Generation and Characterization of α9 and α10 Nicotinic Acetylcholine Receptor Subunit Knockout Mice on a C57BL/6J Background

Abstract

We generated constitutive knockout mouse models for the α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits by derivation from conditional knockouts by breeding with CRE deleter mice. We then backcrossed them onto a C57BL/6J genetic background. In this manuscript, we report the generation of the strains and an auditory phenotypic characterization of the constitutive α9 and α10 knockouts and a double α9α10 constitutive knockout. Although the α9 and α10 nAChR subunits are relevant to a number of physiological measures, we chose to characterize the mouse with auditory studies to compare them to existing but different α9 and α10 nAChR knockouts (KOs). Auditory brainstem response (ABR) measurements and distortion product otoacoustic emissions (DPOAEs) showed that all constitutive mouse strains had normal hearing. DPOAEs with contralateral noise (efferent adaptation measurements), however, showed that efferent strength was significantly reduced after deletion of both the α9 and α10 subunits, in comparison to wildtype controls. Animals tested were 3–8 weeks of age and efferent strength was not correlated with age. Confocal studies of single and double constitutive KOs showed that all KOs had abnormal efferent innervation of cochlear hair cells. The morphological results are similar to those obtained in other strains using constitutive deletion of exon 4 of α9 or α10 nAChR. The results of our physiological studies, however, differ from previous auditory studies using a α9 KO generated by deletion of the exon 4 region and backcrossed onto a mixed CBA/CaJ X 129Sv background.

Efferent feedback enforces bilateral coupling of spontaneous activity in the developing auditory system

“…Note that this observation is based on central responses from the IC using wide-field calcium imaging, which are difficult to detect with the ABR method ( Figure 5C-E). Thresholds determined with the only visible ABR waves I-II are largely normal in the α9 nAChR knockouts compared to controls ( Figure 5A-B), consistent with data from adults (Morley et al, 2017). Our results suggest that central circuits downstream to the CN, rather than the peripheral machinery, are undermined in the knockout, which are in line with previous reports of impaired tonotopic refinement and synaptic functions in the MNTB with either the α9 nAChR knockout (Clause et al, 2014) or the enhanced α9 nAChR knock-in (Di Guilmi et al, 2019).…”

“…Intriguingly, the strength of bilateral coupling matched the time course of a transient cholinergic modulation imposed on inner hair cells by the medial-olivocochlear (MOC) efferent system (Katz et al, 2004, Elgoyhen and Katz, 2012, Kearney et al, 2019, Frank and Goodrich, 2018. Mice lacking the a9/a10 nicotinic acetylcholine receptor (nAChR), a necessary constituent of efferent modulation (Elgoyhen et al, 1994, Elgoyhen et al, 2001, Morley et al, 2017, displayed severly disrupted bilateral correlations. Chemogenetic and pharmacological experiments in vivo, used to acutely manipulate pre-and post-synaptic components of the efferent circuits, indicate that the MOC system was necessary and sufficient to enforce bilateral coupling.…”

“…Although MOC-OHC synapses are already functional at this age (Vattino et al, 2020), the deficit is probably not caused by loss of acute MOC inhibition on the cochlear amplifier. In fact, eliminating the efferent modulation does not change auditory sensitivity as knocking out the α9 nAChR or the coupled SK2 channel does not alter ABR thresholds in mature animals (Ryugo et al, 2011, Morley et al, 2017, while strengthening efferent inhibitions leads to threshold elevation (Taranda et al, 2009). On the other hand, animals should have gained very limited auditory experience at this early age, as auditory thresholds are extremely high before P14.0 (Song et al, 2006) (see also Supplementary Figure 10E).…”

“…In the nervous system, expression of α9/α10 nAChRs is limited to sensory hair cells (Morley et al, 2018). We therefore examined bilateral coupling of spontaneous activity in constitutive α9/α10 knockout mice on the same genetic background (Morley et al, 2017) as the "wildtype" SNAP25-G6s mice. At P6-P7, we saw similar bilateral correlation patterns in the α9/α10 double heterozygous animals ( Figure 2B) as in wild-type mice ( Figure 1J).…”

Deletion of nicotinic acetylcholine receptor alpha9 in mice resulted in altered bone structure

“…No significant differences in ultrastructure were found for α10KO compared to α10WT. Therefore, it would be interesting to investigate the bone status of the recently generated double KO mice of α9α10 nAChR [ 53 ] and bone cell specific KO mice in an up-coming study. One limitation of our study is the usage of constitutive gene KOs that could have systemic impacts, e.g.…”