Presentation of a predator, like a cat, or cues associated with it, such as fur, odor, and excretions, have been found to elicit both an endocrine stress response (R. J. Blanchard et al., 1998; File, Zangrossi, Sanders, & Mabbutt, 1993) and behavioral changes in rodents (R. J. Blanchard & Blanchard, 1971; R. J. Blanchard et al., 1998; Dielenberg, Arnold, & McGregor, 1999; Dielenberg, Hunt, & McGregor, 2001; File et al., 1993; McGregor & Dielenberg, 1999; McGregor, Schrama, Ambermoon, & Dielenberg, 2002). However, many laboratory rat strains, like Sprague–Dawley, show highly variable stress responses to cat odor (Andrews et al., 1993; File et al., 1993). Indeed, responders and nonresponders to cat odor exist in rat strains that overall as a group respond to cat odor (Hogg & File, 1994). A component of fox feces, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), has also been used as a predator odor stress model with mixed results (Falconer & Galea, 2003; Holmes & Galea, 2002; Morrow, Redmond, Roth, & Elsworth, 2000). Some researchers have suggested that TMT may produce its endocrine and behavioral changes because of its noxious rather than predatory characteristics (Blanchard, Markham, et al., 2003; Dielenberg & McGregor, 2001; Lowry & Kay, 2004; McGregor et al., 2002). A predatory odor that does not have a noxious component and produces reliable endocrine and behavioral effects would offer researchers an additional alternative for studying processive stress.

The current studies examined ferret odor as a naturalistic stressor in the rat. The ferret, which is a member of the carnivorous family Mustelidae, is a natural predator of rats (Apfelbach, 1978; Rusiniak, Gustavson, Hankins, & Garcia, 1976). Researchers have previously determined that rats exposed to live ferrets exhibit elevated plasma corticosterone and adrenocorticotropin hormone (ACTH; Anisman et al., 1997; McIntyre, Kent, Hayley, Merali, & Anisman, 1999; Plata-Salaman et al., 2000) and behavioral changes (Plata-Salaman et al., 2000) indicative of a stress response. To our knowledge, ferret odor has not previously been used as a stressor.

For the purposes of this work, a psychological stress model was defined as a processive stressor that produces activation of the hypothalamo-pituitary-adrenocortical (HPA) axis and behavioral avoidance of the stimuli. In addition, regional c-fos messenger RNA (mRNA) induction was assessed to determine the similarity of brain activity elicited by ferret odor and previously reported processive stressors like loud noise and restraint stress. Specifically, plasma levels of ACTH and corticosterone were measured after exposure to ferret or control odors. Since Selye (1956), adrenocortical hormones have been routinely used as a primary indicator of an acute stress response (Endroczi, 1983; Levine, 2000). The rats' behavioral responses to ferret odors were also measured. Avoidance, immobility, and other defensive or risk assessment behaviors have been reported for other predator odors (see Dielenberg & McGregor, 2001, for a review).

Therefore, the hypothesis that ferret odor can be used as a processive stress model was examined in the current set of experiments. Endocrine (ACTH and corticosterone), behavioral (defensive withdrawal paradigm), and neuroanatomical (c-fos mRNA induction) reactions to presentations of ferret odor were examined. In addition, endocrine responses to ferret feces, urine, anal scent gland secretions, and fur were examined to determine their capacity to elicit stress reactions. Some of these data have been presented in abstract form (Campeau & Masini, 2003).

We examined the endocrine stress response produced by presenting towels that had been in contact with ferrets or control odor. The endocrine response was assessed by measuring plasma ACTH and corticosterone after 30 min of odor exposure. The brains of these rats were removed, and c-fos mRNA expression in several brain areas was measured by in situ hybridization. Currently used widely as a functional mapping tool, c-fos is an immediate early gene that is rapidly and transiently expressed in response to extracellular stimuli (Kovacs, 1998; Senba & Ueyama, 1997). Its induction was used in the current experiment as a marker of regional brain activation, which is often reported to peak at about 30 min (see Cullinan, Herman, Battaglia, Akil, & Watson, 1995).

Subjects Twenty male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were used. They were housed in a dedicated colony facility and placed in groups of 4 to 5 in clear polycarbonate cages (48 × 27 × 20 cm) containing floor wood shavings and covered with wire lids providing food (rat chow) and water ad libitum. Rats were housed for a period of at least 10 days after arrival from the supplier before any experimental manipulations were conducted. They were kept on a controlled light– dark cycle (lights on 7:00 a.m. and off at 7:00 p.m.) under constant humidity and temperature conditions. All procedures were performed between 9:00 a.m. and 12:00 p.m. to reduce variability resulting from normal circadian hormonal variations. This initial experimental time of day was chosen because the endocrine hormones (corticosterone and ACTH) are at their circadian trough and c-fos mRNA is also found at low abundance at this time of day in untreated rats. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Colorado and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Odor presentation Ferret odor was collected by placing a bath towel in a cage with 1 male and 1 female undescented adult ferrets for approximately 1 month.¹ The towel was cut into 5 × 5 cm squares, sealed in a plastic bag, express mailed overnight, and then placed in an −80 °C freezer until used. The control towel was soaked in 10% chlorine bleach, rinsed with water, air dried, and sealed in a plastic bag until used. The towels were transported to the experimental rooms immediately before testing inside sealed glass bell jars.

Rats were placed individually in 28 × 18 × 14 cm plastic cages with wire lids and transported to remote experimental rooms (kept on the same light cycle with overhead fluorescent lighting) on the afternoon before odor presentation to avoid manipulation and transport of the rats immediately before odor exposure. Rats had access to food and water during the entire experiment. The next day two pieces of towel with ferret odor (n = 12) or control odor (n = 8) were carefully placed at each end of the cage without disturbing the rats by hooking the towels to the wire cage lid with paper clips, so the towels hung inside the cage. Separate rooms were used for ferret and control towel presentation, and the control rats were always run before ferret odor rats to ensure that the control rats were not exposed to any ferret odor. Dividers were also placed between each cage so that rats could not see each other, but vocal communication was still possible. Immediately after the 30-min exposure, rats were taken to an adjacent room and rapidly decapitated. Trunk blood was collected, and brains were quickly removed and frozen. The same experimental room was used only once a day to ensure dissipation of any residual smell.

Corticosterone and ACTH radioimmunoassays Blood was collected into ice-chilled tubes containing ethylenediaminetetraacetic acid (20 mg/ml). Blood samples were centrifuged at 1,500 rpm for 10 min, and the plasma was pipetted into 0.5 ml Ependorf microcentrifuge tubes and stored at −80 °C until assayed.

Corticosterone was measured by radioimmunoassay using a specific rabbit antibody,² with less than 3% cross-reactivity with other steroids. Plasma samples were diluted 1:100 in 0.05 M sodium phosphate buffer containing 0.25% bovine serum albumin pH 7.4 and corticosterone separated from binding protein by heat (70 °C, 30 min). Duplicate samples of 200 μl to which 50 μl of trace (³H-corticosterone; Amersham 50 Ci/mmol, 10,000 cpm/tube) and 50 μl of antibody (final concentration 1:12800) were incubated at 4 °C overnight. Separation of bound from free corticosterone was achieved by adding 0.5 ml of chilled 1% charcoal–0.1% dextran mixture in buffer for 10 min at 4 °C and centrifuged for 10 min at 3,000 rpm (Eppendorf/Brinkman 5810R). The supernatant was poured into 5 ml scintillation fluid and bound ³H-corticosterone counted on a Packard Instruments (Model 1600TR) liquid scintillation analyzer and compared with a standard curve (range: 0–80 μg/dl). All samples from an experiment were measured simultaneously to reduce interassay variability; within-assay variability between duplicates was less than 8%.

ACTH was measured with a kit (ACTH 130T kit, catalog no. 40–2195) from Nichols Institute Diagnostics (San Clemente, CA) according to the manufacturer's protocol. The sensitivity of the assay ranged from 5 to 1,400 pg/ml. All samples from an experiment were measured simultaneously to reduce interassay variability.

In situ hybridization histochemistry After rapid decapitation, brains were removed and frozen in isopentane chilled to −30 °C and stored at −80 °C. Ten-micron sections were then cut on a cryostat (Leica Model 1850, Wetzlar, Germany), thaw-mounted onto polylysine-coated slides, and stored at −80 °C until further processed. Slides were fixed in a buffered 4% paraformaldehyde solution for 1 hr and rinsed in three changes of 2× standard saline citrate (SSC) buffer. The slides were then acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride for 10 min, rinsed for an additional 5 min in H₂O, and dehydrated in a progressive series of alcohols.

We generated sulfur 35-labeled cRNA probes for c-fos from cDNA subclones in transcription vectors using standard in vitro transcription methodology. The rat c-fos cDNA clone³ was subcloned in pGem3Z and cut with HindIII to yield a 680-nt cRNA probe. Riboprobes were labeled in a reaction mixture consisting of 1 μg linearized plasmid, 1× T7 or SP6 transcription buffer (Promega), 125 μCi ³⁵S-UTP, 150 μmol/L NTPs (CTP, ATP, and GTP), 12.5 mM dithiothreitol, 20 U RNase inhibitor, and 6 U RNA polymerase (T7). The reaction was allowed to proceed for 120 min at 37 °C, and probe was separated from free nucleotides over a Sephadex G50–50 column. Riboprobes were diluted in hybridization buffer to yield approximately 1.5 × 10⁶ dpm/65 μl buffer. The hybridization buffer consisted of 50% formamide, 10% dextran sulfate, 2× SSC, 50 mM sodium phosphate buffer (pH = 7.4), 1× Denhardt's solution, and 0.1 mg/ml yeast tRNA. Diluted probe (65 μl) was applied to each slide, and sections were coverslipped. Slides were placed in sealed plastic boxes lined with filter paper moistened with 50% formamide in distilled water and were subsequently incubated overnight at 55 °C. Coverslips were then removed, and slides were rinsed several times in 2× SSC. Slides were then incubated in RNase A (200 μg/ml) for 60 min at 37 °C, washed successively in 2×, 1×, 0.5×, and 0.1× SSC for 5–10 min each, and washed in 0.1× SSC for 60 min at 70 °C. Slides were subsequently rinsed in fresh 0.1× SSC, dehydrated in a graded series of alcohols, and exposed to Kodak MR x-ray film.

Control experiments were performed on tissue sections pretreated with RNase A (200 μg/ml at 37 °C for 60 min) before hybridization; this treatment prevented labeling. Alternatively, some control sections were hybridized with the sense cDNA strands, which in all cases did not lead to significant hybridization to tissue sections.

Three to five slides for a given brain region from each rat included in the study were processed simultaneously to allow direct comparisons in the same regions. Multiple in situ hybridizations were thus performed at different levels of the brain; all rats were represented to reduce the effects of technical variations within regions. Sections of all rats in the same region were all exposed on the same x-ray film to further minimize variations. Semiquantitative analyses were performed on digitized images from x-ray films in the linear range of the gray values obtained from our acquisition system (Northern Light lightbox Model B 95, a SONY TV camera model XC-ST70 fitted with a Navitar 7000 zoom lens, connected to an LG3–01 frame grabber [Scion Corp., Frederick, MD] inside a Dell Dimension 500, captured with Scion Image beta rel. 4.02). Signal pixels of a region of interest were defined as being 3.5 standard deviations above the mean gray value of a cell poor area close to the region of interest. The number of pixels and the average pixel values above the set background were then computed for each region of interest and multiplied, giving an integrated mean gray value measure. An average of four to eight measurements were made on different sections (which included bilateral counts made in all cases) for each region of interest, and these values were further averaged to obtain a single integrated mean gray value per region for each rat.

Data analysis Student's two-tailed t tests were performed on mean corticosterone and ACTH values (p < .05). Two-tailed t tests were also computed on the mean integrated densities obtained from each region where c-fos mRNA induction was measured (p < .01).

Figure 1 A: Mean plasma levels of corticosterone (CORT; ± SEM) for rats exposed to ferret odor (n = 12) and control odor (n = 8) for 30 min. B: Mean plasma levels of adrenocorticotropin hormone (ACTH; ± SEM) for rats exposed to ferret odor (n = (more ...)

The differences in c-fos mRNA expression produced by 30 min of ferret odor exposure compared with control odor exposure are shown in Table 1. Presentation of ferret odor for 30 min produced significantly higher c-fos mRNA expression in 26 of the 35 brain regions analyzed compared with control odor exposure. These regions included several cortical areas (cingulate, motor, orbitofrontal, piriform, and prelimbic), forebrain regions (fusiform and oval nuclei of bed nucleus of stria terminalis, caudate/putamen, and lateral septum), hypothalamic regions (dorsomedial, paraventricular, posterior, ventromedial, anterodorsal preoptic, premammillary, and supramammillary nuclei), regions of the amygdala (basolateral, posterodorsal and posteroventral medial, and posterolateral cortical), and periaqueductal gray (dorsomedial, dorsolateral, and ventrolateral). In addition, hippocampal CA3 subfield and the brainstem nucleus of the solitary tract of ferret odor exposed rats exhibited higher c-fos expression. Representative photomicrographs are shown in Figure 2.

Table 1 Mean Integrated Densities/100 (± SEM) of c-fos mRNA Expression

Figure 2 Representative photomicrographs depicting c-fos expression in the brain after ferret odor or control odor exposure for 30 min. Note the high c-fos induction in cortex, lateral septum (LS), the oval and fusiform nuclei of the bed nucleus of stria terminalis (more ...)

Several brain areas expressed higher levels of c-fos mRNA after exposure to ferret odor compared with control odor. Not surprisingly, areas of the brain that have previously been associated with HPA axis activity were activated by ferret odor exposure but not control odor. These areas included the bed nucleus of stria terminalis, cingulate cortex, hippocampus, lateral septum, and paraventricular nucleus of hypothalamus (Campeau & Watson, 1997; Cullinan, Herman, Battaglia, Akil, & Watson, 1995; Herman & Cullinan, 1997; Kovacs, 1998; Larsen & Mikkelsen, 1995). The main purpose of examining c-fos mRNA expression in rats exposed to ferret odor was to determine whether areas activated were the same as those activated by other known processive stressors. Additional areas examined that show similar induction as other processive stressors included orbitofrontal cortex, piriform cortex, anterodorsal preoptic, dorsomedial and ventromedial nuclei of the hypothalamus, premammillary nucleus, supramammillary nucleus, basolateral and posterodorsal medial amygdala, and periaqueductal gray (Campeau et al., 1997; Campeau & Watson, 1997; Chen & Herbert, 1995; Cullinan et al., 1995; Dielenberg et al., 2001; Imaki, Shibasaki, Hotta, & Demura, 1993; Melia, Ryabinin, Schroeder, Bloom, & Wilson, 1994). Overall, compared with cat odor-induced Fos induction, ferret towel-induced c-fos mRNA induction was remarkably similar. Areas such as the lateral septum, dorsomedial hypothalamus, ventromedial hypothalamus, premammillary nucleus, posteroventral medial nucleus of amygdala, and periaqueductal gray (dorsolateral, dorsomedial, and ventrolateral parts) had high levels of c-fos induction produced by cat and ferret odors (Dielenberg et al., 2001). On the other hand, little induction was observed in the lateral hypothalamic area, anterodorsal medial, posteromedial cortical amygdala, and central amygdala. Areas that were different between the odors include prelimbic cortex, basolateral amygdala, and cuneiform nucleus. Thus, in addition to being relatively similar to cat odor, ferret odor elicits a pattern of c-fos mRNA induction that includes many of the same brain regions activated by forced swim, restraint, and loud noise.

The second study examined the endocrine stress response to ferret odor during the rats' dark phase of their light– dark circadian cycle and behavior in a defensive withdrawal paradigm. It can be difficult to detect endocrine stress hormone differences during the dark–active phase of rodents because of higher basal levels, but rats' exploratory tendencies are much higher in their active phase, which allows for more sensitive detection of behavioral differences induced by predatory cues. After 4 days of habituation, rats were exposed to either towels with ferret or control (clean and strawberry) odors in an open-field apparatus with a small hide box in it for 10 min. Behavior was videotaped and later analyzed. A separate group of subjects were used to examine corticosterone and ACTH responses after acute exposure to ferret or control odors.

Subjects Forty-nine male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were used. They were group housed in a manner similar to that described in Experiment 1. They were kept on a controlled reverse light– dark cycle (lights on 7:00 p.m. and off at 7:00 a.m.) under constant humidity and temperature conditions. All procedures were performed between 9:00 a.m. and 12:00 p.m. to reduce variability from normal circadian hormonal variations.

Odor presentation Ten days after arrival, the rats were exposed to odors using the same procedure as in Experiment 1 but during the dark phase of the rats' light– dark circadian cycle. Also, in addition to ferret (n = 8) and control (n = 8) towels, an additional group (n = 8) was exposed to strawberry-scented towels (50 μl strawberry extract/towel, McCormick & Co., Inc., Hunt Valley, MD). This additional control group was used as an extra precaution to test for possible effects of novel odors on defensive behaviors and endocrine hormone release. Immediately after the 30-min exposure, rats were taken to an adjacent room and rapidly decapitated; trunk blood was collected.

Corticosterone and ACTH radioimmunoassays The same procedures were used as in Experiment 1.

Behavioral apparatus The apparatus was a 58 × 58 × 39 cm Plexiglas open-field chamber with a metal 41 × 41 × 20 cm chamber in one corner. The floor and sides of the open field were painted black, and white tape was used to delineate 16 equal-sized squares on the floor. The open field was elevated to a height of 53 cm. The room that contained the apparatus was dimly lit by a 75-W light, and white noise (60 dB sound pressure level) was provided by an AM7 Grass Medical Instruments audio monitor (Quincy, MA). Behavior was videotaped (Sony VHS recorder) by a Panasonic WV-BP130 video camera (Ontario, Canada) mounted directly above the apparatus (approximately 2.4 m) to the ceiling.

Habituation Ten days after arrival from the supplier, rats were individually habituated to the experimental apparatus. The rats were carted to a room opposite the behavioral testing room approximately 1 hr before testing, during which they had access to both food and water. The rats were then individually carried to the behavior room. Each rat was placed in the apparatus for 10 min on 4 consecutive days at approximately the same time each day. All behavioral testing was conducted during the rats' dark phase.

Test The day after completion of habituation, a 5 × 5 cm square piece of towel with ferret odor (n = 9), control towel odor (n = 8), or strawberry (100 μl/towel) odor (n = 8) was taped to the floor of the apparatus in the diagonal corner opposite the small metal chamber. The rat was then placed in the center of the apparatus and behavior was videotaped for 10 min. The apparatus was carefully cleaned with a 10% chlorine bleach solution between each rat's testing to reduce olfactory cues. New pieces of towel were used for each subject.

Video analysis Two researchers unaware of the experimental conditions analyzed the videotaped behavior. Behaviors analyzed include latency to enter the small metal chamber for the first time (seconds), number of visits to the small chamber, time spent in small chamber (seconds), number of visits to the corner containing the towel, time in towel corner (seconds), number of bouts of chewing on towel, grooming bouts, and number of rears (front paws off floor). The scores of the two researchers were averaged. Scorers achieved a level of 95% to 99% agreement depending on the measure.

Data analysis One-way analyses of variance (ANOVAs) were performed on mean corticosterone and ACTH values and behavioral measures (p < .05). These were followed by Tukey's honestly significant difference (HSD) multiple means comparisons.

Figure 3 A: Mean plasma levels of corticosterone (CORT; ± SEM) for rats exposed to ferret odor (n = 8), strawberry odor (n = 8), and clean odor (n = 8) for 30 min during the dark phase of the light– dark circadian cycle. B: Mean plasma levels of (more ...)

Behavior was examined in a defensive withdrawal paradigm during exposure to ferret odor or control odors. Significant differences between groups exposed to different odors were revealed for visits to chamber, F(2, 22) = 4.89, p = .02, visits to towel, F(2, 22) = 4.20, p = .03, time spent in the area of the towel, F(2, 22) = 7.85, p = .003, and chewing bouts on towel, F(2, 22) = 10.03, p = .001 (Figure 4). Post hoc Tukey's HSD comparisons revealed that ferret odor exposure led to significantly fewer visits to the small defensive withdrawal chamber, t(14) = 2.79, p = .01, fewer visits to the towel, t(14) = 2.42, p = .03, less time in the area of the towel, t(14) = 2.61, p = .02, and less chewing on the towel, t(14) = 4.83, p = .0002, compared with strawberry odor exposure. Tukey's HSD comparisons also revealed that ferret odor exposure led to significantly fewer visits to the small defensive withdrawal chamber, t(14) = 2.86, p = .01, less time in the area of the towel, t(14) = 3.91, p = .001, and less chewing on the towel, t(14) = 3.54, p = .003, than control towel odor exposure.

Figure 4 Graphs showing mean (± SEM) behavior in defensive withdrawal apparatus during exposure to ferret odor (n = 9), strawberry odor (n = 8), or clean towel odor (n = 8) for 10 min. A: Number of visits by the rats to the area where the towel stimulus (more ...)

Behavioral measures associated with the small metal defensive withdrawal chamber, latency to enter the chamber the first time, F(2, 22) = 1.22, p = .32, and total time spent in chamber, F(2, 22) = 0.04, p = .97, were not different between groups. Number of rears, F(2, 22) = 1.59, p = .23, and grooming bouts, F(2, 22) = 0.18, p = .83, also did not differ between groups.

Behavior was investigated using a defensive withdrawal paradigm (R. J. Blanchard, Kelley, & Blanchard, 1974; Dielenberg & McGregor, 2001; Takahashi, Kalin, & Baker, 1990). Rats exposed to ferret odor showed several differences from those exposed to the control odors. Behaviors directly associated with the odor stimuli, number of visits to the towel, time spent in the area of the towel, and chewing bouts on towel were the behaviors that were found to be significantly different between groups. Rats exposed to ferret odor avoided the odor stimulus much more compared with the control groups. Rats exposed to ferret odor did not, however, show species-typical defensive reactions like crouching, risk assessment, or freezing, as is sometimes seen with predator presentation (R. J. Blanchard & Blanchard, 1989a). This is not uncommon with predator odors and led previous researchers to suggest that predator odors represent a partial predator stimulus (D. C. Blanchard & Blanchard, 1988; R. J. Blanchard & Blanchard, 1989b; Dielenberg & McGregor, 2001). Avoidance of the odor stimulus is the most common behavior seen with predator odor (Dielenberg & McGregor, 2001; File et al., 1993; McGregor & Dielenberg, 1999; Zangrossi & File, 1992).

Experiment 3 was conducted to determine the source of the ferret-exposed towels' odor in producing a stress response. The towels collected from the ferret cages may have come into contact with ferret feces. This experiment was, therefore, designed to test the capacity of ferret feces to induce stress reactions. Behavioral reactions to ferret or conspecific rat fecal matter were assessed in the defensive withdrawal paradigm. HPA activation was also assessed by measuring plasma corticosterone after exposure to ferret or conspecific rat feces.

Subjects Eleven male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were used. They were housed as described in Experiment 1.

Behavioral procedure Ten days after arrival from the suppliers, rats were individually habituated to the experimental apparatus for 4 days as described in Experiment 2. The rats were then tested in the apparatus in the same manner as described in Experiment 2, during the dark phase of their light– dark cycle. Rats in this experiment were exposed to towels with either 0.35 g ferret feces (n = 5) or 0.35 g rat feces (n = 6) spread on them. The ferret fecal matter was collected from the same 2 ferrets as the towels were, sealed in a plastic bag, express mailed overnight, and then placed in a −80 °C freezer until used. Rat fecal matter was collected from rats of the same litter of the experimental subjects. Immediately before testing, the fecal matter was unthawed, weighed, spread on pieces of clean 5 × 5 cm towel, and transported to the behavioral room in a sealed bell jar. Ferret and rat feces were transported separately.

Feces exposure and blood collection One day before or 1 day after behavioral scoring (counterbalanced), rats were exposed to 0.35 g ferret feces (n = 6) or 0.35 g rat feces (n = 5) for 30 min in the same manner as described in Experiment 1. Immediately after exposure, the rats were taken to an adjacent room, and blood was collected via tail nicks.

Corticosterone radioimmunoassay The same procedures were used as in Experiment 1.

Data analysis Student's two-tailed t tests were performed on mean corticosterone values and behavioral measures analyzed from videotape (p < .05).

There was a significant difference between groups for plasma corticosterone release, t(9) = 2.31, p = .05, but the examination of the means (1.37 μg/dl for ferret feces-exposed rats and 3.14 μg/dl for rat feces-exposed rats) revealed that rats exposed to conspecific feces had slightly higher corticosterone levels. However, even this value is relatively low and suggests that ferret feces do not play a significant role in activating the HPA axis.

Experiment 3 showed that fecal matter was not the source of the odor on the ferret-exposed towels. However, the towels also may have come into contact with the ferrets' urine, which may have been the source of the odor responsible for defensive avoidance and HPA axis activation observed in Experiments 1 and 2. Thus, HPA axis activation was assessed by measuring plasma corticosterone after 30 min of exposure to ferret urine on towels.

Subjects Fourteen male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were used. They were housed as described in Experiment 1.

Urine collection Urine was sterile collected from an adult breeding male ferret by veterinarians at Marshall Farms (North Rose, NY). After collection, the urine was frozen and shipped on dry ice overnight. The urine was then stored in a −80 °C freezer until use.

Urine exposure and blood collection Seven days after arrival, the rats were exposed to substances using the same procedure as in Experiment 1. The substances used in this test were 100 μl ferret urine per towel (n = 8) or control/100 μl distilled water per cleaned towel (n = 6). Immediately after the 30-min exposure, rats were taken to an adjacent room and rapidly decapitated; trunk blood was collected.

Corticosterone radioimmunoassay The same procedures were used as in Experiment 1.

Data analysis Student's two-tailed t tests were performed on mean corticosterone values (p < .05).

The previous experiments suggest that the odor from the ferret towels that produced HPA activation was not due to fecal matter or urine. Experiment 5 tested the hypothesis that the odor originated from their anal scent gland secretions. Ferrets have scent glands located on both sides of their anus that secrete odorous sulfur-containing compounds (Crump, 1980; Woodley & Baum, 2003). These compounds are generally used for scent marking (Clapperton, 1989; Clapperton, Minot, & Crump, 1988). HPA activation was assessed by measuring plasma corticosterone after 30 min of exposure to ferret anal gland secretions.

Subjects Twenty-two male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were used. They were housed as described in Experiment 1.

Gland collection and dissection The anal sac scent glands from an adult breeding male ferret were surgically removed by veterinarians at Marshall Farms (North Rose, NY). After collection, the glands were frozen and shipped on dry ice overnight. The glands were than stored in a −80 °C freezer until use. The glands were thawed and the contents removed by making a lengthwise cut with a scalpel and then scraping the secretions out with a small spatula. The secretions were then diluted with mineral oil and stored in a −20 °C freezer until use (Woodley & Baum, 2003).

Secretion exposure and blood collection Seven days after arrival, the rats were exposed to substances using the same procedure as in Experiment 1. Rats were exposed to mineral oil (n = 6) or to 100 μl/towel anal gland secretions diluted with mineral oil in the following doses: 1:400 (n = 4), 1:100 (n = 4), 1:50 (n = 4), and 1:1 (n = 4). Immediately after the 30-min exposure, rats were taken to an adjacent room and rapidly decapitated, and trunk blood was collected.

Corticosterone radioimmunoassay The same procedures were used as in Experiment 1.

Data analysis One-way ANOVAs were performed on mean corticosterone values (p < .05).

One final experiment was performed to test the hypothesis that the odor on the towels was indeed from the skin or fur of the ferrets. The odors of all other possible sources in the ferret cage were ruled out (feces, urine, anal scent gland secretions) as the source of the odor that induced elevated corticosterone and ACTH levels. In Experiment 6, rats were exposed to ferret fur for 30 min and plasma corticosterone levels were measured.

Subjects Sixteen male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were used and housed as described in Experiment 1.

Fur exposure and blood collection Seven days after arrival, the rats were exposed to substances using the same procedure as in Experiment 1. Fur was collected from the cage of 2 shedding undescented male ferrets⁴ and mailed overnight. On arrival, the fur was stored in a −80 °C freezer until use. The fur (0.175 g/towel; n = 10) or bedding on clean towel (0.175 g/towel; n = 8) was stuck to pieces of duct tape and hung in the cages. Immediately after the 30-min exposure, rats were taken to an adjacent room and rapidly decapitated; trunk blood was collected.

Corticosterone radioimmunoassay The same procedures were used as in Experiment 1.

Data analysis Student's two-tailed t tests were performed on mean corticosterone values (p < .05).

Figure 5 Graph showing mean (± SEM) plasma levels of corticosterone (CORT) for rats exposed to ferret fur (n = 8) or control odor (n = 8) for 30 min. Asterisk indicates a significant difference from control odor (p < .05).

The levels of plasma corticosterone in each rat were also more variable than in Experiment 1. There was 1 control subject with higher than usual corticosterone level, and there was 1 rat that was unresponsive to the ferret fur and had a very low corticosterone level. However, this experiment did show that ferret fur–skin odor significantly increased corticosterone levels compared with control odor exposure.

Endocrine, behavioral, and neurochemical tests were conducted to determine the usefulness of ferret odor as a processive stress model. Predator odor stress has many advantages over other types of processive stressors like forced restraint, conditioned fear, mild footshock, and social defeat. Predator odor exposure does not involve any physical pain or injuries that may involve behavioral responses and neural circuits associated with physical stress. This is particularly important for determining brain circuits associated with processive stress. Responses to predator odors are also innate. Rats do not need to be trained to respond to predators and their odors that they have never before encountered. Predator odors have been shown to serve as unconditioned stimuli (see D. C. Blanchard, Griebel, & Blanchard, 2003, for review). Their responses are also very resistant to habituation (R. J. Blanchard, Blanchard, Weiss, & Meyer, 1990; R. J. Blanchard et al., 1998; File et al., 1993; Zangrossi & File, 1992).

Ferret odor successfully produced HPA axis activation in Sprague–Dawley rats as determined from plasma corticosterone and ACTH during both light and dark phase of their light– dark circadian cycle. It was further determined that the odor from the towels originated from the fur or skin of the ferrets. Ferret fecal matter, urine, and anal scent gland secretions exposure did not produce HPA activation. D. C. Blanchard, Griebel, and Blanchard (2003) have previously found that behavioral avoidance and species-typical defensive behaviors in the rat after exposure to feces–anal odorants and urine of cats are less clear than with exposure to fur or skin odors of cats. They have suggested that the fur or skin odor is a much more straightforward danger. The other odors are not as good indicators that the predator is in close proximity to the prey.

Rats exposed to ferret odor in a defensive withdrawal paradigm exhibited behavioral differences from control odor-exposed rats that suggested the ferret odor was aversive to the rats. The rats visited and spent time with the ferret towel stimulus significantly less than the control odor towels. The rats also chewed on the ferret odor towels much less than the control towels. These results suggest that the rats were avoiding the ferret odor towels. Risk assessment behaviors like crouching, stretch-attend, or flat-back approach as described by researchers examining defensive behavior were not seen (R. J. Blanchard & Blanchard, 1989a; Dielenberg & McGregor, 2001). However, researchers have previously found that when shelter is available (as in the defensive withdrawal paradigm), less risk assessment and more avoidance and hiding are seen (R. J. Blanchard et al., 1990; R. J. Blanchard, Shepherd, Rodgers, Magee, & Blanchard, 1993; R. J. Blanchard, Yang, Li, Gervacio, & Blanchard, 2001; Dielenberg et al., 1999; Dielenberg & McGregor, 1999; File et al., 1993; Zangrossi & File, 1992).

A single 30-min exposure to ferret odor induced a broad induction of c-fos mRNA expression in the rat brain. Elevated c-fos mRNA was found in numerous areas previously associated with defensive behaviors and other processive stressors like audiogenic and restraint stress. Forebrain areas like the bed nucleus of the stria terminalis (especially fusiform and oval nuclei) and lateral septum showed high c-fos expression compared with control odor-exposed rats. These areas have direct projections to the paraventricular nucleus of the hypothalamus (Swanson & Cowan, 1979), which also exhibited elevated c-fos after ferret odor exposure. These areas have previously been shown to display high c-fos mRNA or Fos protein after audiogenic stress, restraint stress, and cat or cat odor exposure (Campeau & Watson, 1997; Canteras, Chiavegatto, Valle, & Swanson, 1997; Chen & Herbert, 1995; Cullinan et al., 1995; Dielenberg et al., 2001; Melia et al., 1994). The hypothalamus has been shown to play an important role in the expression of defensive behaviors (Fuchs, Edinger, & Siegel, 1985a, 1985b; Lammers, Kruk, Meelis, & van der Poel, 1988). The current study found high c-fos mRNA expression in several hypothalamic nuclei, including dorsomedial, posterior, ventromedial, premammillary, and supramammillary regions, and in periaqueductal gray areas (dorsomedial, dorsolateral, and ventrolateral). These areas previously were found to have high levels of Fos protein after exposure to another rat predator, the cat, and to cat skin or fur odor (Canteras et al., 1997; Dielenberg et al., 2001).

Studies are now being conducted to determine the ability of ferret odor to serve as an unconditioned stimulus in conditioning experiments. Also, the behavioral and endocrine responses to ferret odor are being tested for their ability to habituate over repeated exposures. These studies will further the utility of ferret odor as a processive stress model.

In conclusion, ferret odor originating from the fur or skin of ferrets produces HPA axis activation in Sprague–Dawley rats. The rats also avoid ferret odor but not control odor in a defensive withdrawal paradigm. In addition, c-fos expression is increased in multiple brain areas that are associated with defensive behaviors and processive stress after exposure to ferret odor.

This work was supported by a National Alliance for Research on Schizophrenia and Affective Disorders Young Investigator Award and National Institute of Mental Health Grants B/START R03MH062471 and R01 MH065327.