Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain (2024)

Drugs and reagents

Boswellia carterii resin was purchased from Pamir (Tel Aviv, Israel). For the extraction and isolation of IA, we followed the procedure described by us previously (11). The material obtained was identified by direct comparisons [of mass spectrometry (MS) and nuclear magnetic resonance (NMR) data] with the compound used previously. Diazepam was purchased from Elkins-Sinn (Cherry Hill, NJ, USA). Desipramine hydrochloride, capsaicin, and camphor were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fura-2 AM and pluronic 127 were purchased from Molecular Probes (Eugene, OR, USA), and 2-APB and ionomycin were purchased from Calbiochem (EMD Biosciences, San Diego, CA, USA). 4α-Phorbol 12,13-didecanoate (4αPDD) was purchased from PKC Pharmaceuticals (Woburn, MA, USA). Pental was purchased from C.T.S. Chemical Industries (Tel Aviv, Israel). Rabbit anti c-Fos, horseradish peroxidase, and diaminobenzidine were purchased from Sigma. Secondary biotinylated donkey anti-rabbit antibody was purchased from Chemicon (Temecula, CA, USA).

IA and 4αPDD were dissolved in ethanol for in vitro assays; 2-APB, ionomycin, and capsaicin were dissolved in dimethyl sulfoxide (DMSO). All solvents were applied in a volume <1.1% of the total volume in the well, a concentration that did not induce effects on calcium mobilization or ion currents.

Cell culture

Human embryonic kidney (HEK) 293 cells stably expressing human TRPV1 were a kind gift from Merck Research Laboratories (Whitehouse Station, NJ, USA). Cells were cultured in minimal essential medium, Dulbecco modified Eagle medium (DMEM), modified with nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, and 1.5 g/l sodium bicarbonate (TACK, Manassas, VA, USA), containing 1% penicillin-streptomycin and 10% FBS. Cells were passaged 3 times a week using trypsin-EDTA 1× (Invitrogen, Carlsbad, CA, USA) and grown under 5% CO₂ at 37°C.

HEK293 cells stably expressing mouse TRPV3-YFP, pcDNA3, and rat TRPV4 were acquired as described previously (20, 22, 23).

HEK293 cells were transiently transfected with a rat TRPV2 plasmid (24), using lipofectamine reagent (Invitrogen) according to manufacturer’s protocol. HEK293 cells expressing mouse TRPV3-YFP, pcDNA3, rat TRPV2, or rat TRPV4 were cultured in 1× DMEM (high glucose) with l-glutamine (Mediatech, Inc., Herndon, VA, USA), containing 1% penicillin-streptomycin (Invitrogen) and 10% FBS. Cells were passaged 3 times a week using 1× trypsin-EDTA (Invitrogen) and grown under 5% CO₂ at 37°C.

Animals and procedures

Female Sabra mice (Harlan, Rehovot, Israel; 15–20 wk old) and wild-type (WT) C57BL/6 or TRPV3 knockout (KO) female mice (18–20 wk old; ref. 18) were used for behavioral assessments. Ten mice were housed in each cage. The animal care and protocols met the guidelines of the U.S. National Institutes of Health, detailed in the Guide for the Care and Use of Laboratory Animals, and were applied in conformity with the Johns Hopkins University and Ariel University Center of Samaria Institutional Ethics Committees. Female Sabra mice were also used for the c-Fos immunostaining. Temperature in the animal room was maintained between 20–22°C; the light cycle was 12 h lights on (8 AM to 8 PM) and 12 h lights off (8 PM to 8 AM). Mice were injected intraperitoneally with IA in a mixture of isopropanol:cremophor:saline (1:1:18) at a volume of 10 μl/g body weight.

Behavioral assays

The different behavioral assays performed were all based on established assays. Preliminary observations indicated similar effects of IA when the time interval between injection and testing was between 15 and 45 min. Therefore, mice were assayed within these time limits.

Elevated plus maze

The elevated plus maze assay is based on the preference of mice for the closed arms of a maze, apparently due to fear of open spaces. The assay was performed as described by others (25, 26). Briefly, mice were placed in the central platform (10×10 cm) between the open (10×45 cm) and closed (10×45×40 cm) arms of a plus maze. The time spent in each of the arms was recorded. An “antianxiety” effect was measured as the time spent on the aversive open arms of the maze (relative to the total time spent in both arms).

Porsolt forced swimming test

The Porsolt forced swimming test was based on previous designs (27, 28): mice were placed in a 2 L glass beaker (11 cm diameter) filled with water (24±1°C) up to 30 cm from the bottom (so that the mouse could not touch the bottom) and 8 cm from the rim (so that the mouse could not escape). Immobility time was defined as the time the animal did not move (except for small movements required to float).

Open field behavior

Mice were placed in a transparent open field of 30 × 40 cm, divided into 20 squares of equal size, and their horizontal (ambulatory) and vertical activity was measured for 8 min by scoring the number of squares crossed (29, 30) and number of rears accordingly.

Cataleptic effect

The cataleptic effect of IA was measured as time of immobility on an elevated ring of 5.5 cm diameter (during 4 min; refs. 29, 30).

c-Fos immunostaining

Mice were deeply anesthetized by an intraperitoneal injection of 200 mg/kg sodium pentobarbital (Pental). Brains were fixed by transcardial perfusion with ice-cold 4% paraformaldehyde containing 4% sucrose (pH 7.4) and by overnight immersion in the same, refrigerated fixative. Brains were then immersed in 12% sucrose in PBS (0.02 M, pH 7.4) and kept refrigerated until sectioning. Brains were cut on a cryostat as coronal sections, 30 μm thick, and were collected floating and kept in a cryopreservation buffer at −18°C until immunohistochemical staining of c-Fos. For each brain region, two fields were sampled containing this region from the right and left hemisphere. After the sections were rinsed in PBS (0.02 M, pH 7.4) and quenched of endogenous peroxidase by incubation in 0.15% hydrogen peroxide for 30 min, they were incubated with rabbit anti c-Fos diluted 1:5000. The secondary antibody was diluted 1:400, and sections were incubated with extravidin conjugated with horseradish peroxidase diluted 1:200. The final color reaction was performed with diaminobenzidine.

Binding assays

All binding assays were performed under license agreement at Cerep (Paris, France; refs. 31, 32).

Calcium imaging of HEK293 cells

Human TRPV1-, mouse TRPV3-YFP-, rat TRPV4-, and pcDNA3-expressing HEK293 cells were plated 24–48 h before imaging in 96-well black walled, clear bottom CellBind plates (Corning, Corning, NY, USA), loaded with 3 μM Fura-2 AM, and imaged as described previously (33). For single-cell calcium imaging, HEK293-rat TRPV2- and -mouse TRPV3-YFP-expressing cells were plated on collagen-coated glass cover slips. Cells were loaded for 60 min with 3 μM Fura-2 AM in 0.05% w/v pluronic 127 in HEPES-Tyrode buffer (pH 7.4) containing the following (in mM): 25 HEPES, 140 NaCl, 2.7 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.4 NaH₂PO₄, and 5 glucose. All experiments were performed at room temperature (∼24°C). A Flexstation II plate reader (Molecular Devices) was programmed with the following settings: excitation wavelength, 340 and 380 nm; emission wavelength, 510 nm. The initial volume per well was 175 μl. After 30 s of baseline recording, 75 μl buffer containing drug or vehicle was added at a rate of up to ∼52 μl/s per well. The total run time was 200 s. For single-cell calcium imaging experiments, coverslips were mounted on an inverted microscope (Nikon TS-100, Tokyo, Japan). Cells were alternately excited at 340 and 380 nm. Emitted light (510 nm) was captured using a Cohu 4920 cooled charge-coupled device (CCD) camera (Cohu, San Diego, CA, USA) and analyzed with the InCyt Im2 image acquisition and analysis software (Intracellular Imaging Inc., Cincinnati, OH, USA).

Calcium imaging of TRPV3^+/+ and TRPV3^−/− keratinocytes

Primary keratinocytes from TRPV3-deficient and TRPV3^+/+ mouse pups (days 1–4) were harvested and cultured as described previously (34). Cells were plated on glass coverslips (10⁵/cm²) and incubated for 48–60 h and then loaded with Fura-2 AM (20 μM, 0.04% pleuronic acid, 32°C for 1 h) in imaging buffer containing the following (in mM): 130 NaCl, 2.5 CaCl₂, 0.6 MgCl₂, 10 HEPES, 1.2 NaHCO₃, and 10 glucose, pH 7.45. Ratiometric Ca²⁺ imaging was performed as described previously (22). Drug was added to the bath after a period of baseline recording. Calcium measurements were made from 30 randomly selected cells per coverslip (the cells were not chosen based on criteria such as morphology or labeling).

Electrophysiological recording

Currents were recorded using whole-cell voltage clamp. Pipettes were pulled from microcapillary glass (A-M Systems, Sequim, WA, USA). A coverslip containing cells was transferred to a 300 μl chamber that was constantly perfused (1–2 ml/min) with external solution. Voltage protocols were generated and data were digitized and recorded using Pulse (HEKA Elektronik, Lambrecht, Germany) software in conjunction with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA, USA). The data were analyzed using an in-house Visual Basic (Microsoft, Redmond, WA, USA) analysis program.

The pipette solution contained the following (in mM): 121.5 K gluconate, 10 HEPES, 17 KCl, 9 NaCl, 1 MgCl₂, 0.2 EGTA, 2 MgATP, and 0.5 NaATP, pH 7.2. The external solution contained the following (in mM): 120 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 20 HEPES, pH 7.4, with NaOH. The measured charge (pC) was defined as the charge elicited between −85 and −45 mV by a ramping voltage stimulus (−85 mV to +35 mV, 0.54 mV/ms; holding potential −55 mV). Currents were sampled at 5 kHz. Experimental and control cells were alternated whenever possible. Control values were obtained from WT HEK293 cells.

Data analysis

In general, for comparisons between two data sets we used an unpaired 2-tailed Student’s t test. For tests of statistical significance involving more than two data sets, we used a 1-way ANOVA with a post hoc test, because this test is appropriate for comparing three groups on one factor. Data obtained from the KO vs. WT mice in ​in​​​​​Fig.Fig. 7 were analyzed by 2-way ANOVA because in this case two factors were investigated (strain: WT vs. KO; and drug: vehicle vs. IA). Behavioral responses in Sabra mice to IA vs. diazepam or desipramine were analyzed by 1-way ANOVA with Newman-Keuls post hoc comparisons. Motor behavior and catalepsy data were analyzed by t tests (comparing vehicle- and IA-injected mice), and behaviors in C57BL/6 (WT) vs. TRPV3^−/− mice were analyzed using 2-way ANOVA with Bonferroni post hoc comparisons (GraphPad 4 Prism; GraphPad, San Diego, CA, USA). Analysis of calcium imaging data was performed using a nonlinear regression curve fit (GraphPad 4 Prism). In the keratinocyte experiments, drug-induced response for each cell was taken as the maximal postdrug measurements over time minus the average of the last 5 predrug measurements. Averaged drug responses over 30 randomly selected cells per coverslip were analyzed with 2-tailed unpaired t tests.

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Figure 7.

IA exhibits an anxiolytic effect in the elevated plus maze test and an antidepressant-like effect in the Porsolt forced swimming test in WT but not TRPV3−/− mice. WT and TRPV3−/− mice (18–20 wk old) were injected with vehicle (isopropanol:emulphor:saline=1:1:18) or IA (75 mg/kg). Thirty to forty min later, the mice were tested in the elevated plus maze for 5 min (A) and the Porsolt forced swimming test for 9 min (B). In the elevated plus maze assay, IA caused WT mice to spend significantly more time in the aversive open arms of the maze (relative to the total time spent in both arms). In the Porsolt forced swimming test, IA significantly reduced immobility in WT mice, whereas TRPV3 KO mice did not respond to IA. No significant difference was noted in WT and TRPV3 KO mice in response to vehicle. Data are means ± se; n = 4–5. *P < 0.05, **P < 0.01 vs. WT vehicle-injected mice (Bonferroni post hoc test).

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Figure 1.

Structure of IA. Structure elucidation is based on NMR and MS data (see ref. 14).

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Figure 2.

Effect of IA on performance in the elevated plus maze and the Porsolt forced swimming test. Female Sabra mice (15-20 wk) were injected with vehicle isopropanol:emulphor (a commercial emulsifier):saline=1:1:18 or IA (50 mg/kg) in vehicle. Diazepam (5 mg/kg, dissolved in ethanol:emulphor:saline=1:1:18) was used as positive control for the plus maze; desipramine (DMI; 15 mg/kg, dissolved in saline) was used as a positive control in the Porsolt forced swimming test. Thirty to 40 min later, the mice were tested in the elevated plus maze for 5 min (A) and the Porsolt forced swimming test for 9 min (B). Data are means ± se; n = 4–6. *P < 0.05, **P < 0.01 vs. WT vehicle-injected mice (Newman-Keuls post hoc test).

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Figure 3.

Effects of IA on motor activity and catalepsy. Female Sabra mice were injected intraperitoneally with 50 mg/kg IA dissolved in a mixture of isopropanol:cremophor:saline = 1:1:18. Fifteen minutes later, the mice were observed for locomotion and rearing in an open field (30×40 cm, divided into 20 squares of equal size) and for catalepsy assay on a ring (7.5 cm diameter) elevated 30 cm above the table top. Data are means ± se; n = 4–5. *P < 0.05; ***P < 0.001 (unpaired t test).

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Figure 4.

IA modulates stress-induced c-Fos expression in several brain areas. Diagrams illustrate brain areas of female Sabra mice (15–20 wk; n=4–5) where IA (50 mg/kg) significantly changed the number of c-Fos-immunoreactive cells, 60 min after intraperitoneal injection of IA or vehicle. Drawings were modified from Paxinos and Franklin (49), Plates 30 (A), 38 (B), 45 (C), and 89 (D). Atlas sections are arranged from anterior (A) to posterior (D). Number under each section indicates its distance (mm) anterior (A) or posterior (P) from the bregma. IA significantly increased c-Fos (red fill) in the lateral septum (LS), central nucleus of the amygdala (CEA), solitary complex (Sol), and bed nucleus stria terminalis (BNST). IA significantly reduced c-Fos (light blue fill) in the motor cortex (MCtx), medial striatum (MSt), and hippocampal CA3 region (CA3). See Table 1 for quantification and Supplemental Fig. 1A, B for representative micrographs.

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Figure 5.

IA is a potent TRPV3 activator. A) IA or 2-APB evoked calcium increases in mouse HEK293 TRPV3-YFP-transfected cells compared with vehicle; #P < 0.001 (n=9). IA-treated HEK293-TRPV3(+) cells show a significantly higher activation than HEK293-pcDNA cells; *P < 0.001 (n=9). B) IA dose dependently induced calcium influx in TRPV3-YFP-transfected HEK293 cells in the presence of calcium in the extracellular media (○); EC50 = 16 μM, Hill slope = 2.2 (n=10). In the absence of calcium (○), the effect of IA was markedly reduced; #P < 0.05 (n=5). C) IA (500 μM) increased intracellular calcium levels in primary keratinocytes from TRPV3+/+ but not TRPV3−/− mice. Camphor (10 mM) showed a similar effect. *^,#P < 0.005 (n=6), t test (2-tailed). D) Representative single-cell calcium traces of HEK293 cells stably expressing mouse-TRPV3-YFP. IA or 2-APB was applied at 60 s (after 60 s of baseline recording; arrow); the drug remained in the bath throughout the recording. E) IA induced a very small influx of calcium in human TRPV1-transfected HEK293 cells compared with vehicle; #P < 0.001, (n=22–29). Capsaicin induced a calcium increase significantly greater than that induced by IA; *P < 0.001 (n=29–35). F) IA did not induce calcium influx in HEK293 cells transiently transfected with rat-TRPV2, whereas 2-APB increased calcium in these cells; #P < 0.001 (n=41–51). G) IA induced a very modest calcium influx in rat-TRPV4-transfected HEK293 cells compared with vehicle; #P < 0.001 (n=26). 4αPDD induced a calcium increase that was significantly larger than the effect of IA; * P < 0.001 (n=26). All error bars indicate se; P values in all subfigures but C represent analysis with 1-way ANOVA with Bonferroni’s post hoc test.

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Figure 6.

IA activates a TRPV3 current when it is stably expressed in HEK293 cells. A) Sample time course shows summed charge of current activated (−85 to −45 mV, in pC) with application of IA (200 μM). B) Sample current response to voltage ramp from same cell as A. C) Dose response for IA shows activation of currents at 200 μM in TRPV3(+) HEK293 cells (▪) but not in TRPV3(−) controls (▴). *P < 0.001 vs. TRPV3(−); 1-way ANOVA with Dunnett’s post hoc test. D) TRPV agonist 2-APB (100 μM) activates currents in TRPV3(+) cells but not in TRPV3(−) cells. **P < 0.001; unpaired 2-tailed Student’s t test. E) IA (200 μM) does not activate currents in TRPV1(+) and TRPV4(+) cells nor does vehicle in TRPV3(+) cells. TRPV3(+) response to IA is shown for reference. Two TRPV3(−) controls were used, one set in WT HEK293 cells and one set in nonglowing cells found among the TRPV3-YFP-expressing HEK293 cells; the results were identical. *P < 0.001 vs. TRPV3(+); 1-way ANOVA with Dunnett’s post hoc test. Error bars indicate se; n = 4–5.

For analysis of c-Fos immunoreactivity, positive nuclei were identified based on their round form and optical density at least twice that of background. The numbers of c-Fos immunoreactive nuclei from the right and left hemispheres were averaged to obtain a representative number for the given region from each mouse. Student t tests were performed comparing the control (vehicle) with the IA group.

Behavioral assays

To study the functional effects of IA on the CNS, we first assayed IA (Fig. 1) in a panel of standard behavioral assays in mice (female Sabra strain, 15–20 wk old): the elevated plus maze, the Porsolt forced swimming test, locomotion in the open field test, and cataleptic response in a ring test. At 50 mg/kg, IA exerted a potent anxiolytic-like effect, causing mice to spend significantly more time in the aversive open arms of the elevated plus maze (Fig. 2A). In the Porsolt forced swimming test, a standard assay for the evaluation of antidepressant effects, IA significantly reduced the immobility recorded over 9 min, thus indicating a reversal of an avolition response (Fig. 2B). We also observed a significant reduction in open field behavior (Fig. 3A, B), as well as increased immobility on a ring (Fig. 3C).

The experiments described in Figs. 2and 3 are representative of 4 to 7 independent experiments, and all gave comparable results. It should be noted that while the experiments presented in Fig. 2 are designed to reflect the emotional state of the examined animal, the open field locomotion and the ring catalepsy tests, presented in Fig. 3, reflect the motor effects of IA.

IA modulates the expression of c-Fos in mice brains in several areas, including areas involved in anxiety and depression

To investigate the activity of IA on different brain regions, we studied its effect on c-Fos immunoreactivity in mouse brains 60 min after administration (50 mg/kg ip). IA significantly increased c-Fos in the lateral septum, central nucleus of the amygdala, and solitary nucleus, while significantly reducing c-Fos in the motor cortex, medial striatum, and hippocampal CA3 region (Fig. 4; Table 1; Supplemental Fig. 1).

Binding assays

IA was assayed for its ability to displace radioligands bound to an array of 24 receptors, ion channels, and transport proteins potentially relevant to its putative emotional and behavioral effects (Supplemental Table 1). No significant displacement of radioligand was observed for any of these targets using up to 10 μM of IA (data not shown).

IA activates TRPV3 channels, but not several other TRPV channels, as determined by calcium imaging

Our attention then turned to the TRPV family of channels, especially as TRPV1 is assumed to mediate behavioral effects that resemble those exhibited by IA (35). We hence proceeded to examine the effect of IA on TRPV1–4. IA significantly increased calcium influx (EC₅₀=16 μM; Hill slope=2.2; Fig. 5A, B, D) in HEK293 cells stably expressing mouse TRPV3-YFP. When calcium was removed from the extracellular medium, the calcium increase in response to IA was significantly reduced (Fig. 5B), providing further evidence for the influx of calcium through TRPV3 channels. The effect of IA on TRPV3-induced calcium influx resembles the effect of the broad-spectrum TRP agonist 2-APB, which served as a positive control (Fig. 5A, D). IA (500 μM) also induced a calcium influx in primary keratinocytes from WT mice but not from TRPV3^−/− mice (Fig. 5C). The effect of IA (500 μM) on TRPV-induced calcium influx in primary keratinocytes resembles the one of camphor (10 mM), a known agonist of TRPV3. Interestingly, at a concentration (100 μM) that was maximally effective in TRPV3 expressing cells, IA did not induce calcium influx in HEK293 cells transiently transfected with rat-TRPV2 (Fig. 5F) and caused only minimal calcium influx in HEK293 cells expressing human TRPV1 and rat TRPV4 (Fig. 5E, G).

IA activates a TRPV3 current in HEK293 cells

IA also activated a cation current in mouse-TRPV3-YFP-expressing HEK293 cells (Fig. 6A–C) with properties consistent with TRPV3 activation (36). This current was lower than the current activated by 2-APB, which served as a positive control (Fig. 6D). IA-induced current was not activated in HEK293 cells not expressing TRPV3 nor in TRPV1 or TRPV4 expressing cells (Fig. 6C, E).

IA exerts anxiolytic-like and antidepressive-like effects in WT but not in TRPV3 KO mice

Given the effect of IA on TRPV3 channels and the observation that IA does not interact with a long list of receptors known to be involved in psychoactivity, we investigated the possibility that its behavioral effects are mediated through CNS TRPV3 channels. We repeated the panel of behavioral assays with WT and TRPV3^−/− mice, which were administered either IA or vehicle. IA (75 mg/kg) exerted a significant anxiolytic-like effect in WT mice, while TRPV3^−/− mice spent identical time on the open arms, regardless of whether they were injected with IA or only vehicle (Fig. 7A; F_strain=6.3, df=1,14, P<0.05; F_interaction=5.0, df=1,14, P<0.05). In the Porsolt forced swimming test, IA significantly reduced the immobility time in WT but not in TRPV3^−/− mice (F_IA=5.5, df=1,16, P<0.04; F_interaction=5.9, df=1,16, P<0.03; Fig. 7B). No significant differences were recorded between vehicle-treated WT mice and vehicle-treated TRPV3^−/− mice in the Porsolt forced swimming and elevated plus maze assays. The decreased locomotion and cataleptic effects of IA were maintained in TRPV3^−/− mice (Fig. 8).

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Figure 8.

IA shows a similar effect on open field behavior in WT and TRPV3−/− mice. WT and TRPV3−/− mice (18–20 wk) were injected with vehicle (isopropanol:emulphor:saline=1:1:18) or IA (75 mg/kg). Fifteen min later, they were introduced into a transparent cage and tested for the effect of IA on locomotion for 8 min: horizontal, number of squares crossed (A); and vertical, number of rears (B). Mice were also tested for the cataleptic effect of IA as time of immobility on an elevated ring of 5.5 cm diameter over 4 min (C). No difference was noted in WT and TRPV3 KO mice in response to vehicle. Data are means ± se; n = 4–5. #P < 0.05, ***P < 0.001 vs. WT vehicle-injected mice (Bonferroni post hoc test).

IA as a novel anxiolytic and antidepressive agent

Anxiety and depression are the most common forms of psychiatric disorders in the United States (37, 38) and pose risk factors for numerous additional diseases (39). The clinical treatment of these high-prevalence disorders often relies on pharmacologic interventions. However, although antianxiety and antidepressive drugs have been developed, new types of such medicaments are needed, as the presently available treatments are not satisfactory in many cases, due to treatment-limiting adverse effects (40, 41). Finding new drugs for the amelioration of anxiety and depression is thus of considerable significance and even more so is the search for new pharmacological targets that can be addressed in this quest.

c-Fos transcription factor is a product of an immediate early gene, and changes in its expression serve as markers of changes in neuronal activity. It is used in histological sections to map out brain regions that are activated or attenuated after treatment with psychoactive drugs, such as anxiolytics and antidepressants (42, 43). The central nucleus of the amygdala and the lateral septum play major roles in the expression of emotions (44, 45), and it is assumed that c-Fos expression in the central nucleus of the amygdala is due to circuits that are engaged by both anxiolytic and anxiogenic drugs (44).

We found that IA shows significant TRPV3-dependent activity in both the elevated plus maze and the Porsolt forced swimming test. Despite minor differences, which are expected in different strains, IA maintains significant behavioral effects on both Sabra mice and WT C57BL/6 mice, generalizing and corroborating its putative psychoactive effect. It should be noted that these behavioral assays are independent from the locomotion open field test. Hence, despite the significant attenuation of locomotion, IA significantly decreased the time mice spend immobile in water. IA also modulates the expression of c-Fos in mice brains in several areas, including areas involved in anxiety and depression (Fig. 4). Thus, the data from the behavioral assays together with the c-Fos immunostaining suggest that IA may represent a novel anxiolytic and antidepressive agent. Our next step was to look for a possible mechanism for these behavioral effects. Considering its effects on animal models, we were surprised to note that IA does not bind to any of 24 related receptors, ion channels, and transport proteins. However, we found that it is a potent TRPV3 channel activator.

IA is a potent TRPV3 activator

In view of the critical role played by natural products in the discovery of the TRP channels and their functions (46), several groups have recently attempted to identify natural products that activate TRPV3 channel. Smith et al. (47) have screened extracts from 50 Chinese herbal plants; no TRPV3 active compounds were identified. Vogt-Eisele et al. (48) examined a library of monoterpenoids for their action on TRPV3 and found some of them to be active, the lowest EC₅₀ reported as 370 μM.

As described above (Fig. 5A, B, D), we found that IA induces calcium influx in HEK293 cells expressing TRPV3 channel. This observation was confirmed by the activation of TRPV3 in primary mouse keratinocytes (Fig. 5C) and by the activation of a TRPV3 current in HEK293 cells stably expressing TRPV3 (Fig. 6A–D). The effect of IA on TRPV3-induced calcium influx resembles the effect of 2-APB, the most potent known TRPV3 activator (Fig. 5D). However, its effect on cell current is lower (Fig. 6). The effect elicited by IA on TRPV3 channels seems to be more specific than that by 2-APB, as it showed no or very modest activity on cells expressing TRPV2, TRPV1, or TRPV4 (Figs. 5E–G and ​and6E).6E). Because the charge response to IA lags the calcium response, it is possible that a calcium-activated current is evoked in the cells. However, this difference can be attributed to the inherent differences between the calcium imaging system and an electrophysiology apparatus, e.g., differences in the ions measured or in drug application (manual application vs. perfusion system).

Terpenoids have been used for centuries as medicinally useful compounds, but still little is known about the mechanism of action of many of them (48). TRP channels as targets for terpenoids can potentially explain several of their described effects. Investigations of novel ligands for members of the TRP channel family are not only relevant for the understanding of this protein family but may also have implications for target-directed drug design (48).

TRPV3 channel may pose a possible novel therapeutic target in the CNS

Our data, in particular the observations that IA has no effect in the elevated plus maze and the Porsolt forced swimming test in TRPV3^−/− mice, indicate that the TRPV3 channels are involved in the effects of IA in animal models for antidepressant and anxiolytic effects. This implies that TRPV3 channels in the brain may play a role in emotional regulation, thus attributing, for the first time, a function for this channel in the CNS. These data also raise hopes that TRPV3 channels in the brain may be targeted in the efforts of ameliorating depressive and anxiety disorders. Our observation that IA reduces activity in the open field and the ring tests to the same extent in both TRPV3^−/− and WT mice implies that the sedation produced by IA is not mediated via TRPV3 channels.

IA is chemically unrelated to compounds that are currently in use as anxiolytic and antidepressive drugs. It belongs to an important group of common natural products, cembranoid diterpenes. Many of these compounds exhibit biological activities; however, to the best of our knowledge, no work has been done on the CNS activities of these compounds to date. The identification of CNS effects by a cembranoid diterpene may lead to a novel group of antidepressive and anxiolytic agents, originating from an ancient drug.

Taken together, our data support our original contention, namely that Boswellia resin may affect sensation and emotional states. These observations also indicate that the TRPV3 channel is involved in emotional and behavioral processes in the CNS in addition to its known effects on the perception of warmth (thermosensation). It is possible that IA augments the euphoric feeling produced during religious functions, due to both positive, presumably mild, emotional effects and the sensation of warmth. Thus the neurobehavioral effect of IA may provide a biochemical basis for the millennial and widespread use of Boswellia-containing incense. However, only direct human trials including the investigation of human dosage and dosage forms may give final, concrete proof.

Thus, IA joins the notable group of plant-derived compounds that are active in the mammalian CNS. It is therefore possible that IA would provide us with novel therapeutic agents and neurochemical insights.

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