyric acid or caproic acid was not different from freezing during the neutral odor. Discussion The results indicate that with equimolar amounts of odors, freezing rates in the presence of TMT were higher than two odors not related to predators (caproic acid and butyric acid). Freezing was not just concentrated during initial exposure to TMT but persisted throughout the entire period in the chamber, and animals did not habituate within the session. The small test chamber selected freezing as the dominant behavior. Thus, by changing the configuration of the environment we were able to easily select and identify a fear-related behavior that is quantifiable and commonly measured in fear-conditioning studies (Fanselow & Helmstetter, 1988; R. J. Blanchard & Blanchard, 1969). However, in contrast to fear conditioning, in which a cue or context must be paired with a footshock before it elicits a fear response, rats in the present experiment were naive to TMT, yet TMT elicited a similar freezing response. Freezing during the first encounter with TMT suggests that TMT is an unconditioned, ecologically relevant fear signal. It appears that in the proper environment there is a propensity for elicitation of a fear response to a predator odor compared with odors that are avoided but not associated with danger. The elicitation of freezing by TMT in the small chamber also supports the assumption that the immobility measured in the larger shoebox chamber was also freezing. Although it is appealing to think that TMT may be an unconditioned fear stimulus, the difference in freezing rates to TMT and other odors may be due to differences in intensity of the odors. TMT may be more volatile and aversive than other molecules, and with sufficient levels of odor other nonpredator odor may become aversive and induce freezing as well. Therefore, the following experiment tested whether response curves could be generated with increasing amounts of TMT, butyric acid, and isoamyl acetate. Experiment 3 In fear conditioning, rats freeze at increasing rates in response to more dense and/or intense footshock (Fanselow & Bolles, 1979; Leaton & Borszcz, 1985). We conducted Experiment 3 to determine whether the same holds true for increasing amounts of predator odor. In addition, in the previous experiment, the difference in freezing rates to TMT, butyric acid, and caproic acid could have been due to a difference in intensity of the odors with the same number of moles, or it may have been due to the type of odor (i.e., predator vs. noxious). Thus, response curves to different amounts of the odors were generated to determine whether rats freeze more to increasing amounts of TMT, butyric acid, and isoamyl acetate. Butyric acid and isoamyl acetate were chosen as a comparison with TMT because of the preference of rats to avoid these odors (Heale et al., 1994, Experiment 1). Method Seventy-four male Sprague-Dawley rats (Harlan) weighing g were tested in this experiment. The same chambers as described in Experiment 2 were used. TMT amounts of 1 (0.07/xl per door; n = 6), 10 (0.70 p,1 per door; n = 5), 100 (6.50 ttl per door; n = 10), 300 (19.38/xl per door; n = 6), and 600 (38.70/xl per door; n = 9) nmol were investigated. Butyric acid amounts of 100 (4.50/xl per door; n = 5), 300 (13.20 txl per door; n = 6), 600 (26.40/xl per door; n = 9), 6,000 ( Ixl per door; n = 4), and 12,000 ( /xl per door; n = 5) nmol were tested. In addition, neutral cage odor (n = 9) was used as a control. Isoamyl acetate was presented to 3 rats each in amounts of 300 (19.53/xl per door), 600 (39.06 p~l per door), and 6,000 (390.60/xl per door) nmol. All rats were handled and were allowed to acclimate to the chamber for 4 to 5 days as in Experiment 2. All subjects were naive to the odor presented and received only one presentation of an odor, except those receiving isoamyl acetate, which were tested with all three amounts of the odor. The odors were presented for 10 min following a 3-min preodor period. Freezing behavior was scored as in Experiment 2; that is, each rat was assessed every 5 sec and determined to be freezing or not. A percentage of time spent freezing was calculated for each rat ([number of freezing observations/total observations] 100). Preodor freezing was used to make sure that freezing levels were not different for each rat before an odor was presented. These data were not used in the statistical analysis. To determine whether there was an amountresponse relationship of the individual odors, separate one-way ANOVAs for TMT and butyric acid were performed, followed by Scheffr's S post hoc test. For isoamyl acetate, a two-way ANOVA (Amount [between factors] Preodor vs. Odor [within factors]) was performed, followed by individual comparison contrasts. Statistical significance was set at.05. To compare whether levels of freezing were different between the odors, we performed a one-way ANOVA, followed by Scheffr's S test, between the amounts of TMT, butyric acid, and isoamyl acetate that induced the highest levels of freezing. These were 600, 12,000, and 6,000 nmol, respectively. Resul~ Response curves for TMT, butyric acid, and isoamyl acetate are presented in Figure 4. There was an overall difference in freezing between the amounts of TMT, F(5, 41) = 31.5, p <.0001, tested. The mean (+SEM) percentage of the time spent freezing to TMT increased significantly as the amount increased (neutral odor: ; 1 nmol TMT: 28, ; 10 nmol TMT: _ 7.0; 100 nmol TMT: 48.8 ± 7.2; 300 nmol TMT: ; 600 nmol TMT: 81.3 ± 3.8). Scheffr's S post hoc analysis revealed that percentages of time spent freezing to the neutral odor and 1 and 10 nmol TMT were not different. Although 100 nmol TMT was different from neutral odor, it was not different from 1 or 10 nmol

7 918 WALLACE AND ROSEN 100 " "~ ~ , ~ 50' '~ 3o N 20 ~ to g. o t I I I I Amount of Odor (nmol) Neutral Figure 4. Amount-response curve to trimethylthiazoline (TMT), butyric acid, isoamyl acetate, and neutral odor. Mean (+_SEM) percentage of time spent freezing to TMT significantly increased with increasing amounts of TMT; freezing to 100 nmol was greater than to the neutral odor, and freezing to 300 and 600 nmol was greater than to 1, 10, and 100 nmol TMT. Freezing to isoamyl acetate was significantly increased with 6,000 nmol compared with preodor levels. Freezing did not increase with greater amounts of butyric acid and was not different from freezing to neutral odor. The solid and dotted lines represent the mean and SEM of the percentage of time spent freezing to the neutral odor, respectively. a s Butyric Acid Isoamyl Acetate A TMT I 1~0~ TMT. Freezing to 300 and 600 nmol TMT was greater than freezing to the neutral odor and 1, 10, and 100 nmol TMT. In contrast to TMT, no differences in freezing were found between the neutral odor and amounts of butyric acid ranging from 100 to 12,000 nmol, F(5, 34) = 0.68, ns. Thus, freezing was not induced by the amount of butyric acid 1,200 times greater than the amount of TMT (100 nmol) that induced significant freezing. This 1,200-fold difference is far greater than the difference in volatility of TMT and butyric acid. TMT is about three times as volatile as butyric acid (Hotsenpiller & Williams, 1997). Three rats were tested with isoamyl acetate; each was tested with 300, 600, and 6,000 nmol. Freezing tended to increase compared with preodor freezing levels but did not reach statistical significance, F(1, 2) = 16.0, p <.06. However, an individual comparison test found significantly more freezing to 6,000 nmol isoamyl acetate compared with preodor freezing (44% vs. 16%, respectively). A final statistical comparison of freezing was made between 600 nmol TMT, 12,000 nmol butyric acid, and 6,000 nmol isoamyl acetate. A one-way ANOVA found an overall difference, F(2, 14) = 54.8, p < A Scheffr's S test revealed that freezing to TMT was greater than freezing to butyric acid and isoamyl acetate. Freezing to isoamyl acetate was also greater than freezing to butyric acid. This analysis indicates that TMT induced more freezing that the other odors at only a fraction of the amount; nevertheless, a large amount of isoamyl acetate (6,000 nmol) also induced appreciable levels of freezing. Discussion The results demonstrate an amount-dependent relationship of TMT to freezing, indicating that the topography of fear behavior is governed by the intensity of the eliciting stimulus. With an unconditioned, ecologically relevant fear stimulus, such as a predator, the closer it is to the prey, the more intense are fear and its associated behaviors (R. J. Blanchard et al., 1989). With footshock-induced fear, the amount of freezing is generated proportionally to the intensity of the shock (Fanselow & Bolles, 1979; Leaton & Borszcz, 1985). In our experiment, the response curve generated by TMT is evidence that the amount of freezing is also proportional to the stimulus intensity and that intensity of the odor was a key factor for selection of freezing as the fear response. The amount of freezing to 300 and 600 nmol TMT (about 80% of the time) is comparable to the amount of contextually conditioned freezing induced in our laboratory by a single 1-sec, 1.5-mA footshock (Malkani & Rosen, 2000; Thompson & Rosen, 2000), suggesting that high levels of fear are induced by TMT. Isoamyl acetate also induced significant freezing. This suggests that isoamyl acetate does have some fear-inducing properties. Increased immobility with isoamyl acetate in Experiment 1 supports this conclusion. A similar finding has been reported before that isoamyl acetate was found to induce arousal and vigilance in rats at a lower rate than TMT but more frequently than other nonpredator odors (Cattarelli and Chanel, 1979). The percentage of time spent freezing to 6,000 nmol isoamyl acetate in our experiment reached only 44%, which is the same level of freezing that 100 nmol TMT produced. Thus, it took 60 times the amount of isoamyl acetate to produce the same levels of freezing as TMT. Apparently, fear-inducing properties of odors are not exclusive to predator odors; however, at least for isoamyl acetate, very large amounts of the odor are needed. In addition to intensity of odor, the configuration of the testing chamber was also a possible influence on the selection of fear behavior. In a large space, rats are able to avoid, escape, or move away from the fear stimulus. When the space was confining, freezing behavior to small amounts of TMT predominated, suggesting that the shape of the environment selected the response. However, Fanselow (1986, 1994) has argued that shape and size of the testing chamber are not critical factors in defensive response selection but that the amount of aversive stimulation and the amount of fear induced are controlling variables. Questions of whether the shape of the testing chamber is an important variable for the elicitation of freezing need to be tested further. Nevertheless, it is clear that the intensity of TMT is a controlling factor because of the amount-response relationship of TMT and freezing. Experiment 4 Rats presented with TMT in the small chamber froze for the entire 9-min test session, demonstrating no within-sessions habituation (see Experiment 2 and Figure 2). TMT remained a powerful inducer of freezing throughout the session, which contrasts with fear-conditioned freezing to a cue that extinguishes or habituates if continuously presented without further footshock (Bouton & King, 1983). Whether freezing will habituate or extinguish on repeated exposure to TMT is not known. Two experiments using a cat as a predator showed no habituation of fear responses after repeated test sessions with a cat (R. J. Blanchard, Blanchard, Rodgers, & Weiss, 1990) or cat odor (Zangrossi & File, 1992). Experiment 4 of the present study was designed to determine whether freezing to TMT would also be maintained with repeated presentations of the odor. The experiment was also designed to determine whether TMT could act as an unconditioned stimulus to support contextual fear conditioning. Footshock can act as an unconditioned stimulus to

8 UNCONDITIONED FREEZING TO A FOX ODOR 919 induce fear of either contextual or specific cues (Kim, Rison, & Fanselow, 1993; Phillips & LeDoux, 1992). If TMT can do the same, it would be an important tool for studying fear conditioning without a painful unconditioned stimulus. We examined whether TMT can support contextual fear conditioning. Method Ten male Sprague-Dawley rats (Harlan) were used in this experiment. All rats were similar to those described in Experiments 2 and 3 and received the same pretesting treatment as in those experiments, with one exception--these subjects were not acclimated to the cylindrical chamber before testing. During the experiment, rats were placed in the chamber as before for a 3-min before-odor period prior to 10 min of odor presentation. This was done for five training sessions over 5 days at the same time of day for each session. In each session, freezing was measured in the 3-min before-odor period and for the following 10 min with TMT (n = 5) or neutral odor (n = 5). Freezing during the 3-rain before-odor period was a measure of contextual conditioning due to the previous day's exposure to odor. Freezing during the 10 rain of odor presentation was a measure of habituation to the odor. All TMT presentations were at 300 nmol (19.38/xl per door) because this amount produced high asymptotic levels of freezing, with the rats freezing about 70% of the time in the chamber (see Experiment 3). We performed a three-way ANOVA (TMT vs. Neutral Odor Before Odor vs. During Odor [repeated measure] X Test Day [repeated measure]), followed by Schefft's S or individual-comparison post hoc analysis, to determine differences in percentage of time spent freezing. Statistical significance for all tests was set at.05. Resul~ Significant main effects were found between TMT and the neutral odor, F(1, 8) = 11.13, p <.01, the test days, F(4, 32) = 2.71, p <.05, and before versus during odor, F(1, 8) = 50.56, p < Because there was not an Odor Test Day interaction, F(4, 32) = 0.17, ns, and post hoc analysis revealed that freezing on Day 2 was greater than on the other days, the main effect of test day was due to more freezing in both groups on only Day 2. There was a significant interaction between the groups and before- versus during-odor comparison, F(1, 8) = 10.16, p <.01. Taking these analyses together, the difference between beforeversus during-odor freezing was significant for TMT but not for the neutral odor (see Figure 5). An interaction effect of Test Day Before Odor versus During Odor was also significant, F(4, 32) = 6.73, p < In the analysis of contextual fear conditioning, individual comparisons of test day in the before-odor condition found that freezing on Day 1 was different from Days 2, 3, and 4. Also, freezing on Day 2 was different from all other days, whereas freezing on Days 3, 4, and 5 did not differ. These effects are presented in Figure 6 as a change in the amount of freezing on each day from freezing on Day 1 in the before-odor condition. In the analysis of habituation, individual comparisons of test day in the during-odor condition, freezing on Day 4 was different from freezing on Days 1, 2, and 5. No other differences were found. These effects are presented in Figure 7 as a change in the amount of freezing on each day from freezing on Day 1 in the during-odor condition. Finally, the three-way interaction of TMT versus Neutral Odor Before Odor versus During Odor Test Day was not significant, F(4, 32) = 0.90, ns, indicating that test-day differences g~ g~ g~ [] Before Odor During Odor Neutral TMT Figure 5. Mean percentage (+-SEM) of time spent freezing before and during odor in Experiment 4. Data are collapsed over the 5 days of exposure. There was no difference in freezing before and during the neutral odor, but freezing was significantly greater during trimethylthiazoline (TMT) exposure than before TMT. *p < in freezing in the before- and during-odor conditions were not due to differences between TMT and neutral odor. Discussion The data from the during-odor condition demonstrate that freezing does not appear to habituate to TMT over five daily exposures. Although there was a decrease in freezing on Day 4 compared with the other days, this occurred in both the TMT and neutral odor groups. In addition, both groups' freezing on Day 5 returned to the same levels as those found on Days 1, 2, and 3. Thus, there was not a pattern of habituation of freezing to TMT, and the decrease on Day 4 seems spurious to both the TMT and neutral odor groups. The lack of habituation resembles what R. J. Blanchard and others (1990, 1998) found with repeated presentation of a cat. A cat was placed briefly in a rat-visible burrow system over successive days, and the same pattern of avoidance behavior in the rats was found on each day of cat presentation. This is also similar to what File, Zangrossi, Sanders, and Mabbutt (1993) found when exposing rats to cat-fur odor--there was no behavioral habituation, although corticosterone levels elevated during the first exposure to the cat odor decreased after repeated exposures. The data from the before-odor condition show that freezing in both the TMT and neutral odor groups was higher on Day 2 than all other days. Although this may be a small contextual conditioning effect, it occurred in both the TMT and neutral odor groups, suggesting the increase is not an effect exclusive to TMT. Any contextual conditioning that occurred in both groups is likely due to the stress of being placed back in the test chamber for a second day and not specifically to the odors. The lack of a specific TMT-conditioned response to the context may be because TMT is not an unconditioned stimulus in the traditional sense that footshock is. Conditioned fear typically occurs following direct exposure to a painful stimulus. Conditioned freezing is a response in anticipation of a subsequent delivery of the painful stimulus (Rosen & Schulkin, 1998). The freezing generated by TMT may also be an anticipatory response to the

9 920 WALLACE AND ROSEN 5O 40 0 & 3O gl "~ 20 l_..~ lo ~ o,ii Neutral 1 TMT ] Day of Exposure Figure 6. Analysis of contextual fear conditioning as shown by freezing in the before-odor condition. Data are presented as a change in freezing on each day from freezing on Day 1 (freezing on each day minus freezing on Day 1). Rats froze significantly less on Day 1 compared with Days 2, 3, and 4 and significantly more on Day 2 compared with the other days. However, these changes in freezing over days did not differ between trimethylthiazoline (TMT) and the neutral odor. possibility of an encounter with a predator. Therefore, just as a light that was previously paired with footshock functions as a cue for another footshock, TMT may act as a cue or signal for an impending attack. At the same time, TMT exposure is not sufficient to generate conditioning. A similar lack of conditioned freezing has also been shown with nontactile exposure to a cat (Fanselow, 1989), suggesting that exposure to signals of danger without direct contact may not be sufficient to produce a conditioned freezing response in rats. Although exposure to a cat does not engender conditioned freezing, Fanselow (1989) hypothesized that cat exposure may reinforce conditioning of other, less intense fear behaviors such as a change in eating patterns in an environment in which a rat previously encountered a cat. Whether TMT can support conditioning of less intense fear responses than freezing would need to be examined in future experiments. General Discussion The results of these experiments demonstrate that a component of fox feces, trimethylthiazoline or TMT, can elicit behaviors classified as defensive and fearful--that is, avoidance and freezing-and can suppress nondefensive behaviors. Because TMT is an odor from a predator and the rats had not encountered this odor previously, the studies suggest that TMT is an ecologically relevant, unconditioned fear stimulus for rats. In addition, a habituation and contextual conditioning experiment indicates that TMT has similar properties to that of a predator; there is no habituation to repeated exposure to TMT or a cat (R. J. Blanchard et al., 1990), and neither supports conditioned freezing behavior (Fanselow, 1989). Thus, TMT may be a very useful stimulus for investigation of environmental and neural mechanisms of unconditioned fear in a controllable and quantifiable manner. Paradigms of learned fear provide rigorous experimental control of stimuli and behavior that have produced advances in understanding the neuroanatomy and biology of fear. Neural circuits that include several nuclei of the amygdala and their afferents and efferents have been delineated with conditioned fear paradigms employing unimodal stimuli (Davis, 1997; Kapp, Whalen, Supple, & Pascoe, 1992; LeDoux, 1995). Unimodal conditioned and aversive, painful stimuli afford delineation of particular sensory pathways mediating these stimuli and sites of convergence for fear conditioning. The measurement of ecologically relevant, simple fear responses, such as freezing and startle, that are mediated through defined neural pathways has been crucial in these endeavors. The study of the neurobiology of unconditioned fear is not well developed, particularly compared with research on conditioned fear. Although amygdala and periaqueductal gray lesions in rats have been shown to block freezing and analgesic responses to cats (D. C. Blanchard & Blanchard, 1973; Fox & Sorenson, 1994; De Oca, DeCola, Maren, & Fanselow, 1998), little else is known about the neural circuits of antipredator behavior. Part of this is due to the imprecise control and knowledge of the stimulusresponse relationship with exposure of a whole predator. It is unclear what stimulus features evoke fear. The advantages of isolating sensory features can be found in experiments of shockinduced fear conditioning with a unimodal sensory conditioned cue, such as a light or tone. Similar types of analyses of unimodal sensory cues of unconditioned fear would allow advances in the study of environmental control of unconditioned fear and the neural circuits underlying it. To remove tactile and painful stimuli of a predator in antipredator paradigms, the "prey" can be separated physically by a barrier while the visual, auditory, and olfactory features of the predator are still present. To isolate olfactory cues specifically, the odor of cat fur has been used as a unimodal unconditioned fear stimulus (R. J. Blanchard et al., 1989). However, the specific odors in cat fur have not been isolated and likely include numerous components. TMT would offer an advantage over cat fur because it is a single molecule and therefore eliminates ambiguity about the inducing stimulus. O~ 30 2o 10 "~ 0.=_ I -.- Neutral] TMT ] Day of Exposure Figure 7. No habituation to trimethylthiazoline (TMT), as shown by analysis of freezing in the during-odor condition. Data are presented as a change in freezing on each day from freezing on Day 1 (freezing on each day minus freezing on Day 1). Although rats froze significantly less on Day 4 compared with Days 1, 2, and 5, changes in freezing over days did not differ between TMT and the neutral odor.

10 UNCONDITIONED FREEZING TO A FOX ODOR 921 Presentation of TMT and the elicitation of freezing satisfy requirements for an experimental analysis of a neurobehavioral system of unconditioned fear. TMT is a "natural," painless, isolated, and unimodal olfactory stimulus that has now been shown to elicit a quantifiable species-specific and ethologicauy relevant fear response, freezing. Freezing to TMT can be controlled in a lawful fashion by varying the amount presented. In addition, because there is no within-sessions or between-sessions habituation, TMT can be tested repeatedly without the diminution or loss of freezing. Furthermore, the lack of associative conditioning of the context to TMT permits analysis of an unfettered, unconditioned stimulusresponse relationship. Additional analyses using other fear-related responses would reinforce the notion that TMT induces unconditioned fear (Hotsenpiller & Williams, 1997). A neuroanatomical analysis of TMT-induced unconditioned fear can be pursued from the sensory organ to the behavioral output system. The central nucleus of the amygdala and periaqueductal gray are assumed to play a central role in mediating unconditioned, fear-related freezing to TMT (D. C. Blanchard & Blanchard, 1973; De Oca et al., 1998; Fox & Sorenson, 1994). The sensory pathways from the main and accessory olfactory systems to the amygdala are well-delineated (Shipley, McLean, & Ennis, 1995) and provide a starting point to investigate the sensory substrates for TMT-induced freezing. In addition, the olfactory receptors that bind TMT and transduce the behaviorally relevant signal can be investigated. Furthermore, comparison between the neuroanatomy of conditioned and unconditioned fear can be made using the same behavioral response for both types of fear. In summary, the predator odor TMT appears to be an ecologically relevant, unconditioned fear stimulus for rats. The intensity of TMT and possibly the configuration of the testing chamber can control the selection and amount of particular species-specific defense reactions. The lack of habituation of the freezing response can be an experimental advantage and allow for repeated analysis without the concern of extinction of the response. Furthermore, the ability to experimentally control unconditioned fear behavior elicited by a painless, unimodal, and isolated sensory stimulus provides a predictable and quantifiable tool for the investigation of behavioral and neural mediation of unconditioned fear. Much progress has been made in elucidating the neural circuitry involved in learned fear by use of cue-specific fear conditioning (Davis, 1997; LeDoux, 1996). The study of TMT-elicited freezing may offer the same advantages for the study of the neural systems and circuitry important for unconditioned fear. References Berton, F., Vogel, E., & Belzung, C. (1998). 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