2026 Volume 51 Issue 7 Pages 393-402
Multiple chemical sensitivity (MCS) is characterized by neuropsychological symptoms including anxiety, depression, and fatigue following exposure to environmental chemicals, yet its underlying mechanisms remain poorly understood. Linalool, a monoterpene alcohol widely used in consumer products, has been suggested as a potential contributor to MCS. To elucidate its neural effects, we examined the behavioral effects of linalool inhalation in mice. Linalool exposure induced both anxiety- and depression-like behaviors. While the depression-like behavior required olfactory input, the anxiety-like behavior occurred independently of olfactory perception. Our previous studies demonstrated that inhaled linalool accumulates in the brain and undergoes cytochrome P450 (P450)-dependent metabolism. We therefore tested whether P450-mediated metabolism contributes to the behavioral effects of linalool. Significantly, inhibition of P450 activity abolished the anxiety-like behavior. These findings reveal an olfactory-independent mechanism by which inhaled linalool induces anxiety-like behavior in mice and suggest that P450 activity is required for this effect, providing a basis for investigating fragrance-induced MCS-like neurobehavioral responses.
Multiple chemical sensitivity (MCS) is a disorder characterized by a wide range of symptoms including anxiety, depression, headache, fatigue, and impaired concentration, which are triggered by exposure to environmental chemicals (Miller, 2001; Zucco and Doty, 2021; Lavric et al., 2024). Its prevalence has been increasing worldwide, and patients often experience a marked decline in quality of life due to heightened sensitivity to chemicals that are harmless to most individuals (Steinemann, 2018a, 2019). Various environmental substances, including building materials, solvents, and pesticides, have been implicated as potential causal factors. Among these, fragrance compounds—widely used in perfumes, detergents, and personal care products—are considered representative triggers due to their ubiquitous use (Steinemann, 2018b, 2018a, 2019). However, the biological mechanisms through which fragrance exposure produces these neuropsychological symptoms remain poorly understood.
Linalool, a monoterpene alcohol abundant in essential oils such as lavender and citrus, is one of the most commonly used fragrance ingredients in consumer products. Owing to its widespread use and diverse biological activities, linalool represents a key candidate for investigating the pathophysiological mechanisms underlying MCS (Aprotosoaie et al., 2014; Kamatou and Viljoen, 2008). Linalool has been reported to exert various effects on the central nervous system (CNS) in both humans and experimental animals. Inhalation of linalool or linalool-rich essential oils has been reported to produce anxiolytic and antidepressant effects in humans, as demonstrated by clinical studies and aromatherapy research (Dos Santos et al., 2022; Yoo and Park, 2023). Similarly, animal studies have demonstrated that inhalation exposure to linalool reduces anxiety-like behavior and produces sedative effects (Harada et al., 2018; Linck et al., 2010; Bi et al., 2024). In contrast, linalool has also been reported to induce anxiogenic effects under certain experimental conditions, suggesting its potential involvement in MCS-related symptoms (Wagner et al., 2024).
The inconsistency among previous findings implies that linalool may exert distinct effects through at least two neural mechanisms. Its positive effects, such as anxiolysis, are mediated by olfactory-dependent pathways (Harada et al., 2018). Conversely, its negative or MCS-like effects, such as anxiety and discomfort, may arise via olfactory-independent mechanisms, possibly through the accumulation and direct action of linalool or its metabolites within the CNS. Our previous studies demonstrated that linalool accumulates rapidly in the mouse brain following inhalation exposure and is metabolized into several derivatives (Oguro et al., 2025). Moreover, a class of these metabolites called furanoid linalool oxides increased c-Fos–positive neurons in the amygdala, a key region regulating emotional responses such as anxiety and aversion (Oguro et al., 2025).
Our earlier work demonstrated that linalool undergoes oxidation by cytochrome P450 (P450) enzymes—major drug-metabolizing catalysts—to form multiple oxidized derivatives, including cis- and trans-furanoid and pyranoid linalool oxides (Oguro et al., 2025). Genetic polymorphisms in P450 enzymes have been associated with interindividual susceptibility to MCS. For example, the active-type polymorphism of CYP2D6 and the inactive-type polymorphism of CYP2C9 or CYP2C19 have been reported to correlate with higher MCS prevalence (McKeown-Eyssen et al., 2004; Caccamo et al., 2013). Although P450 enzymes are most abundant in the liver, they are also expressed in the brain, where they metabolize exogenous chemicals (Hedlund et al., 2001; Durairaj and Liu, 2025). P450 enzymes are known to generate biologically active intermediates that can contribute to toxic reactions, such as allergic inflammation or carcinogenesis. For instance, P450 oxidizes a fragrance compound called geraniol into reactive metabolites that exacerbate allergic contact dermatitis (Hagvall et al., 2008). Similarly, P450-mediated oxidation of linalool may yield metabolites with enhanced bioactivity, exerting stronger effects on the CNS than the parent compound (Zehetner et al., 2019; Bråred Christensson et al., 2016). Taken together, these findings suggest that P450-mediated biotransformation of linalool within the brain contributes to its central neurobehavioral effects, potentially linking fragrance exposure to MCS-related symptoms.
In this study, we aimed to elucidate the mechanisms by which linalool inhalation induces MCS-like behavioral changes in mice. We first evaluated whether inhaled linalool produces anxiety- and depression-like behaviors in standard behavioral tests. To dissect the underlying pathways, we examined the roles of olfactory input, P450-dependent metabolism, and gamma-aminobutyric acid (GABA)A receptor–mediated modulation.
C57BL/6J mice were purchased from SLC (Shizuoka, Japan). Male mice (8-12 weeks old) were exposed to linalool (Tokyo Chemical Industry, Tokyo, Japan) at concentrations of 0.67 or 4 ppm as a mist with particle sizes of 4-6 µm using the inExpose system (emka Technologies, France). The linalool exposure concentrations were based on our previous inhalation study and preliminary observations. In our previous study, inhaled linalool accumulated in the mouse brain at 4 ppm under the same exposure system (Oguro et al., 2025). In the present study, linalool inhalation exposure was performed at 4 ppm and at a lower concentration of 0.67 ppm. After 15 min of exposure, the mice were immediately used for behavioral experiments or decapitated for tissue collection. At decapitation, samples of the olfactory bulb (OB), remaining brain tissue, liver, and whole blood collected by exsanguination were obtained. All animal experiments were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The Animal Care and Use Committee of Hiroshima University approved the experimental protocol (No. A24-166).
Pharmacological treatmentsAll compounds were dissolved in sterile physiological saline. Methimazole (MTZ; Sigma-Aldrich, USA) was administered intraperitoneally at 75 mg/kg 4 days before exposure to induce temporary anosmia, following a previously established protocol for chemical ablation of the olfactory epithelium (Håglin et al., 2021). The nonspecific P450 inhibitor 1-aminobenzotriazole (1-ABT; Tokyo Chemical Industry) was administered intraperitoneally at 100 mg/kg once daily for 3 consecutive days to inhibit systemic P450 activity, with the dosing schedule informed by previous studies in rodents (de Montellano, 2018). To assess the contribution of GABAA receptor modulation at the benzodiazepine binding site, flumazenil (FLU; Tokyo Chemical Industry) was administered intraperitoneally at 10 mg/kg 15 min before exposure, following previously reported protocols (McNamara and Skelton, 1993). Control animals received an equivalent volume of saline.
Open field testThe open field test (OFT) was conducted immediately after linalool exposure to evaluate anxiety-like behavior and locomotor activity. Each mouse was placed individually in a square gray acrylic arena (48 × 48 × 40 cm) under illumination of 20-100 lux. Behavior was recorded for 30 min using an overhead video camera and analyzed with Smart 3.0 tracking software (Panlab Harvard Apparatus, Spain). The central zone was defined as a 24 × 24 cm square in the center of the arena. The time spent in the central zone and the total distance traveled were calculated as indices of anxiety-like behavior and locomotor activity, respectively. The apparatus was wiped with 70% ethanol between trials to eliminate odor cues.
Tail suspension testThe tail suspension test (TST) was performed to assess depression-like behavior. Each mouse was suspended by the tail using a clip attached to the distal end of the tail and hung 10 cm above the floor under illumination of 20-100 lux. The entire session was recorded by video camera for 6 min from the side, and immobility time was manually measured by visual inspection of the video. Immobility was defined as the absence of active movement except for passive swinging. An increase in immobility time is considered an indicator of depression-like or motivationally passive behavior.
Odor preference testThe odor preference test (OPT) was conducted to verify methimazole-induced anosmia. A 30 × 20 cm cage was used, and a 2 × 2 cm filter paper spotted with 20 μL of either water (control) or female mouse urine (attractive odor) was placed at one short side of the cage. Under normal conditions, male mice show a strong preference for female urine and spend more time sniffing it than water. To assess olfactory function, mice were introduced from the opposite side of the cage, and their behavior was recorded by a lateral video camera for 3 min. Sniffing time was quantified by manually scoring video frames in which the mouse’s nose was close to the filter paper. Reduced preference for female urine relative to water was taken to indicate impaired olfactory ability.
Quantification of linalool and linalool oxide in the mouse brainBrain and other tissues from mice exposed to linalool were homogenized by sonication in hexane and centrifuged at 14,000 g for 15 min. After dehydration with anhydrous sodium sulfate, the samples were analyzed using a gas chromatography system equipped with tandem quadrupole mass spectrometry (GC-MS) (8860/5977C GCMS system, Agilent, USA) and an HP-5ms Ultra Inert column (30 m × 250 μm × 0.25 μm, Agilent). The column temperature was set to 40°C for the initial 5 min, then increased to 130°C at a rate of 5°C /min, followed by a further increase to 240°C at a rate of 20°C/min, where it was held for 20 min. Additional temperatures were set as follows: transfer line at 250°C, source at 230°C, and the quadrupole at 150°C. Helium was used as the carrier gas at a constant flow rate of 1 mL/min, and electron ionization (EI) mode was employed. Detection was conducted in SIM mode with m/z: 71.0, 93.0, and 121.0 for linalool, and with m/z: 59.1, 68.1, and 94.1 for furanoid linalool oxide. The cis/trans isomers of furanoid linalool oxide were assigned based on the previously reported GC/MS elution order, in which the trans-isomer elutes before the corresponding cis-isomer (Matich et al., 2010). Data were analyzed using MassHunter software (Agilent), and absolute quantification was achieved using a calibration curve prepared with analytical standards of linalool and 2-(2-hydroxy-2-propyl)-5-methyl-5-vinyltetrahydrofuran (a mixture of isomers; furanoid linalool oxide) obtained from Tokyo Chemical Industry.
Statistical analysisStatistical analyses were performed using Student’s t-test for two-group comparisons. For experiments involving multiple groups, one-way ANOVA followed by Dunnett’s post hoc test was used to compare each treatment group with the corresponding control. Probability (p) values < 0.05 were considered statistically significant.
Because no established animal model exists for MCS, we first examined whether frequently reported emotional symptoms could be reproduced in mice. Inhalation exposure to linalool significantly affected mouse behavior in the OFT and TST (Fig. 1). In the OFT, linalool-exposed mice spent significantly less time in the central zone compared with controls, indicating enhanced anxiety-like behavior, while total distance traveled did not differ between groups (Fig. 1A, B). In the TST, immobility time was significantly increased in mice exposed to 4 ppm linalool, suggesting a depression-like state (Fig. 1C). These results demonstrate that linalool inhalation exposure induces both anxiety- and depression-like behaviors without affecting general locomotor activity.
Effects of linalool inhalation on anxiety- and depression-like behaviors in mice. Mice were exposed to water or linalool vapor for 15 min using an inhalation exposure apparatus. Behavioral tests were conducted immediately after exposure. (A, B) The OFT was performed to assess anxiety-like behavior and locomotor activity. Mice were exposed to 0.67 ppm linalool for 15 min, and the time spent in the central zone (A) and the total distance traveled (B) during a 30-min session were recorded. (C) The TST was conducted for 6 min to evaluate depression-like behavior following exposure to 0.67 ppm or 4 ppm linalool vapor for 15 min. Data are presented as mean ± SD for each group (OFT: n = 10 per group; TST: control, n = 17; linalool (0.67 ppm), n = 15; linalool (4 ppm), n = 13). Statistical analysis used Student’s t-test for the OFT and one-way ANOVA followed by Dunnett’s post hoc test for the TST (*p < 0.05; **p < 0.01 vs. control).
To determine whether the behavioral effects of linalool inhalation exposure depend on olfactory perception, mice were pretreated with MTZ to induce anosmia (Fig. 2). Olfactory loss was confirmed using the OPT, in which MTZ-treated mice failed to show the typical preference for female mouse urine over water, which was taken to indicate successful suppression of olfactory function (Fig. 2A, B). Even under MTZ treatment, linalool-exposed mice spent significantly less time in the central zone of the OFT compared with controls, indicating that the anxiogenic effect of linalool inhalation exposure is independent of olfactory input (Fig. 2C). In contrast, the increase in immobility time observed in intact mice disappeared under MTZ treatment, suggesting that the depression-like effect of linalool inhalation exposure requires olfactory signaling (Fig. 2E). Locomotor activity was unaffected by either MTZ or linalool treatment (Fig. 2D).
Behavioral tests under olfactory blockade with methimazole treatment. Mice were intraperitoneally injected with MTZ (75 mg/kg) to induce temporary olfactory loss. Behavioral tests were conducted 4 days later to evaluate olfaction’s involvement in the effects of linalool. (A, B) The OPT was performed to confirm olfactory blockade. Mice were presented with water and urine for 3 min, and total sniffing time toward each odor source was measured. (C, D) The OFT was conducted after exposure to 0.67 ppm linalool vapor for 15 min. The time spent in the central zone (C) and the total distance traveled (D) during a 30-min session were recorded. (E) The TST was conducted for 6 min to evaluate depression-like behavior following exposure to 4 ppm linalool vapor for 15 min. Data are presented as mean ± SD for each group (preference test: n = 3 per group; OFT: n = 10 per group; TST: control, n = 15; linalool, n = 10). Statistical analysis used Student’s t-test (*p < 0.05 vs. control).
The tissue distributions of linalool and its major metabolites were quantified using GC-MS (Fig. 3). Our previous work showed that 4 ppm linalool exposure for 2 hr leads to detectable accumulation in the brain (Oguro et al., 2025). In the present study, even under milder exposure conditions—short duration (15 min) and lower concentration (0.67 ppm)—both linalool and its metabolites were clearly detected in the OB and remaining brain tissue. Linalool was strongly enriched in the OB, and its concentration was higher in MTZ-treated mice than in controls (Fig. 3A). This increase is likely attributable to MTZ-induced damage to the olfactory epithelium, which may have enhanced passive diffusion of linalool into the OB. The major oxidative metabolites, cis- and trans-furanoid linalool oxides, were primarily detected in the OB, remaining brain tissue, and blood, whereas their concentrations in the liver were negligible (Fig. 3B, C). These findings indicate that inhaled linalool readily enters the OB and other brain regions, where local metabolism occurs predominantly within these regions.
Brain transfer and metabolism of linalool under olfactory blockade. Mice treated with saline or MTZ were exposed to 0.67 ppm linalool vapor for 15 min using an inhalation exposure apparatus. The concentrations of linalool and its metabolites were quantified in each tissue by GC-MS. (A) Linalool levels in the olfactory bulb (OB), remaining brain tissue (Brain), liver, and blood. (B, C) Concentrations of cis- (B) and trans- (C) furanoid linalool oxides in each tissue. Data are presented as mean ± SD (control, n = 6; MTZ, n = 3). Statistical analysis used Student’s t-test (**p < 0.01 vs. control).
To evaluate the role of P450-mediated metabolism, mice were pretreated with the nonspecific P450 inhibitor 1-ABT (Fig. 4). 1-ABT pretreatment abolished the anxiogenic effect of linalool inhalation exposure, as the groups showed no significant difference in the time spent in the central zone (Fig. 4A). In contrast, total distance traveled tended to increase in linalool-exposed mice (Fig. 4B). To confirm the inhibitory effect of 1-ABT on linalool metabolism, OB, remaining brain tissue, liver, and blood were analyzed by GC-MS (Fig. 4C–E). 1-ABT pretreatment markedly reduced the formation of cis-furanoid linalool oxide, while trans-furanoid linalool oxide showed a similar decreasing tendency. Because the absolute abundance of the trans metabolite was lower, this reduction did not reach statistical significance (Fig. 4D, E). The concentration of linalool itself remained unchanged in the brain and liver but was significantly reduced in blood after 1-ABT pretreatment (Fig. 4C). These findings suggest that P450-dependent metabolic activation is necessary for the anxiogenic behavioral effects of linalool inhalation exposure.
Effects of cytochrome P450 inhibition on linalool exposure‒induced behaviors and linalool metabolism in the brain. Mice were intraperitoneally injected with the nonspecific P450 inhibitor 1-ABT at a dosage of 100 mg/kg once daily for 3 consecutive days before the behavioral or metabolic tests. On the test day, mice were exposed to 0.67 ppm linalool vapor for 15 min using an inhalation exposure apparatus. (A, B) The OFT was performed. The time spent in the central zone (A) and the total distance traveled (B) during a 30-min session were recorded. (C–E) Tissue concentrations of linalool and its metabolites were determined by GC-MS. (C) Linalool, (D) cis-furanoid linalool oxide, and (E) trans-furanoid linalool oxide in the OB, remaining brain tissue (Brain), liver, and blood. Data are presented as mean ± SD (OFT: n = 10 per group; metabolic test: control, n = 6; 1-ABT, n = 3). Statistical analysis used Student’s t-test (*p < 0.05; **p < 0.01 vs. control).
To determine whether the anxiogenic effect of linalool involves GABAergic modulation through the benzodiazepine (BDZ) binding site of the GABAA receptor, mice were pretreated with flumazenil (FLU), a selective antagonist of the BDZ site, 15 min before exposure (Fig. 5). In the OFT, FLU pretreatment completely abolished the linalool inhalation‒induced reduction of the time spent in the central zone (Fig. 5A), indicating that the anxiogenic effect of linalool inhalation exposure depends on BDZ site–mediated modulation of GABAA receptor signaling. FLU treatment did not significantly affect total distance traveled (Fig. 5B), suggesting that the behavioral change was specific to anxiety-related responses. These findings indicate that linalool or its metabolites alter emotional behavior via allosteric regulation of the GABAA receptor at the BDZ binding site.
Behavioral effects of linalool inhalation under GABAA receptor benzodiazepine-site blockade by flumazenil. Mice were intraperitoneally injected with FLU (10 mg/kg), a selective antagonist at the BDZ binding site of the GABAA receptor, 15 min before linalool exposure. Mice were then exposed to 0.67 ppm linalool vapor for 15 min using an inhalation exposure apparatus. (A, B) The OFT was performed. The time spent in the central zone (A) and the total distance traveled (B) during the 30-min session were recorded. Data are presented as mean ± SD (OFT: control, n = 9; linalool, n = 10). Statistical analysis used Student’s t-test.
Linalool inhalation induced depression-like behavior in mice (Fig. 1C), and this effect was abolished by MTZ-induced anosmia (Fig. 2E), indicating that it depends on olfactory input. Fragrance compounds are well known to modulate brain activity through olfactory pathways, and previous studies have shown that linalool produces anxiolytic and sedative effects via olfactory-dependent mechanisms (Harada et al., 2018). The olfactory-dependent increase in immobility time in the TST observed here may therefore reflect an extension of linalool’s sedative or anxiolytic actions, in which olfactory stimulation dampens emotional responsiveness and manifests as a mild depression-like effect.
On the other hand, the anxiogenic effect observed in the OFT (Fig. 1A) occurred independently of olfactory input (Fig. 2C), indicating the involvement of a distinct mechanism. In MCS, several studies have reported no significant differences in olfactory thresholds between patients and healthy controls (Doty et al., 1988; Azuma et al., 2016), suggesting that olfaction itself is unlikely to be the primary cause of fragrance-induced symptoms.
Our previous work demonstrated that linalool accumulates in the CNS following inhalation exposure (Oguro et al., 2025). Based on this, we hypothesized that the anxiety-like behavioral effects observed in the present study might arise from direct actions of linalool within the CNS. GC-MS analysis confirmed that both linalool and its metabolites were detectable in the brain as early as 15 min after exposure, with particularly high concentrations in the OB (Fig. 3). In contrast, hepatic levels of linalool were relatively low, and its metabolites were undetectable, suggesting that nasal uptake and local brain metabolism, rather than systemic circulation, constitute the primary route of entry. Chemicals absorbed through the nasal epithelium may pass between epithelial cells, enter the perineural space surrounding the olfactory nerve bundles, traverse the cribriform plate, and diffuse into the cerebrospinal fluid (Koo et al., 2024). Nevertheless, systemic absorption through the nasal or respiratory epithelium cannot be ruled out, as both linalool and its metabolites were detected in blood, and the high lipophilicity of linalool permits potential diffusion across the blood–brain barrier. Thus, although the anxiogenic behavior is likely driven by linalool accumulation in the CNS, potential contributions from peripheral tissues or peripheral nervous system pathways cannot be fully ruled out.
We used 1-ABT, a nonspecific P450 inhibitor, to assess the contribution of P450-dependent metabolism to the behavioral effects of linalool inhalation. Previous studies have shown that systemic administration of 1-ABT can inhibit P450 activity not only in the liver but also in the brain (Zong et al., 2016). In the present study, 1-ABT pretreatment suppressed the formation of cis- and trans-furanoid linalool oxides in the brain (Fig. 4D, E) and abolished the linalool inhalation-induced increase in anxiety-like behavior (Fig. 4A). These findings suggest that the production of P450-derived metabolites contributes to the anxiogenic behavioral effects of linalool inhalation. Polymorphisms in CYP2D6 and CYP2C9 have been associated with MCS susceptibility (McKeown-Eyssen et al., 2004; Caccamo et al., 2013), suggesting that P450 enzymes may play a role in the manifestation of MCS-related symptoms. In our previous study, linalool was shown to be metabolized by human CYP2A6, CYP2D6, and CYP3A4 to form furanoid linalool oxides, which increased c-Fos–positive neurons in the amygdala (Oguro et al., 2025). Based on these findings, we hypothesized that after entering the CNS, linalool may be metabolized by brain-expressed P450 enzymes, including CYP2A-, CYP2D-, and CYP3A-family enzymes, to generate active metabolites that modulate emotional neural circuits such as the amygdala, thereby altering behavior. These findings further raise the possibility that changes in P450 metabolic ability, for example due to concomitant medications, may modify the metabolism and biological effects of fragrance compounds such as linalool, thereby influencing fragrance-induced behavioral responses.
Several brain-expressed P450 enzymes are known to participate in the metabolism of endogenous neuroactive substrates; for example, CYP2D enzymes have been implicated in monoaminergic pathways, CYP2J/CYP2C enzymes in bioactive lipid metabolism, and CYP19A1/aromatase in local estrogen synthesis in the brain (Kuban and Daniel, 2021; Lu et al., 2019). Therefore, because 1-ABT is a nonspecific P450 inhibitor, its administration may affect not only linalool metabolism but also the metabolism of such endogenous substrates, thereby influencing locomotor activity and emotional behavior. The increase in total locomotor activity observed in linalool-exposed mice pretreated with 1-ABT (Fig. 4B) may also be related to such effects on endogenous metabolic processes. In addition, 1-ABT treatment decreased the whole-blood concentration of linalool (Fig. 4C). Although inhibition of P450-dependent metabolism alone would not directly explain this decrease, this finding may indicate that 1-ABT affected the systemic disposition of linalool, such as its tissue distribution or retention in blood. Consistent with this possibility, linalool accumulation in the liver was slightly increased after 1-ABT treatment. 1-ABT may also have affected the expression of factors that influence the retention of linalool in blood, although the underlying mechanism remains unclear. Therefore, although P450-dependent metabolism appears to play an important role in the expression of anxiety-like behavior induced by linalool inhalation, potential physiological and neurochemical confounding effects resulting from broad metabolic inhibition, as well as altered systemic distribution of linalool, should be considered.
Both hydroxylinalool and hydroxylinalyl acetate have been suggested to attenuate GABA-evoked currents through allosteric modulation of GABAA receptor function in vitro (Milanos et al., 2017). It is therefore plausible that other P450-derived metabolites, including furanoid linalool oxides, act through a similar allosteric mechanism to reduce receptor responsiveness to GABA. The central nucleus of the amygdala, which communicates extensively with other limbic regions and plays a pivotal role in the regulation of fear and anxiety, is composed predominantly of GABAergic neurons (Wang et al., 2023; Jie et al., 2018). Taken together, these observations suggest that P450-derived metabolites may disrupt the inhibitory balance within emotion-related brain regions, leading to the manifestation of anxiety-like behaviors in mice. Consistent with this, pretreatment with FLU, an antagonist acting at the BDZ binding site of GABAA receptors, abolished the linalool inhalation‒induced anxiogenic behavior in the OFT (Fig. 5A). This finding supports the involvement of BDZ site–mediated GABAergic modulation in the behavioral effects of linalool metabolites. Because FLU blocks both positive and negative allosteric modulators at this site, the disappearance of anxiety-like behavior suggests that P450-derived metabolites may act as negative allosteric modulators, reducing GABAA receptor sensitivity and thereby inducing an anxiogenic state. Such a mechanism resembles that of β-carboline derivatives, which function as inverse agonists at the BDZ site and produce anxiogenic or convulsant effects (Thiébot et al., 1988).
In conclusion, this study identifies an olfactory-independent component of linalool inhalation-induced anxiety-like behavior in mice. In contrast, the depression-like response appeared to depend on olfactory input. The detection of linalool and its metabolites in the brain, together with the disappearance of anxiety-like behavior after P450 inhibition, suggests that P450 activity is required for this olfactory-independent effect. These findings indicate that inhaled fragrance compounds can influence emotional behavior through non-olfactory mechanisms and provide a basis for investigating fragrance-induced MCS-like neurobehavioral responses.
FundingThis study was supported by grants from the Japan Science and Technology Agency (JST) Fusion Oriented Research for Disruptive Science and Technology (FOREST) (no. JPMJFR2119), the Japan Society for the Promotion of Science (JSPS) KAKENHI (no. 23K18401), and JST SPRING (no. JPMJSP2132).
Conflict of interestThe authors declare no competing financial interests or personal relationships that could be perceived to have influenced the work reported in this paper.
Data availabilityData are available from the corresponding author upon reasonable request.
Author contributionsHideaki Sato: Data curation, Investigation, Methodology, and Writing ‒ original draft.
Ami Oguro: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, and Writing ‒ review & editing.
Masatsugu Miyara: Resources and Writing ‒ review and editing.
Yaichiro Kotake: Funding acquisition, Resources, Supervision, and Writing ‒ review and editing.
Ethical approval and consent to participateAll animal experiments were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The Animal Care and Use Committee of Hiroshima University approved the experimental protocol (No. A24-166).
Patient consent for publicationNot applicable.
