Pharmacology Unit, Group of Pharmacology, School of Medicine, Western Sydney University, Penrith, NSW, 2751, Australia.
Center for Molecular and Behavioral Neuroscience, Rutgers The State University of New Jersey, Newark, NJ, 07102, USA.
BMC Neurosci. 2023 Jan 17;24(1):4. doi: 10.1186/s12868-023-00773-9.
A historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), a synthesizing enzyme for acetylcholine, (ACh), which can be found in high concentrations in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, is produced in the body of the neuron, transported to the nerve terminal (where its concentration is highest), and catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding ACh. The creation of bacterial artificial chromosome (BAC) transgenic mice that express promoter-specific fluorescent reporter proteins (green fluorescent protein-[GFP]) provided an enormous advantage for neuroscience. Both in vivo and in vitro experimental methods benefited from the transgenic visualization of cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which enhanced GFP (eGFP) is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing eGFP in all cholinergic neurons and fibers in the central and peripheral nervous systems as well as in non-neuronal cells.
This project systematically compared the exact distribution of the ChAT-eGFP expressing neurons in the brain with the expression of ChAT by immunohistochemistry using mapping and also made comparisons with in situ hybridization (ISH).
We qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain by comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression did not overlap, such as the cortex, striatum, thalamus and hypothalamus. We found a complete overlap between the transgenic expression of eGFP and the immunohistochemical staining in the areas of the cholinergic basal forebrain. However, in the cortex and hippocampus, we found small neurons that were only labeled with the antibody and not expressed eGFP or vice versa. Most importantly, we found no transgenic expression of eGFP in the lateral dorsal, ventral and dorsomedial tegmental nuclei cholinergic cells.
While the majority of the forebrain ChAT expression was aligned in the transgenic animals with immunohistochemistry, other areas of interest, such as the brainstem should be considered before choosing this particular transgenic mouse line.
胆碱乙酰转移酶（ChAT）是乙酰胆碱（ACh）的合成酶，是胆碱能神经元的一个历史明确标志物，在中枢和周围神经系统中的胆碱能神经元中都有高浓度存在。ChAT 由神经元体产生，被运送到神经末梢（那里的浓度最高），并催化辅酶乙酰辅酶 A 上的一个乙酰基转移到胆碱上，生成 ACh。 细菌人工染色体（BAC）转基因小鼠的创建，这些小鼠表达启动子特异性荧光报告蛋白（绿色荧光蛋白-[GFP]），为神经科学提供了巨大的优势。体内和体外实验方法都受益于胆碱能神经元的转基因可视化。通过将 BAC 克隆添加到 ChAT 基因座中，在 ChAT 起始密码子处的外显子 3 中插入增强型 GFP（eGFP），从而在中枢和周围神经系统以及非神经元细胞中的所有胆碱能神经元和纤维中强烈且据称选择性地表达 eGFP，从而创建了这些小鼠。
本项目通过映射，系统地比较了 ChAT-eGFP 表达神经元在大脑中的精确分布与免疫组织化学中 ChAT 的表达，并且还与原位杂交（ISH）进行了比较。
通过将 ChAT-eGFP 神经元的分布与免疫反应性神经元和 ISH 数据的分布进行比较，我们定性地描述了小鼠大脑中 ChAT-eGFP 神经元的分布，特别注意了表达不重叠的区域，如皮质、纹状体、丘脑和下丘脑。我们发现，在胆碱能基底前脑的区域，转基因表达的 eGFP 与免疫组织化学染色完全重叠。然而，在皮质和海马体中，我们发现了仅用抗体标记而不表达 eGFP 的小神经元，或者反之亦然。最重要的是，我们在外侧背侧、腹侧和背内侧脑桥核的胆碱能细胞中没有发现 eGFP 的转基因表达。
虽然大多数前脑 ChAT 表达与免疫组织化学在转基因动物中一致，但在选择特定的转基因小鼠品系之前，应考虑其他感兴趣的区域，如脑干。
