Anxiolytic and Antidepressant Effects of Organic Polysulfide, Dimethyl Trisulfide Are Partly Mediated by the Transient Receptor Potential Ankyrin 1 Ion Channel in Mice

Abstract

Background/Objectives: Dimethyl trisulfide (DMTS) is a naturally occurring polysulfide with known antioxidant and neuroprotective properties. DMTS is a lipophilic transient receptor potential ankyrin 1 (TRPA1) ligand that reaches the central nervous system (CNS). Its role in the CNS, particularly regarding depression-like behaviour, has yet to be explored. This study investigates the influence of DMTS on stress responses and whether this effect is mediated through the TRPA1 ion channel, known for its role in stress adaptation. Using a mouse model involving three-week exposure, we examined the impact of DMTS on depression-like behaviour and anxiety and identified the involved brain regions. Methods: Our methods involved testing both Trpa1-wild-type and gene-knockout mice under CUMS conditions and DMTS treatment. DMTS was administered intraperitoneally at a dose of 30 mg/kg on days 16 and 20 of the 21-day CUMS protocol-in hourly injections seven times to ensure sustained exposure. Various behavioural assessments-including the open field, marble burying, tail suspension, forced swim, and sucrose preference tests-were performed to evaluate anxiety and depression-like behaviour. Additionally, we measured body weight changes and the relative weights of the thymus and adrenal glands, while serum levels of corticosterone and adrenocorticotropic hormone were quantified via ELISA. FOSB (FBJ murine osteosarcoma viral oncogene homolog B) immunohistochemistry was utilised to assess chronic neuronal activation in stress-relevant brain areas. Results: Results showed that CUMS induces depression-like behaviour, with the response being modulated by the TRPA1 status and that DMTS treatment significantly reduced these effects when TRPA1 channels were functional. DMTS also mitigated thymus involution due to hypothalamic-pituitary-adrenal (HPA) axis dysregulation. Conclusions: Overall, DMTS appears to relieve depressive and anxiety symptoms through TRPA1-mediated pathways, suggesting its potential as a dietary supplement or adjunct therapy for depression and anxiety.

Keywords: TRPA1; anxiety; chronic stress; depression; dimethyl trisulfide; ion channels; mechanism.

Figure 13

Summary of possible mechanisms underlying the effects of chronic stress and DMTS treatment on depression-like behaviour and anxiety. The antidepressant-like effects of DMTS (e.g., in the sucrose preference test) were abolished in Trpa1-knockout mice, suggesting a TRPA1-dependent mechanism, whereas its anxiolytic-like effects (e.g., increased peripheral zone activity in the open field test) were preserved, indicating TRPA1-independent pathways, possibly involving serotonergic circuits in limbic regions, such as the basolateral amygdala. Red arrows = inhibition; green arrows = stimulation; blue arrow = modulation; black arrow = potential regulation. HPA = hypothalamic–pituitary–adrenal axis; TRPA1 = transient receptor potential ankyrin 1; ACTH = adrenocorticotropic hormone; CRH = corticotropin-releasing hormone; DMTS = dimethyl trisulfide; CRF = corticotropin-releasing factor; 5-HT1A = serotonin 1A; UCN1 = urocortin 1; PAG = periaqueductal grey matter; DR = dorsal raphe nucleus; EWcp = centrally projecting Edinger–Westphal nucleus; BST = bed nucleus of the stria terminalis; BLA = basolateral amygdala; LS = lateral septum; PVT = paraventricular nucleus of the thalamus; PVN = paraventricular nucleus of the hypothalamus.

Figure 1

Experimental timeline of control (A) and chronic-stress-exposed (B) animals. IHC: immunohistochemistry; ACTH: adrenocorticotropic hormone; CORT: corticosterone; CUMS: chronic unpredictable mild stress; DMTS: dimethyl trisulfide; ELISA: enzyme-linked immunosorbent assay.

Figure 2

Establishing the dose of dimethyl trisulfide (DMTS) via open field tests. Symbols represent, from left to right, untreated (naïve), vehicle-treated, 30 mg/kg (DMTS30), and 40 mg/kg DMTS-treated (DMTS40) Trpa1 WT mice. The distance travelled (A) and mobility time (B) are plotted on the left y-axes (one-way analysis of variance; ** p < 0.01; n = 8–10 per group).

Figure 3

The efficacy of chronic unpredictable mild stress (CUMS) exposure and dimethyl trisulfide (DMTS) treatment. We found that the body weight change (A), relative adrenal weight (C), and relative thymus weight (D) mirrored the somatic changes induced by chronic stress. Panel (B) presents absolute body weights of mice at the end of the in vivo experiment. We assessed the hypothalamic–pituitary–adrenal axis activity by determining the serum corticosterone (E) and adrenocorticotropic hormone (ACTH) concentrations (F) (three-way analysis of variance followed by Tukey’s post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n = 8–16 per group). Only the relevant significance levels have been indicated to facilitate clarity.

Figure 4

Effect of chronic unpredictable mild stress (CUMS) and dimethyl trisulfide (DMTS) treatment on anxiety and depression-related behaviour. In order to assess the level of anxiety, we evaluated the time spent (A) and the distance travelled (B) in the periphery part of the arena in the open field test, and we counted the number of hidden marbles in the buried marble test (C). To detect depression-like behaviour, we determined the presence of anhedonia with the sucrose preference test (D); moreover, we assessed the inactive duration in the forced swim test (E) and tail suspension test (F) (three-way analysis of variance followed by Tukey’s post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001; n = 8–16 per group). Only the relevant significance levels have been indicated to facilitate clarity.

Figure 5

FOSB immunohistochemistry in centrally projecting Edinger–Westphal nucleus (EWcp), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: * p < 0.05; ** p < 0.01; n = 5–6 per group. Black line: scale bar 200 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 6

FOSB immunohistochemistry in dorsal raphe nucleus (DR), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: * p < 0.05; ** p < 0.01; n = 5–6 per group. Black line: scale bar 200 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 7

FOSB immunohistochemistry in periaqueductal grey matter (PAG), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: *** p < 0.001; n = 5–6 per group. Black line: scale bar 500 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 8

FOSB immunohistochemistry in paraventricular nucleus of the thalamus (PVT), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: * p < 0.05; n = 5–6 per group. Black line: scale bar 200 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 9

FOSB immunohistochemistry in lateral septal nucleus (LS), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: * p < 0.05, ** p < 0.01; n = 5–6 per group. Black line: scale bar 500 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 10

FOSB immunohistochemistry in paraventricular nucleus of the hypothalamus (PVN), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: ** p < 0.01, *** p < 0.001; n = 5–6 per group. Black line: scale bar 200 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 11

FOSB immunohistochemistry in bed nucleus of the stria terminalis (BST), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: * p < 0.05, ** p < 0.01; n = 5–6 per group. Black line: scale bar 500 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.

Figure 12

FOSB immunohistochemistry in basolateral amygdala (BLA), representative micrographs. Bar graphs show the number of FOSB-immunoreactive neurons in each group. Three-way analysis of variance followed by Tukey’s post hoc test: n = 5–6 per group. Black line: scale bar 500 μm. Control = non-stressed; CUMS = chronic unpredictable mild stress; DMTS = dimethyl trisulfide. Only the relevant significance levels have been indicated to facilitate clarity.