CAT ODOR, BUT NOT TRIMETHYLTHIAZOLINE (FOX ODOR), ACTIVATES ACCESSORY OLFACTORY AND DEFENSE-RELATED BRAIN REGIONS IN RATS L. G. STAPLES, a * I. S. MCGREGOR, a R. APFELBACH b AND G. E. HUNT c a School of Psychology, University of Sydney, Griffith Taylor Building (A18), NSW 2006, Australia b Tierphysiologie, Universitat Tubingen, D-72076 Tubingen, Germany c Department of Psychological Medicine, University of Sydney, Con- cord Hospital, NSW 2139, Australia Abstract—Cat odor and trimethylthiazoline (TMT, a compo- nent of fox feces) are two stimuli widely used in rodent models of fear and anxiety. Recent studies suggest that these odorants have distinct behavioral effects, raising questions as to whether TMT is a true “predator odor.” Here we used c-Fos immunohistochemistry to compare patterns of neural activation produced by cat odor and TMT. Rats were exposed to either (1) three pieces of a collar that had been worn by a domestic cat, (2) three collar pieces impregnated with TMT (30 l/piece), (3) three collar pieces impregnated with 4% formaldehyde (200 l/piece, an acrid but non-predatory odor), or (4) three control (no odor) collar pieces. Odors were presented in a small well-ventilated plastic box. All odorants (cat odor, TMT and formaldehyde) produced in- creased defecation in rats compared with the control group, and formaldehyde exposure also decreased rearing. Cat odor increased contact with the stimulus relative to all other groups, while TMT increased contact compared with the formaldehyde and clean air groups. Only cat odor de- creased grooming and elicited escape attempts. In addi- tion, only cat odor caused pronounced activation of Fos in the accessory olfactory bulb and its projection areas, an- terior olfactory nucleus, medial prefrontal cortex, striatum, and a medial hypothalamic circuit associated with defen- sive behavior. In contrast, the only areas activated by TMT were the internal granular layer of the main olfactory bulb and central amygdala, while both cat odor and TMT acti- vated the glomeruli of the main olfactory bulb, piriform cortex, ventral orbital cortex and anterior cortical amyg- dala. Results indicate that the effects of cat odor and TMT are easily distinguished both behaviorally and at a neural level, and suggest that TMT lacks the “pheromone-like” quality of cat odor that engages key hypothalamic sites involved in defensive behavior. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: anxiety, olfaction, predator, c-Fos, AOB, amyg- dala. An encouraging development in preclinical research on anxiety has been the increasing use of naturalistic stimuli to probe the neurobiology of anxiety states. As human anxiety disorders are frequently elicited by primordial anx- iogenic stimuli such as snakes and spiders, there is un- doubtedly value in modeling this with stimuli that have innate aversive qualities in rodents. One such approach is the use of predator-related stimuli to induce defensive responses in rats. Laboratory rats show robust defensive response to cats and to associated stimuli such as the odor of a cat, despite having never encountered a cat previously. When exposed to the cat odor, these rats exhibit a variety of risk assessment behaviors directed toward the monitoring of the predatory stimulus, while adaptive behaviors such as grooming and feeding are suppressed (Blanchard et al., 1990; Zangrossi et al., 1992; Dielenberg et al., 1999). Rats will also readily learn to avoid stimuli and places that are associated with the odor (Blanchard et al., 2001; Hubbard et al., 2004; Staples et al., 2005). Another widely used predator odor is trimethylthiazo- line (TMT). This component of fox feces elicits avoidance and freezing in rats (Wallace and Rosen, 2000; Fendt et al., 2005). However, it does not elicit characteristic defensive responses such as risk assessment or fear con- ditioning (McGregor et al., 2002; Blanchard et al., 2003; Staples and McGregor, 2006). As TMT has a strong aver- sive odor, its limited behavioral effects may be due more to its noxious qualities, rather than its abilities to signal a predator threat. Thus in the present experiment we had the relatively straightforward aim of comparing patterns of neural acti- vation produced by cat odor and TMT. Cat odor activates the accessory olfactory system of the rat, its projection areas in the extended amygdala, and a medial hypotha- lamic circuit strongly associated with defensive behavior (Dielenberg et al., 2001; McGregor et al., 2004; Staples et al., 2005). If TMT is a predator odor, we would expect to see a similar activation in these regions. Alternatively, if TMT is simply a “nasty smell,” it may be expected to engage brain substrates associated with aversion rather than anxiety. For purposes of comparison we also exam- ined the brain regions activated by formaldehyde, an acrid odor that tends to be avoided by rats (McGregor et al., 2002). *Correspondence to: L. G. Staples, Prince of Wales Medical Research Institute, Barker St. Randwick 2031, NSW, Australia. Tel: +61-2-9399- 1037; fax: +61-2-9399-1082. E-mail address: l.staples@unsw.edu.au (L. G. Staples). Abbreviations: ACo, anterior cortical amygdala; ANOVA, analysis of variance; AOB, accessory olfactory bulb; CeA, central amygdala; CnF, cuneiform nucleus; HDB, horizontal limb of the diagonal band of Broca; MOB, main olfactory bulb; PAG, periaqueductal gray; PB, phosphate buffer; PBH, phosphate-buffered horse serum; PBS, phos- phate-buffered saline; PMd, dorsal premammillary nucleus; TMT, tri- methylthiazoline; VO, ventral orbital cortex. Neuroscience 151 (2008) 937–947 0306-4522/08$32.00+0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.11.039 937