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

Abstract

Background 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. 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 also made comparisons with in situ hybridization (ISH). Results 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. Conclusion 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.

Acetylcholine Neurons Become Cholinergic during Three Time Windows in the Developing Mouse Brain

“…Consistent with previous work, we found that the expression of the ACh neuron marker ChAT changes over time or is low in specific brain regions, such as the lateral septum, cerebral cortex, or cerebellum ( Clos et al, 1989 ; Li et al, 2018 ; Gamage et al, 2023 ). Therefore, the well-characterized ChAT-IRES-Cre driver mouse line was used in combination with Cre reporter lines to express tdTomato in ACh neurons to ensure robust soma detection.…”

“…Firstly, our data provide novel insights into the timeline of when ACh neurons become cholinergic. We report additional or revised ACh neuron populations in the brainstem, the midbrain, and the hypothalamus compared with those previously described in other recent studies ( Li et al, 2018 ; Gamage et al, 2023 ). Secondly, we describe the changes in cholinergic innervation of the cerebellum as well as the cholinergic projections from the habenula to the hindbrain.…”

“…Despite recent breakthroughs, such as reconstruction of individual ACh neuron projection fields, the generation of whole-brain atlases of ACh neurons or input/output mapping to/from the basal forebrain ACh neurons, and RNA expression patterns in the developed brain, very little progress has been made regarding a comprehensive assessment of developmental changes in ACh neurons ( Oh et al, 2014 ; Wu et al, 2014 ; Gielow and Zaborszky, 2017 ; Li et al, 2018 ; Cizeron, 2020 ; Wang et al, 2020 ; Tomas-Roca et al, 2022 ; Gamage et al, 2023 ). While there is a trove of literature about nAChR mRNA or protein level changes in many brain regions in the pre- or postnatal rodent brain, there is little data about how onset of cholinergic signaling overlaps with the nAChR expression patterns in these brain regions ( Cimino, 1995 ; Jaarsma et al, 1997 ; Zhang, 1998 ; Abreu-Villaca et al, 2011 ; Zhang et al, 2016 ; Broide et al, 2019 ; Abbasi et al, 2020 ; Alzu’bi et al, 2020 ; Colangelo et al, 2022 ; Saad et al, 2022 ).…”

Chronic neuroinflammation during aging leads to cholinergic neurodegeneration in the mouse medial septum

“…The ChAT (BAC) -eGFP mice were created by adding a bacterial artificial chromosome (BAC) clone into the ChAT locus, in which enhanced green fluorescent protein (eGFP) is inserted into exon 3 at the ChAT initiation codon, strongly and selectively express eGFP in all of the CNS and PNS cholinergic neurons, as well as in the non-neuronal cells [ 40 ]. There was almost perfect overlap of both ChAT (BAC) -eGFP and Gt-a-ChAT-labelled neurons in the entire expansion of the BF including the MS, validating the use of the ChAT (BAC) -eGFP transgenic strain or C57BL/6 stain as the age-matched control for this study [ 44 ].…”

An AAV capsid increases transduction of striatum and a ChAT promoter allows selective cholinergic neuron transduction

“…Prior studies using the ChAT promoter to drive transgene expression in transgenic mice have resulted in selective expression in cholinergic neurons. 12 , 13 More recently Martel et al. used a canine adenovirus vector packaging a ChAT-promoter-driven transgene cassette, yielding transduction of ChAT neurons in rat and NHP striatum.…”