Peptidergic neurons of the Edinger-Westphal nucleus express TRPA1 ion channel that is downregulated both upon chronic variable mild stress in male mice and in humans who died by suicide

Abstract

Background: Transient receptor potential ankyrin 1 (TRPA1), a cation channel, is expressed predominantly in primary sensory neurons, but its central distribution and role in mood control are not well understood. We investigated whether TRPA1 is expressed in the urocortin 1 (UCN1)-immunoreactive centrally projecting Edinger-Westphal nucleus (EWcp), and we hypothesized that chronic variable mild stress (CVMS) would reduce its expression in mice. We anticipated that TRPA1 mRNA would be present in the human EWcp, and that it would be downregulated in people who died by suicide.

Methods: We exposed Trpa1 knockout and wild-type mice to CVMS or no-stress control conditions. We then performed behavioural tests for depression and anxiety, and we evaluated physical and endocrinological parameters of stress. We assessed EWcp Trpa1 and Ucn1 mRNA expression, as well as UCN1 peptide content, using RNA-scope in situ hybridization and immunofluorescence. We tested human EWcp samples for TRPA1 using reverse transcription polymerase chain reaction.

Results: Trpa1 mRNA was colocalized with EWcp/UCN1 neurons. Non-stressed Trpa1 knockout mice expressed higher levels of Ucn1 mRNA, had less body weight gain and showed greater immobility in the forced swim test than wild-type mice. CVMS downregulated EWcp/Trpa1 expression and increased immobility in the forced swim test only in wild-type mice. We confirmed that TRPA1 mRNA expression was downregulated in the human EWcp in people who died by suicide.

Limitations: Developmental compensations and the global lack of TRPA1 may have influenced our findings. Because experimental data came from male brains only, we have no evidence for whether findings would be similar in female brains. Because a TRPA1-specific antibody is lacking, we have provided mRNA data only. Limited access to high-quality human tissues restricted sample size.

Conclusion: TRPA1 in EWcp/UCN1 neurons might contribute to the regulation of depression-like behaviour and stress adaptation response in mice. In humans, TRPA1 might contribute to mood control via EWcp/UCN1 neurons.

Figure 1

Timeline of animal experiments. Behavioural tests (MBT, OFT, SPT, TST, FST) and CVMS protocol with midday (SHAKE, REST, TILT, DARK, WD) and overnight (WET, SI, GH) stressors. CVMS = chronic variable mild stress; DARK = dark room exposure; FST = forced swim test; GH = group holding; MBT = marble burying test; OFT = open field test; REST = restraint stress; SHAKE = shaker stress; SI = social isolation; SPT = sucrose preference test; TILT = tilted cage; TST = tail suspension test; WD = water deprivation; WET = wet bedding.

Figure 2

Representative images of Trpa1 mRNA–expressing urocortinergic cells from the mouse Edinger–Westphal nucleus. (A) Sections were counterstained with DAPI (blue) for nuclei. (B) Trpa1 mRNA (red) was visualized by RNAscope. (C) The UCN1 peptide (white) was detected by immunofluorescence (IHC). (D) Note the colocalization of Trpa1 mRNA and UCN1 peptide. DAPI = 4′,6-diamidino-2-phenylindole; IHC = immunohistochemistry; UCN1 = urocortin 1.

Figure 3

Findings from behavioural assessments. (A) To assess anxiety levels, we calculated the number of hidden marbles in the marble burying test. (B) We evaluated the ratio of time spent in the peripheral part of the arena in the open field test. (C) To reveal locomotor effects, we evaluated distance travelled during the open field test. (D) We determined anhedonia levels with the sucrose preference test. (E and F) We evaluated depression-like behaviour as immobility time in the tail suspension test and the forced swim test. Two-way analysis of variance followed by a Fisher post hoc test: *p < 0.05, ***p < 0.001. n = 14–16 per group. Grey bars represent wild-type mice; black bars represent Trpa1 knockout mice. CVMS = chronic variable mild stress.

Figure 4

Efficacy of exposure to CVMS. We assessed the activity of the hypothalamic–pituitary–adrenal axis by determining (A) serum ACTH and (B) corticosterone titres. We found that (C) relative total adrenal weight, (D) relative thymus weight and (E) body weight change mirrored somatic changes induced by chronic stress. (F) Absolute body weight of mice at end of the in vivo experiment. Two-way analysis of variance followed by a Fisher post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001. n = 14–16 per group. Grey bars represent wild-type mice; black bars represent Trpa1 knockout mice. ACTH = adrenocorticotropic hormone; CVMS = chronic variable mild stress.

Figure 5

Trpa1, Ucn1 mRNA and UCN1 peptide expression in EWcp neurons upon CVMS. (A and B) Trpa1 mRNA (red) was downregulated in wild-type mice exposed to CVMS, as also shown in (C) the histogram. (A′ and B′) Trpa1 mRNA transcripts (red) were localized to cells containing UCN1 peptide (white). (D to H) Ucn1 mRNA (green) was expressed at higher levels in control Trpa1 knockout mice (black bars). CVMS increased Ucn1 mRNA expression in wild-type mice only (grey bars). (I to M) UCN1 peptide content (white) of EWcp neurons. The UCN1 peptide SSD was elevated in Trpa1 knockout mice upon exposure to CVMS. DAPI (blue) labelling was performed to mark the nuclei of cells. #p < 0.05 in a Student t test. *p < 0.05, Fisher post hoc test following 2-way analysis of variance. CVMS = chronic variable mild stress; DAPI = 4′,6-diamidino-2-phenylindole; EWcp = centrally projecting division of the Edinger–Westphal nucleus; SSD = specific signal density; UCN1 = urocortin 1.

Figure 6

Electrophoretograms of RT-PCR products. We studied 3 human EWcp samples (1 to 3) and used a TRPA1-expressing human oral squamous cell carcinoma culture (PECA) as a positive control. The housekeeping gene (DNA-directed RNA polymerase II subunit RPB1; POLR2A, size 152 bp) and the gene of interest (TRPA1, size 115 bp) were expressed in all samples, including the PECA cell culture. The UCN1 (size 123 bp) RT-PCR products proved that all brain samples contained the EWcp area. We also used a no reverse transcription control and a no template control. EWcp = centrally projecting Edinger–Westphal nucleus; no RT = no reverse transcription control; NTC = no template control; PECA = human oral squamous cell carcinoma cell line ( PE/CAPJ41; clone D2); RT-PCR = reverse transcription polymerase chain reaction; TRPA1 = transient receptor potential ankyrin 1; UCN1 = urocortin 1.

Figure 7

Relative TRPA1 gene expression in human centrally projecting Edinger–Westphal nucleus samples from male controls (n = 3) and people who died by suicide (n = 3) as determined by Taq-Man quantitative reverse transcription polymerase chain reaction (*p < 0.05; Student t test).