Evaluation of eGFP expression in the ChAT-eGFP transgenic mouse brain

Abstract

Abstract Background: A historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, can be found in high concentration in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, produced in the body of the neuron and is transported to the nerve terminal, where its concentration is highest, catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding acetylcholine (Ach). The creation of the bacterial artificial chromosome (BAC) transgenic mice expressing 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 the cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which eGFP is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing enhanced GFP (eGFP) in all cholinergic neurons and fibers in the central and peripheral nervous systems, as well as in non-neuronal cells. Methods: 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 comparison with in situ hybridization (ISH). Results: We qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression is not overlapping, such as the cortex, the striatum, the thalamus and the hypothalamus. We found 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 labelled 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. Conclusion: While the majority of the forebrain choline acetyltransferase 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.