Romain Hacquemand

Comparative behavioral effects between synthetic 2,4,5-trimethylthiazoline (TMT) and the odor of natural fox (Vulpes vulpes) feces in mice

Synthetic 2,4,5-trimethylthiazoline (TMT)--a component of red fox (Vulpes vulpes) feces--is frequently used to induce unconditioned fear in rodents. Surprisingly, direct comparison between TMT and natural fox feces odor is almost nonexistent. In this study, Experiment 1 compared the avoidance in relation to TMT concentration, natural fox feces, and gender of fox and mice. Results show that the avoidance is (a) higher with either pure or 50% TMT as compared to natural fox feces, whereas the difference is slight with 10% TMT, and (b) significantly higher for the female mouse group compared to the male mouse group with TMT as well as natural fox feces. In addition, no clear difference in effect was observed between male and female fox feces. Experiment 2 compared behavioral parameters recorded as an index of fear and anxiety, general activity, and avoidance in elevated plus-maze and open-field chamber between 10% TMT and natural fox feces in relation to the estrus cycle of the mice. Results show no cycle period effect--except for the avoidance parameter distance to odorant--and no different effects between 10% TMT and natural fox feces except for freezing.

Comparison between low doses of TMT and cat odor exposure in anxiety- and fear-related behaviors in mice

Few comparisons were made between cat odor and synthetic fox odor (TMT) to study fear and anxiety in rodents. TMT is frequently used are at high concentration while the stimulus should be as possible closer to natural conditions. The aim of this work was to compare behavioral responses of mice exposed to cat odor and low doses of TMT (i.e. 10 μl of a solution containing 1%, 0.1% or 0.01% TMT). Behavioral parameters were recorded in elevated plus-maze and in open field. Results showed that 1% TMT and 0.1% TMT induced similar responses to cat odor, contrary to water and 0.01% TMT which failed to elicit fear or anxiety-related behaviors. Additionally, behavioral changes were more marked in EPM - e.g. time spent in open arms - than in open field - e.g. freezing. These findings are discussed in terms of a possible continuum of mild anxiety-like behaviors to strong fear-like behaviors linked to predator odor intensity.

Fear-like behavioral responses in mice in different odorant environments: Trigeminal versus olfactory mediation under low doses

Odors can have repulsive effects on rodents based on two complementary adaptive behaviors: the avoidance of predator odors (potentially dangerous) and the avoidance of trigeminal stimulants (potentially noxious). The present study aimed to compare the behavioral effects on mice of odors according to their trigeminal properties and ecological significance. We used three different odors: 2,4,5-trimethylthiazoline (TMT: a fox feces odor frequently used to elicit fear-induced behaviors), toluene (a strong stimulant of the trigeminal system) and phenyl ethyl alcohol (PEA: a selective stimulant of the olfactory system). First, we checked preference and avoidance behaviors in mice with and without anosmia towards these odors to ensure their olfactory/trigeminal properties. Secondly, we used a standard test (open-field and elevated plus-maze) to assess the behaviors of mice when exposed to these odors. The results show that the anosmic and control mice both avoided TMT and toluene odors. In the open-field and the elevated plus-maze, mice exhibited anxious behaviors when exposed to TMT. Conversely, exposure to PEA induced anxiolytic effects confirmed by low blood corticosterone levels resulting from completion of the elevated plus-maze. Compared with TMT exposure, toluene exposure induced moderate anxious effects.

Comparative fear-related behaviors to predator odors (TMT and natural fox feces) before and after intranasal ZnSO 4 treatment in mice

The possibility that synthetic 2,4,5-trimethylthiazoline (TMT), frequently used to induce unconditioned fear in rodents, could be more a pungent odor activating intranasal trigeminal nerve fibers rather than a predator odor index is currently discussed. In order to explore this question, the present study compared fear-related behaviors to predator odors (synthetic 10% TMT and natural fox feces) and toluene (as an irritant compound without ecological significance) before and after intranasal ZnSO4 perfusion which is known to provoke transient anosmia. Results show that natural fox feces could be consider as a pure olfactory (CN I) nerve stimulant while 10% TMT appeared to be a mixed olfactory (CN I) and trigeminal (CN V) nerves stimulant with a great olfactory power and a low trigeminal power. These findings suggest that behavioral neuroscience studies should use concentrations lower than 10% TMT to obtain fear-related behaviors similar to those obtained with natural fox feces odor.

Inhalation exposure to acetone induces selective damage on olfactory neuroepithelium in mice

Due to their specific position in the nasal cavity, the cells of olfactory neuroepithelium can be damaged by exposure to environmental airborne chemicals. However, few studies have been focused on selective damage, i.e. olfactory sensory neurons, basal cells, supporting and duct cells. As solvents are known to induce critical effects on olfactory neuroepithelium (OE), this study was designed to characterize histological and immunohistological effects induced by acetone exposure on OE in mice. Behavioral tests were conducted to evaluate olfactory sensitivity. Moreover, olfactory neuroepithelium was examined to evaluate the thickness and the total number of cells. Finally, different markers, olfactory marker protein (OMP) and proliferating cell nuclear antigen (PCNA), were used to characterize respectively olfactory sensory neurons and basal cells, and secondly to evaluate the dynamic of the tissue turnover. Results showed structural modifications, since the thickness and the number of cells in the OE were modified according to the time course of the exposure. Additionally, no changes for OMP-positive cells were observed whereas significant differences appeared for the density of PCNA-positive cells in relation to their location (main-body or basal layer of OE). These findings indicate that acetone exposure induces selective damage in olfactory neuroepithelium.

Postnatal exposure to synthetic predator odor (TMT) induces quantitative modification in fear-related behaviors during adulthood without change in corticosterone levels

Environmental stimuli and adverse experiences in early life may result in behavioral and physiological changes in adulthood. In several animal species, the odors cues are crucial in the setting of adaptive behaviors, especially towards predators. However, little is known about the effects of postnatal exposure to predator odor on the later physiological and behavioral responses to this natural stressor. Thus, the aim of this study was to investigate the effects of a postnatal exposure to synthetic predator odor (TMT) in mice pups on later adult fear-related behaviors and corticosterone levels in response to this specific stimulus. Pups postnatally exposed to only water showed later in adult life behavioral responses when exposed to TMT that were statistically different from mice that were exposed as neonates to TMT. In addition, mice exposed as neonates to TMT showed a decrease of fear-related behaviors while no differences occurred in the corticosterone levels between both groups.

Effects of CO2 inhalation exposure on mice vomeronasal epithelium.

Nasal epitheliums are the first sites of the respiratory tract in contact with the external environment and may therefore be susceptible to damage from exposure to many toxic volatile substances (i.e., volatile organic components, vapors, and gases). In the field of inhalation toxicology, a number of studies have considered the main olfactory epithelium, but few have dealt with the epithelium of the vomeronasal organ (VNO). However, in several species such as in rodents, the VNO (an organ of pheromone detection) plays an important role in social interactions, and alterations of this organ are known to induce adaptative behavioral disturbances. Among volatile toxicants, health effects of inhaled gases have been thoroughly investigated, especially during CO2 inhalation because of its increasing atmospheric concentration. Therefore, this work was designed to examine the effects of 3% CO2 inhalation on VNO in two different exposure conditions (5xa0h/day and 12xa0h/day) in mice. Behavioral sensitivity tests to urine of congener and histological measurements of VNO were conducted before, during (weeksu20091–4), and after (weeksu20095–8) CO2 inhalation exposures. Results showed no significant modifications of behavioral responses to urine, but there were significant changes of both cell number and thickness of the VNO epithelium. Moreover, the findings indicated a selectively dose-dependent effect of CO2, and further research could use other gases in the same manner for comparison.

Postnatal exposure to predator odor (TMT) enhances spatial learning in mice adulthood.

Adult behavioral and physiological responses are partly dependent on neonatal experiences. In several animal species, enriched/aprovished environments and stressful/appeasing events are crucial in the setting of adaptative behaviors. However, little is known about the effects of postnatal exposure to predator odor (as unconditioned fear-related stimulus) on spatial learning at adulthood. Thus, the aim of the present study was to investigate the effects of a postnatal exposure to 2,4,5-trimethylthiazoline (TMT, as a predator odor) on radial arm maze (RAM), Tolman maze (TM) and Morris water maze (MWM) in mice at adulthood. The results showed that a TMT group constituted by mice exposed postnatally during 3 weeks to TMT presented significantly better spatial learning achievements in adulthood compared to a water group, postnatally exposed to water only, as well as compared to a butanol group (butanol used as an odor without ecological significance) exposed postnatally to butanol during 3 weeks.

Carbon dioxide effects on olfactory functioning: behavioral, histological and immunohistochemical measurements.

Most studies on toxic inhalation focus on solvent effects and few have dealt with gases on olfactory functioning. Among gases, the effects of carbon dioxide on general physiology have been well investigated contrary to the impact on olfactory neuroepithelium. Thus, this work was designed to evaluate in mice the possible effects of 3% CO(2) in two exposure periods: a 5h/day and a 12h/day conditions. Behavioral, histological and immunohistochemical observations were conducted every 2 weeks, i.e. before (W0), during (W2, W4) and after exposure (W6, W8). Firstly, behavioral evaluations of odor sensitivity showed differences in relation to the odor tested, i.e. no effect with congener urine odor and a reinforcement of 2,4,5-trimethythiazoline (TMT) (predator odor) repulsion. Secondly, histological evaluations showed a similar evolution of the epithelium thickness, i.e. a decrease along the exposure as well as during the post-exposure period and an increase of cell number (whatever the phenotype) although the kinetic appeared different in both experimental conditions. Thirdly, immunohistochemical quantification of olfactory marker protein (OMP)- and proliferating cell nuclear antigen (PCNA)-positive cells revealed that the number of mature olfactory neurons increased at the early beginning of exposure period in both conditions. While a decrease was observed in the following weeks (W4-W8) for the 12h/day condition, a stable amount of OMP-positive cells was maintained in the 5h/day condition. In contrast, the number of PCNA-positive cells followed a similar evolution, i.e. a constant decrease along the experiment. These findings indicate that the effects of CO(2) inhalation exposure are selectively dose-dependent.

Effects of pyridine inhalation exposure on olfactory epithelium in mice

Olfactory neurons in the nasal mucosa have the capacity to regenerate continuously along the lifespan by neurogenesis processes starting with progenitor cells close to the basal lamina. The cellular turnover into olfactory neuroepithelium may be modified by environmental stimuli insofar as nasal mucosa is directly in contact with airborne chemicals. However, few studies have been focused on selective changes, especially those concerning mature olfactory neurons and basal cells during specific inhalation exposure. Among chemicals, solvents are known to induce changes in smell abilities and concomitant histological and cellular modifications related to the type of molecule, concentration and time of exposure. This study was designed to characterize smell sensitivity (using behavioral tests) and immunohistochemical effects on olfactory neuroepithelium induced by pyridine exposure in mice. Olfactory marker protein (OMP) and proliferating cell nuclear antigen (PCNA) were used to characterize respectively mature olfactory neurons and basal cells. Results showed that inhalation exposure to pyridine had no impact on smell sensitivity whatever the concentration used and the time of exposure. These findings were in agreement with immunohistochemical measurements showing the same cellular kinetic whatever the condition of exposition to pyridine. Indeed, OMP-positive cells increased and PCNA-positive cells decreased as early as the beginning of exposure and cell amounts remained stable at this level until the end of exposure. These findings suggest that pyridine could have the property to rapidly activate a cellular turnover from basal cell progenitors. Rather than toxic effects, the present findings suggest that the metabolites of pyridine might have cell cycle activation properties.