Al-Omari Ammar, Kecskés Miklós, Gaszner Balázs, Biró-Sütő Tünde, Fazekas Balázs, Berta Gergely, Kuzma Mónika, Pintér Erika, Kormos Viktória
Department of Pharmacology and Pharmacotherapy, Centre for Neuroscience, Szentágothai Research Centre, Medical School and Molecular Pharmacology Research Group, University of Pécs, Pécs, Hungary.
Medical School, Institute of Physiology, University of Pécs, Pécs, Hungary.
Front Cell Dev Biol. 2023 Jan 10;10:1046559. doi: 10.3389/fcell.2022.1046559. eCollection 2022.
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. 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 hybridization combined with immunofluorescence. 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 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. We proved the presence of functional TRPA1 receptors on urocortin 1 neurons of the centrally projecting EWcp. Decreased 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.
向中枢投射的动眼神经副核（EWcp）通过其共表达尿皮质素1（UCN1）和可卡因及苯丙胺调节转录物（CART）的肽能神经元，参与对酒精摄入的控制。我们团队最近发现，向中枢投射的含尿皮质素能的EWcp是中枢神经系统瞬时受体电位锚蛋白1（TRPA1）阳离子通道mRNA表达的主要位点。在此，我们推测穿过血脑屏障的酒精及其代谢产物可能通过激活TRPA1离子通道，影响向中枢投射的EWcp中尿皮质素能细胞的功能。我们旨在研究向中枢投射的EWcp中TRPA1的功能活性及其在急性酒精暴露小鼠模型中的可能作用。对含有向中枢投射的EWcp的C57BL/6J雄性小鼠的急性脑片进行电生理测量，使用选择性、强效、共价激动剂JT010来证明TRPA1的功能活性。在急性酒精处理后的形态学研究中，对雄性TRPA1基因敲除（KO）小鼠和野生型（WT）小鼠进行了比较。在两种基因型中，一半动物腹腔注射1 g/kg 6%乙醇，另一半注射生理盐水作为对照。处理后2小时进行经心灌注。在向中枢投射的EWcp区域，进行FOS免疫组织化学以评估神经元激活情况。通过RNAscope杂交结合免疫荧光对TRPA1、CART和尿皮质素1 mRNA表达以及尿皮质素1和CART肽含量进行半定量分析。JT010激活了急性脑片中尿皮质素能细胞的TRPA1通道。酒精处理在两种基因型中均导致显著的FOS激活。酒精降低了WT小鼠中的mRNA表达。尿皮质素1肽免疫反应性评估显示，与WT小鼠相比，KO小鼠的基础尿皮质素1水平较低。尿皮质素1肽含量受酒精的基因型依赖性影响：WT小鼠中的肽含量降低，而KO小鼠中的肽含量增加。酒精暴露既不影响CART和尿皮质素1 mRNA表达，也不影响向中枢投射的EWcp/CART肽含量。我们证明了在向中枢投射的EWcp中尿皮质素1神经元上存在功能性TRPA1受体。急性酒精处理后mRNA表达降低，同时神经元尿皮质素1肽含量减少，这表明该阳离子通道可能参与尿皮质素1释放的调节。
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