Generation of a ChAT mouse line without the early onset hearing loss typical of the C57BL/6J strain.

The development of knockin mice with Cre recombinase expressed under the control of the promoter for choline acetyltransferase (ChAT) has allowed experimental manipulation of cholinergic circuits. However, currently available ChAT mouse lines are on the C57BL/6J strain background, which shows early onset age-related hearing loss attributed to the Cdh23 mutation (a.k.a., the ahl mutation). To develop ChAT mice without accelerated hearing loss, we backcrossed ChAT mice with CBA/CaJ mice that have normal hearing. We used genotyping to obtain mice homozygous for ChAT and the wild-type allele at the Cdh23 locus (ChAT). In the new line, auditory brainstem response thresholds were ∼20 dB lower than those in 9 month old ChAT mice at all frequencies tested (4-31.5 kHz). These thresholds were stable throughout the period of testing (3-12 months of age). We then bred ChAT animals with Ai14 reporter mice to confirm the expression pattern of ChAT. In these mice, tdTomato-labeled cells were observed in all brainstem regions known to contain cholinergic cells. We then stained the tissue with a neuron-specific marker, NeuN, to determine whether Cre expression was limited to neurons. Across several brainstem nuclei (pontomesencephalic tegmentum, motor trigeminal and facial nuclei), 100% of the tdTomato-labeled cells were double-labeled with anti-NeuN (n = 1896 cells), indicating Cre-recombinase was limited to neurons. Almost all of these cells (1867/1896 = 98.5%) also stained with antibodies against ChAT, indicating that reporter label was expressed almost exclusively in cholinergic neurons. Finally, an average 88.7% of the ChAT+ cells in these nuclei were labeled with tdTomato, indicating that the Cre is expressed in a large proportion of the cholinergic cells in these nuclei. We conclude that the backcrossed ChAT mouse line has normal hearing and expresses Cre recombinase almost exclusively in cholinergic neurons. This ChAT mouse line may provide an opportunity to manipulate cholinergic circuits without the confound of accelerated hearing loss associated with the C57BL/6J background. Furthermore, comparison with lines that do show early hearing loss may provide insight into possible cholinergic roles in age-related hearing loss.