Functionally active TRPA1 ion channel is downregulated in peptidergic neurons of the Edinger-Westphal nucleus upon acute alcohol exposure

Abstract

Introduction: The centrally projecting Edinger-Westphal nucleus (EWcp) contributes to the control of alcohol consumption by its urocortin 1 (UCN1) and cocaine- and amphetamine-regulated transcript (CART) co-expressing peptidergic neurons. Our group recently showed that the urocortinergic centrally projecting EWcp is the primary seat of central nervous system transient receptor potential ankyrin 1 (TRPA1) cation channel mRNA expression. Here, we hypothesized that alcohol and its metabolites, that pass through the blood-brain barrier, may influence the function of urocortinergic cells in centrally projecting EWcp by activating TRPA1 ion channels. We aimed to examine the functional activity of TRPA1 in centrally projecting EWcp and its possible role in a mouse model of acute alcohol exposure. Methods: Electrophysiological measurements were performed on acute brain slices of C57BL/6J male mice containing the centrally projecting EWcp to prove the functional activity of TRPA1 using a selective, potent, covalent agonist JT010. Male TRPA1 knockout (KO) and wildtype (WT) mice were compared with each other in the morphological studies upon acute alcohol treatment. In both genotypes, half of the animals was treated intraperitoneally with 1 g/kg 6% ethanol vs. physiological saline-injected controls. Transcardial perfusion was performed 2 h after the treatment. In the centrally projecting EWcp area, FOS immunohistochemistry was performed to assess neuronal activation. TRPA1, CART, and urocortin 1 mRNA expression as well as urocortin 1 and CART peptide content was semi-quantified by RNAscope in situ hybridization combined with immunofluorescence. Results: JT010 activated TRPA1 channels of the urocortinergic cells in acute brain slices. Alcohol treatment resulted in a significant FOS activation in both genotypes. Alcohol decreased the Trpa1 mRNA expression in WT mice. The assessment of urocortin 1 peptide immunoreactivity revealed lower basal urocortin 1 in KO mice compared to WTs. The urocortin 1 peptide content was affected genotype-dependently by alcohol: the peptide content decreased in WTs while it increased in KO mice. Alcohol exposure influenced neither CART and urocortin 1 mRNA expression nor the centrally projecting EWcp/CART peptide content. Conclusion: We proved the presence of functional TRPA1 receptors on urocortin 1 neurons of the centrally projecting EWcp. Decreased Trpa1 mRNA expression upon acute alcohol treatment, associated with reduced neuronal urocortin 1 peptide content suggesting that this cation channel may contribute to the regulation of the urocortin 1 release.

Keywords: JT010; alcohol; centrally projecting Edinger-Westphal nucleus; cocaine-and amphetamine-regulated transcript; transient receptor potential ankyrin 1; urocortin 1.

FIGURE 1

JT010 increases spontaneous firing frequency of UCN1-immunoreactive neurons in the EWcp nucleus. Representative confocal images of UCN1-immunoreactive (red) cells (A) and a biocytin (green) filled patched neuron (B) and the merged image (C). Insets shows the magnified soma of the patched neuron. Scale bars: 40 µm. Response of the recorded cell (D) to 1 s current injection (−100 and +100 pA). Representative current clamp recordings (E, upper panel) showing the spontaneous activity of UCN1-immunoreactive neuron. Black bar represents JT010 application (5 µM). Instantaneous firing frequency (E), lower panel] of each action potential in the upper recording is plotted. Statistics (F) showing the firing frequency at baseline (2 min before drug application) during JT010 application and after washing out the drug (n = 9 from 4 mice). ***p < .001; Tukey’s post hoc test upon one-way ANOVA.

FIGURE 2

Urine alcohol concentration. Quantitative evaluation of alcohol concentration in the urine of wildtype (WT) and Trpa1 knockout (KO) mice, 2 h after i.p., saline (control) or 1 g/kg ethanol injection. Columns show means ± SEM of alcohol concentration (g/L) (n = 6–8; **p = .002; ***p = .0009; Tukey’s post hoc test upon two-way ANOVA).

FIGURE 3

Quantitation of FOS immunoreactivity in the centrally projecting Edinger–Westphal nucleus (EWcp). (A) Representative immunohistochemical images showing the expression of FOS, as a marker of early neural activation, in the EWcp of wildtype (WT) and Trpa1 knockout (KO) mice 2 h after i.p., saline (control) and 1 g/kg ethanol injection. Neuronal activation is represented by brown colored nuclei. (B) Quantitative evaluation of FOS immunostaining in the EWcp of WT and Trpa1 KO mice, 2 h after i.p., saline (control) and 1 g/kg ethanol injection. Columns show means ± SEM of FOS positive neurons in the EWcp (n = 6–8; ***p = .0001; Tukey’s post hoc test upon two-way ANOVA).

FIGURE 4

Trpa1 mRNA expression in the centrally projecting Edinger–Westphal nucleus (EWcp) of control and ethanol-treated mice. (A) Representative fluorescence images showing the expression of Trpa1 mRNA (red) by RNAscope in situ hybridization and its co-localization with the urocortin1 (UCN1) peptide (green) by immunofluorescence, in the EWcp of Trpa1 wildtype (WT) mice 2 h after i.p., saline (control) and 1 g/kg ethanol injection. For nuclei, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Quantitative evaluation of Trpa1 mRNA expression in the EWcp of WT mice, 2 h after i.p., saline (control) and 1 g/kg ethanol injection. Columns show means ± SEM of Trpa1 mRNA transcripts in the EWcp (n = 6–8; ***p = .0001; Student’s t-test).

FIGURE 5

UCN1 mRNA and peptide content of the centrally projecting Edinger–Westphal nucleus (EWcp). Representative fluorescence images showing the expression of urocortin1 (Ucn1) mRNA (green) by RNAscope in situ hybridization (A) and the UCN1 peptide (green) by immunofluorescence (B), in the EWcp of wildtype (WT) and Trpa1 knockout (KO) mice 2 h after i.p., saline (control) and 1 g/kg ethanol injection. For nuclei, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Quantitative evaluation of Ucn1 mRNA (C) and UCN1 peptide (D) specific signal density (SSD) in the EWcp of Trpa1 WT and Trpa1 KO mice, 2 h after i.p., saline (control) and 1 g/kg ethanol injection. Columns show means ± SEM of Ucn1 mRNA (C) and UCN1 peptide (D) SSD in the EWcp (n = 6–8; *p = .03; ***p = .0001; two-way ANOVA and Tukey’s post hoc test).

FIGURE 6

CART mRNA and peptide content of the centrally projecting Edinger–Westphal nucleus (EWcp). Representative fluorescence images showing the expression of cocaine- and amphetamine-regulated transcript (Cart) mRNA (red) by RNAscope in situ hybridization (A) and the CART peptide (red) by immunofluorescence (B), in the EWcp of wildtype (WT) and Trpa1 knockout (KO) mice 2 h after i.p., saline (control) and 1 g/kg ethanol injection. For nuclei, the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Quantitative evaluation of Cart mRNA (C) and CART peptide (D) specific signal density (SSD) in the EWcp of WT and Trpa1 KO mice, 2 h after i.p., saline (control) and 1 g/kg ethanol injection. Columns show means ± SEM of Cart mRNA (C) and CART peptide (D) SSD in the EWcp (n = 6–8; *p = .05; two-way ANOVA and Tukey’s post hoc test).