Pyrazine analogues from wolf urine induced unlearned fear in rats

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

Pyrazine analogues are active components of wolf urine that induce avoidance and freezing behaviours in mice.

The common grey wolf (Canis lupus) is found throughout the entire Northern hemisphere and preys on many kinds of mammals. The urine of the wolf contains a number of volatile constituents that can potentially be used for predator-prey chemosignalling. Although wolf urine is put to practical use to keep rabbits, rodents, deer and so on at bay, we are unaware of any prior behavioural studies or chemical analyses regarding the fear-inducing impact of wolf urine on laboratory mice.Three wolf urine samples harvested at different times were used in this study. All of them induced stereotypical fear-associated behaviors (i.e., avoidance and freezing) in female mice. The levels of certain urinary volatiles varied widely among the samples. To identify the volatiles that provoked avoidance and freezing, behavioural, chemical, and immunohistochemical analyses were performed. One of the urine samples (sample C) had higher levels of 2,6-dimethylpyrazine (DMP), trimethylpyrazine (TMP), and 3-ethyl-2,5-dimethyl pyrazine (EDMP) compared with the other two urine samples (samples A and B). In addition, sample C induced avoidance and freezing behaviours more effectively than samples A and B. Moreover, only sample C led to pronounced expression of Fos-immunoreactive cells in the accessory olfactory bulb (AOB) of female mice. Freezing behaviour and Fos immunoreactivity were markedly enhanced when the mice were confronted with a mixture of purified DMP, TMP, and EDMP vs. any one pyrazine alone.The current results suggest that wolf urinary volatiles can engender aversive and fear-related responses in mice. Pyrazine analogues were identified as the predominant active components among these volatiles to induce avoidance and freezing behaviours via stimulation of the murine AOB

Comparison of freezing (immobilization) duration in mice during a 3 min exposure to (a) undiluted wolf urine samples and (b) 5-fold diluted wolf urine samples.

<p>The statistical significance of the differences between the freezing duration in response to wolf urine samples compared with control (water) was assessed by ANOVA followed by Fisher’s PLSD post-hoc test (*P<0.05, **P<0.01 vs. control).</p

Density of Fos-immunoreactive cells (number of cells/mm²) in the MTC and GC layers in the anterior and posterior regions of the AOB after exposure to undiluted wolf urine sample C (sample C/1) or 5-fold diluted samples A, B or C (sample A/5, sample B/5, sample C/5).

<p>The statistical significance of the differences between sample C vs. samples A and B was assessed by ANOVA followed by Fisher’s PLSD post-hoc test. The density of Fos-immunoreactive (Fos-ir) cells in the anterior GC layer was higher after exposure to diluted sample C relative to diluted sample A (P<0.01) or diluted sample B (P<0.01); similarly, the density of Fos-immunoreactive cells in the posterior GC layer was higher after exposure to diluted sample C vs. diluted sample A (P<0.05) or diluted sample B (P<0.01). Furthermore, the density of Fos-immunoreactive cells was higher after exposure to undiluted sample C vs. diluted sample A (P<0.01) or diluted sample B (P<0.005). AMT and PMT, anterior and posterior mitral cell layer; AGC and PGC, anterior and posterior granule cell layer.</p

Sagittal section of the AOB of female mice stained with an antibody against Fos after exposure to female murine urine (A) or 5-fold diluted wolf urine sample C (B).

<p>PTC, periglomerular cell layer; MTC, mitral cell layer; GC, granule cell layer.</p

Chromatograms from GC-MS analyses of wolf urine samples.

<p>Numbers refer to the following compounds: (8) Δ³-isopentenyl methyl sulphide; (9) 1-(methylthio)-2-methylbut-2-ene; (10) 3-buten-1-ol, 3-methyl-; (11) 4-methyl-3-heptanone; (12) 2,4-dithiapentane*; (13) 1-pentanol, 2-methyl-; (14) pyrazine, 2,6-dimethyl- (DMP)*; (15) dimethyl trisulphide*; (16) pyrazine, trimethyl- (TMP)*; (17) pyrazine, 3-ethyl-2,5-dimethyl (EDMP)*; (18) acetic acid*. *Identified by GC-MS (n = 6) and a comparison with the retention times of identified chemicals.</p

Avoidance rate during exposure of mice to wolf urine samples A, B and C.

<p>The avoidance rate was defined as the amount of time spent in the short arm of the Y maze with the control odour (water) divided by the total amount of time spent in both short arms with the wolf urine odour or the control odour. The statistical significance of the differences between the avoidance rates elicited by each of the wolf urine samples was assessed by repeated-measured ANOVA followed by Fisher’s PLSD post-hoc test.</p