Kazumi Osada

The scent of age.

In many species, older males are often preferred mates because they carry ‘good’ genes that account for their viability. How females discern a males age is a matter of question. However, for animals that rely heavily on chemical communication there is some indication that an animals age can be determined by its scent. To investigate whether there are changes in body odours with age, and if so their composition, mice were trained in a Y–maze to discriminate urine odours of donor mice of different ages: Adult (3–10 months old) and Aged (more than 17 months old). Trained mice could discriminate between these two age groups by odour alone. To determine the chemical basis for these discriminations, studies were performed using gas chromatography and mass spectrometry. These analyses demonstrated differences in the ratio of urinary volatiles with age. The most prominent differences involved significantly greater amounts of 2–phenylacetamide and significantly lower amounts of methylbutyric acids in Aged animals relative to Adult animals. Fractionating and manipulating the levels of these compounds in the urine demonstrated that the mice can distinguish age based on variation in amounts of these specific compounds in the combined urine.

Hard-diet feeding recovers neurogenesis in the subventricular zone and olfactory functions of mice impaired by soft-diet feeding.

The subventricular zone (SVZ) generates an immense number of neurons even during adulthood. These neurons migrate to the olfactory bulb (OB) and differentiate into granule cells and periglomerular cells. The information broadcast by general odorants is received by the olfactory sensory neurons and transmitted to the OB. Recent studies have shown that a reduction of mastication impairs both neurogenesis in the hippocampus and brain functions. To examine these effects, we first measured the difference in Fos-immunoreactivity (Fos-ir) at the principal sensory trigeminal nucleus (Pr5), which receives intraoral touch information via the trigeminal nerve, when female adult mice ingested a hard or soft diet to explore whether soft-diet feeding could mimic impaired mastication. Ingestion of a hard diet induced greater expression of Fos-ir cells at the Pr5 than did a soft diet or no diet. Bromodeoxyuridine-immunoreactive (BrdU-ir) structures in sagittal sections of the SVZ and in the OB of mice fed a soft or hard diet were studied to explore the effects of changes in mastication on newly generated neurons. After 1 month, the density of BrdU-ir cells in the SVZ and OB was lower in the soft-diet-fed mice than in the hard-diet-fed mice. The odor preferences of individual female mice to butyric acid were tested in a Y-maze apparatus. Avoidance of butyric acid was reduced by the soft-diet feeding. We then explored the effects of the hard-diet feeding on olfactory functions and neurogenesis in the SVZ of mice impaired by soft-diet feeding. At 3 months of hard-diet feeding, avoidance of butyric acid was reversed and responses to odors and neurogenesis were recovered in the SVZ. The present results suggest that feeding with a hard diet improves neurogenesis in the SVZ, which in turn enhances olfactory function at the OB.

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

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice.

Mice secrete substantial amounts of protein, particularly proteins called the major urinary proteins (MUPs), in urine. One function of MUPs is to sequester volatile pheromone ligands, thereby delaying their release and providing a stable long-lasting signal. Previously, only MUPs isolated from male mice have been used to identify ligands. Here, we tested the hypothesis that MUPs derived from females may also sequester volatile organic compounds. We identified butylated hydroxytoluene (BHT), a synthetic antioxidant present in the laboratory rodent diet, as a major ligand bound to urinary proteins derived from C57BL/6J female urine. BHT was also bound to the male-derived proteins, but the binding was less prominent than that in female urine, even though males express approximately 4 times more proteins than females. We confirmed that the majority of BHT in female urine was associated with the high molecular weight fraction (>10 kDa) and the majority of the proteins that sequestered BHT were MUPs as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequestration of BHT by MUPs was further confirmed by employing the recombinant MUP8 whose natural analogue has been reported in both sexes. Therefore, our data indicate that MUPs expressed in both sexes can bind, transport, and excrete xenobiotics into urine and raise the possibility that in addition to the known role in chemical communication, MUPs function as a defense mechanism against exogenous toxins.

Profiles of Volatiles in Male Rat Urine: The Effect of Puberty on the Female Attraction

Rat urine contains many volatile constituents that may be used for chemical communication. The levels of certain urinary volatiles are strongly dependent on the sex and endocrine status (e.g., puberty). We performed chemical and behavioral studies to identify the volatiles in adult male rat urine that attract mature females. Our results demonstrated that adult male rats have higher levels of 2-heptanone (2-HP), 4-methylphenol (4-MP), and 4-ethylphenol (4-EP) than prepubescent male rats; furthermore, female rats are more attracted to the odor of adult male rat urine than that of prepubescent males. When prepubescent rat urine was supplemented with 2-HP, 4-MP, and 4-EP to the levels found in adult male urine, the attractiveness of the urine to females was markedly enhanced. Our results suggested that this attraction is due to an increased level of chemosignaling.

Syntheses of 2-Isopropyl-4,5-dihydrothiazole and 6-Hydroxy-6-methyl-3-heptanone, Pheromone Components of the Male Mouse, Mus musculus

Syntheses of 2-isopropyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus, were achieved to provide sufficient amounts of samples for biological studies.

Pyrazine analogs are active components of wolf urine that induce avoidance and fear-related behaviors in deer

Our previous studies indicated that a cocktail of pyrazine analogs, identified in wolf urine, induced avoidance and fear behaviors in mice. The effects of the pyrazine cocktail on Hokkaido deer (Cervus nippon yesoensis) were investigated in field bioassays at a deer park in Hokkaido, Japan. A set of feeding bioassay trials tested the effects of the pyrazine cocktail odor on the behavior of the deer located around a feeding area in August and September 2013. This odor effectively suppressed the approach of the deer to the feeding area. In addition, the pyrazine cocktail odor provoked fear-related behaviors, such as “tail-flag”, “flight” and “jump” actions, of the deer around the feeding area. This study is the first experimental demonstration that the pyrazine analogs in wolf urine have robust and continual fearful aversive effects on ungulates as well as mice. The pyrazine cocktail might be suitable for a chemical repellent that could limit damage to forests and agricultural crops by wild ungulates.

Experimental study of fatigue provoked by biotin deficiency in mice

We investigated the relationships among behavioral parameters, forced-swimming test parameters, and plasma and organ biotin concentrations in biotin-deficient mice. Male ddY mice were divided into four groups: early biotin deficiency group (ED group; biotin-free diet for three weeks), progressive biotin-deficiency group (PD group; biotin-free diet for seven weeks), and two age-matched control groups. The dermatological symptoms of frank biotin deficiency were observed in most mice in the PD groups (72.3%) but in only 27% of ED group mice. The liver biotin level was greatly decreased in ED and PD groups, and the plasma biotin level was also significantly decreased in the PD group, but the biotin levels were quite stable in muscle and brain. There were significant decreases in swimming time in ED and PD groups and in struggling behavior in the PD group, suggesting that biotin-deficient mice become depressed and/or fatigued without biotin deficiency being apparent in brain and muscle. One single-injection biotin administration led to a prompt recovery in swimming time. Biochemical data revealed a decrease in liver glucokinase activity and an increase in ketone bodies in both liver and plasma in biotin-deficient mice. In addition, simultaneous biotin deficiency and forced walking synergistically provoked significant increases in total ketone bodies in both plasma and liver. These results suggest that depression and/or fatigue are induced in mice by biotin deficiency.

The scent of wolves: pyrazine analogs induce avoidance and vigilance behaviors in prey.

The common gray wolf (Canis lupus) is an apex predator located at the top of the food chain in the Northern Hemisphere. It preys on rodents, rabbits, ungulates, and many other kinds of mammal. However, the behavioral evidence for, and the chemical basis of, the fear-inducing impact of wolf urine on prey are unclear. Recently, the pyrazine analogs 2, 6-dimethylpyrazine, 2, 3, 5-trimethylpyrazine and 3-ethyl-2, 5-dimethyl pyrazine were identified as kairomones in the urine of wolves. When mice were confronted with a mixture of purified pyrazine analogs, vigilance behaviors, including freezing and excitation of neurons at the accessory olfactory bulb, were markedly increased. Additionally, the odor of the pyrazine cocktail effectively suppressed the approach of deer to a feeding area, and for those close to the feeding area elicited fear-related behaviors such as the “tail-flag,” “flight,” and “jump” actions. In this review, we discuss the transfer of chemical information from wolf to prey through the novel kairomones identified in wolf urine and also compare the characteristics of wolf kairomones with other predator-produced kairomones that affect rodents.

Alteration of Mouse Urinary Odor by Ingestion of the Xenobiotic Monoterpene Citronellal

Body odors provide a rich source of sensory information for other animals. There is considerable evidence to suggest that short-term fluctuations in body odor can be caused by diet; however, few, if any, previous studies have demonstrated that specific compounds can directly mask or alter mouse urinary odor when ingested and thus alter another animals behavior. To investigate whether the ingestion of citronellal, a monoterpene aldehyde that produces an intense aroma detected by both humans and mice, can alter mouse urinary odor, mice (C57BL6J) were trained in a Y maze to discriminate between the urinary odors of male donor mice that had ingested either citronellal in aqueous solution or a control solution. Trained mice could discriminate between urinary odors from the citronellal ingestion and control groups. A series of generalization tests revealed that citronellal ingestion directly altered mouse urinary odor. Moreover, trained mice that had successfully discriminated between urinary odors from donor mice of different ages failed to detect age-related changes in urine from male mice that had ingested 50 ppm of citronellal. This study is the first to show that ingestion of a xenobiotic can alter mouse urinary odor and confuse the behavioral responses of trained mice to age-related scents.