Pyrazine analogues from wolf urine induced unlearned fear in rats

Abstract

Urine excreted from the common grey wolf (Canis lupus) contains a kairomone, inducing fear-related behaviors in various mammals. Numerous fear-inducing substances activate neurons at the main and/or accessory olfactory bulb (AOB), medial and central amygdala, and hypothalamus. Our previous study showed that the mixture of pyrazine analogues (P-mix) contained in wolf urine induced avoidance and fear-related behaviors in laboratory mice and Hokkaido deer (Cervus nippon yesoensis), a species native to Japan. Exposure to wolf urine or P-mix induced expression of Fos, a marker of neuronal excitation, in the AOB of mice. In the present study, we explored the effects of P-mix on fear-related behaviors and Fos-expression in rats. Exposure to P-mix induced avoidance and immobilization in rats, while that to a mixture of i-amyl acetate, linalool and R(+)-limonene (O-mix), which generate floral and fruity odors, induced avoidance but not immobilization. P-mix but not O-mix increased Fos-immunoreactivity of the AOB, medial and central amygdala, and hypothalamus of rats. The present results suggest that P-mix odor induces unlearned fear-related behaviors in rats.

Keywords: Neuroscience.

Fig. 1

Avoidance and immobilization induced by P-mix or O-mix in rats. A schematic of the experimental arena and time spent in the odorized zone (A). (A) Time spent in the cage halves containing petri dishes without odor mixture (white), or with P-mix (red) or O-mix (blue) in the two corners over 5 min. (B) Time spent immobilized over a 2-min period beginning at 3–5 min after exposure to P-mix (red), O-mix (blue), or no odorants (white). n: 6 (control), 6 (P-mix), 7 (O-mix). Values are the mean ± SE of data. *p < 0.05.

Fig. 2

Fos-immunoreactivities at the AOB of rats after exposure to P-mix or O-mix. Sagittal sections of the AOB after exposure to no odorants (A and D), P-mix (B and E), and O-mix (C and F). The frame in the mitral cell layer in the rostral AOB (A, B and C) indicates the area shown in D, E and F, respectively. Scale bar: 200 μm (C), 50 μm (F). The densities of Fos-ir cells (number of cells/mm³) in the mitral cell layer (MT) and granule cell layer (GC) at the rostral and caudal regions of the AOB after exposure to no odorants (white), P-mix (red) and O-mix (blue) are also shown. Density was analyzed using a one-factor ANOVA with Tukey-Kramer's post-hoc test. n: 4 (control), 4 (P-mix), 5 (O-mix). Values are the mean ± SE. *p < 0.05.

Fig. 3

Fos-immunoreactivities at the amygdaloid nucleus and ventromedial hypothalamic nucleus of rats after exposure to P-mix or O-mix. Sagittal sections at the amygdaloid nucleus (A, B and C) and enlarged sections at the MePV (D, E and F) after exposure to no odorants (A and D), P-mix (B and E), or O-mix (C and F). Scale bar: 200 μm (C), 50 μm (F). Density of Fos-ir cells (number of cells/mm³) after exposure to no odorants, P-mix or O-mix in the MePV (G), MePD (H), MeAV (I), MeAD (J), CeM (K), VMHdm (L), AHN (M) and PMD (N). n: 4 (control), 4 (P-mix), 5 (O-mix). Values are the mean ± SE.