Supplementary Material for: SELENOT deficiency in the mouse brain impacts catecholaminergic neuron density: an immunohistochemical, in situ hybridization and 3D light-sheet imaging study

Background: Selenoprotein T (SELENOT), a PACAP-regulated thioredoxin-like protein, plays a role in catecholamine secretion and protects dopaminergic neurons. However, the role of SELENOT in the establishment of the catecholaminergic (CA) neuronal system is not known yet. Methods: We analyzed by immunohistochemistry and RNAscope in situ hybridization the distribution of SELENOT and the expression of its mRNA, respectively. In addition, 3D imaging involving immunostaining in toto, clearing through the iDISCO+ method, acquisitions by light sheet microscopy and processing of 3D images was performed to map the CA neuronal system. A semi-automatic quantification of 3D images was carried out. Results: SELENOT protein and mRNA are widely distributed in the mouse brain, with important local variations. Three-dimensional mapping, through tyrosine hydroxylase (TH) labeling, and semi-automated quantification of CA neurons in brain-specific SELENOT knockout mice showed a significant decrease in the number of TH-positive neurons in the area postrema (AP-A2), the A11 cell group (A11) and the zona incerta (ZI-A13) of SELENOT-deficient females, and in the hypothalamus (Hyp-A12-A14-A15) of SELENOT-deficient females and males. Conclusion: These results showed that SELENOT is diffusely expressed in the mouse brain and that its deficiency impacts CA neuron distribution in different brain areas including Hyp-A12-A14-A15, in both male and female mice.

背景：硒蛋白T（Selenoprotein T, SELENOT）是一种受垂体腺苷酸环化酶激活肽（PACAP）调控的硫氧还蛋白样蛋白，参与儿茶酚胺分泌过程并对多巴胺能神经元具有保护作用。然而，SELENOT在儿茶酚胺能（CA）神经系统建立过程中所发挥的作用尚未明确。方法：本研究分别通过免疫组织化学技术与RNAscope原位杂交技术，分析了SELENOT在小鼠脑内的分布情况及其mRNA的表达水平。此外，本研究采用整体免疫染色、iDISCO+组织透明化方法、光片显微镜成像以及三维图像处理技术，完成了CA神经系统的三维图谱绘制。同时，对三维图像进行了半自动定量分析。结果：SELENOT蛋白及其mRNA在小鼠脑内广泛分布，且存在显著的脑区特异性差异。通过酪氨酸羟化酶（tyrosine hydroxylase, TH）标记完成的三维图谱绘制，结合对脑特异性SELENOT敲除小鼠中CA神经元的半自动定量分析结果显示：在SELENOT缺陷雌性小鼠的最后区（AP-A2）、A11细胞群（A11）以及未定带（ZI-A13）中，TH阳性神经元数量显著减少；同时在SELENOT缺陷雌雄小鼠的下丘脑（Hyp-A12-A14-A15）内，TH阳性神经元数量同样出现显著下降。结论：本研究结果表明，SELENOT在小鼠脑内呈弥散性表达，且其缺失会对雌雄小鼠不同脑区（包括Hyp-A12-A14-A15）内的CA神经元分布产生影响。