Kashiwadani et al. Behav Brain Funct (2021) 17:3 https://doi.org/10.1186/s12993-021-00176-y Behavioral and Brain Functions SHORT PAPER Open Access Linalool odor‐induced analgesia is triggered by TRPA1-independent pathway in mice Hideki Kashiwadani1*† , Yurina Higa1,2†, Mitsutaka Sugimura2 and Tomoyuki Kuwaki1 Abstract We had recently reported that linalool odor exposure induced signifcant analgesic efects in mice and that the efects were disappeared in olfactory-deprived mice in which the olfactory epithelium was damaged, thus indicating that the efects were triggered by chemical senses evoked by linalool odor exposure. However, the peripheral neuronal mechanisms, including linalool receptors that contribute toward triggering the linalool odor-induced analgesia, still remain unexplored. In vitro studies have shown that the transient receptor potential ankyrin 1 (TRPA1) responded to linalool, thus raising the possibility that TRPA1 expressed on the trigeminal nerve terminal detects linalool odor inhaled into the nostril and triggers the analgesic efects. To address this hypothesis, we measured the behavioral pain threshold for noxious mechanical stimulation in TRPA1-defcient mice. In contrast to our expectation, we found a signifcant increase in the threshold after linalool odor exposure in TRPA1-defcient mice, indicating the analgesic efects of linalool odor even in TRPA1-defcient mice. Furthermore, intranasal application of TRPA1 selective antagonist did not alter the analgesic efect of linalool odor. These results showed that the linalool odor-induced analgesia was triggered by a TRPA1-independent pathway in mice. Keywords: Linalool, Analgesia, TRPA1, TRPA1-defcient mice, TRPA1-selective antagonist Introduction Odorous volatile compounds inhaled into the nostril We had recently demonstrated that odor exposure of are detected by two sensory systems, the main olfactory linalool (3,7-dimethylocta-1,6-dien-3-ol), one of the system and the trigeminal sensory system. In the main monoterpene alcohols found in lavender extracts, olfactory system, olfactory sensory neurons express- induces analgesic efects in mice [1]. We observed that ing a given odorant receptor is translated into electri- the efects were disappeared in olfactory-deprived mice cal signals. Tese signals are then transmitted to the in which the olfactory epithelium was damaged, thus main olfactory bulb, the frst relay station of the main indicating that the efects were triggered by chemi- olfactory system, and further to the olfactory cortices cal senses evoked by linalool odor exposure. However, to perceive the olfactory input [2, 3]. In addition to the the peripheral neuronal mechanisms, including linalool classical main olfactory pathway, the trigeminal nerve receptors that contribute toward triggering the linalool pathway contributes to the detection of odorous com- odor-induced analgesia, have not yet been explored. pounds in the nostril [4]. Te ethmoidal nerve arising from the ophthalmic division of the trigeminal nerve pro- jects into the nasal cavity and makes free end terminals in the epithelium [5–8]. Te central projections of the *Correspondence: [email protected] ethmoidal nerve terminate on the superfcial laminae of †Hideki Kashiwadani and Yurina Higa contributed equally to this study 1 Department of Physiology, Graduate School of Medical the medullary dorsal horn [9, 10] with collateral branches and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, which reach directly to the olfactory bulb [7, 11]. In Kagoshima 890-8544, Japan spite of the individual peripheral receptors, the olfactory Full list of author information is available at the end of the article and trigeminal input mutually afect each other in the © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kashiwadani et al. Behav Brain Funct (2021) 17:3 Page 2 of 7 perception of odors [12–15], indicating that the informa- ((Z)-4-(4-chlorophenyl)-3-methylbut-3-en-2-oxime, tion detected by the two chemosensory systems is func- gifted from Prof. Mori at Kyoto University [26]) was dis- tionally integrated in our central nervous system. Te solved in polyethylene glycol (PEG#400, Nacalai, Kyoto, exact location of the interaction between the two systems Japan) at 60 mM. is not yet determined. But several anatomical areas such as the olfactory epithelium, the olfactory bulb, the medi- odorsal thalamus, the piriform cortex, the orbitofrontal Linalool odor exposure cortex and the insula cortex have been proposed as the We used a custom-made olfactometer for linalool odor site [5, 7, 11, 16–18]. exposure as described previously [1]. Briefy, 0.5 mL of Chemosensors expressed on the free end terminals linalool (> 96 %) was dispensed into an uncapped glass can be activated by the chemical compounds that are vial (diameter: 27.5 mm, content: 20 mL). Te vial was inhaled into the nostril and drive the trigeminal pathway placed in an odor chamber (0.32 L), and then linalool was [19]. Among the chemosensors, the transient receptor vaporized at room temperature (24 °C ± 1 °C). Clean air potential ankyrin 1 (TRPA1), which was frst identifed as deodorized using a charcoal flter and double-distilled the thermosensor [20], detects a range of odorous com- water was introduced into the odor chamber (top diame- pounds [21], and plays a key role in triggering the allyl ter: 8 cm, base diameter: 11.5 cm, height: 15 cm, content: isothiocyanate odor-induced bradypnea in mice [22]. In 1 L) at a constant fow rate (1 L/min). After 20 min of addition to pungent odorous compounds, linalool acti- pre-ventilation of the linalool odor, a mouse was placed vates the TRPA1 in dissociated dorsal root ganglia neu- in the observation chamber and exposed to linalool odor rons and in human embryonic kidney 293 cells expressing for 5 min. Because the humidity of the carrier gas and the the wild-type TRPA1 channels [23, 24]. Tese observa- temperature of the odor chamber were maintained con- tions raise a hypothesis that the TRPA1 on the trigeminal stant, the concentration of linalool odor was considered nerve terminal in the nasal cavity detects the inhaled lin- to be constant. alool and triggers the analgesic efects induced by linalool odor. To investigate this hypothesis, we measured the threshold for the behavioral pain response immediately Acclimatization for pain assay after linalool odor exposure and compared the threshold For acclimatization to the experimental condition, the between TRPA1-defcient (KO) mice and wild-type mice. animals were moved to the experiment room for 2 h, handled and gently touched for 3 min, and their head and Materials and methods body were covered with a towel and loosely restrained Animals and housing conditions for 3 min. Tis series of acclimatization procedures was Male wild-type (WT) mice (C57BL/6, weighing 26.6– repeated for 6 days. On the experiment day, the mice were moved to the experiment room 2 h before the 34.6 g, n = 77) and TRPA1 KO mice (weighing 22.7– experiment. 27.4 g, n = 43) were used in this study. Te mutant mice were originally purchased from the Jackson Laboratory and genotyped as previously described [25]. Tey were maintained as heterozygotes in our facility and crossed Tail pincher test to obtain null mutants and WT littermates. Te mutant For evaluating the analgesic efects of linalool odor, we mice were backcrossed with C57BL/6J mice (Clea Japan measured the mechanical nociceptive threshold for tail Inc., Tokyo, Japan) for more than 10 generations. All pinch using calibrated forceps (Rodent Pincher-analge- animals were maintained under a constant temperature sia meter, Bioseb, Pinellas Park, USA) [27, 28]. Immedi- (24 °C ± 1 °C) with free access to food and water. Te ately after 5 min of odor exposure, we gently restrained animals were housed with lights on at 7:00 A.M. and of the mouse with a towel and pressured on the marking at 7:00 P.M. All experiments were conducted during the of the tail using the calibrated forceps. We recorded the light cycle, between 1:00 P.M. and 5:00 P.M. Te animals latency of the ficking, tail withdrawal, or struggling of were naive to linalool odor, and each mouse was used the mouse in the cotton towel. We repeatedly measured only once to avoid carryover efects. the threshold for fve times (trial interval: 10–15 s). We then excluded the maximum and minimum values from Chemicals the sets of fve measurements and used the average value Linalool (CAS#: 78-70-6) was purchased from Tokyo of another three trials as the threshold value [28]. To pre- Chemical Industry (Tokyo, Japan), stored at 4 °C, and vent the mouse from being injured, a cut-of pressure dispensed into a glass vial during each trial to pre- point was set at 500 g. Each animal was used only once to vent degradation. A TRPA1 selective antagonist AP18 prevent hyperalgesia.