Elucidating the role of muscarinic acetylcholine receptor (mAChR) signaling in efferent mediated responses of vestibular afferents in mammals

Abstract

The peripheral vestibular system detects head position and movement through activation of vestibular hair cells (HCs) in vestibular end organs. HCs transmit this information to the CNS by way of primary vestibular afferent neurons. The CNS, in turn, modulates HCs and afferents via the efferent vestibular system (EVS) through activation of cholinergic signaling mechanisms. In mice, we previously demonstrated that activation of muscarinic acetylcholine receptors (mAChRs), during EVS stimulation, gives rise to a slow excitation that takes seconds to peak and tens of seconds to decay back to baseline. This slow excitation is mimicked by muscarine and ablated by the non-selective mAChR blockers scopolamine, atropine, and glycopyrrolate. While five distinct mAChRs (M1-M5) exist, the subtype(s) driving EVS-mediated slow excitation remain unidentified and details on how these mAChRs alter vestibular function is not well understood. The objective of this study is to characterize which mAChR subtypes drive the EVS-mediated slow excitation, and how their activation impacts vestibular physiology and behavior. In C57Bl/6J mice, M3mAChR antagonists were more potent at blocking slow excitation than M1mAChR antagonists, while M2/M4 blockers were ineffective. While unchanged in M2/M4mAChR double KO mice, EVS-mediated slow excitation in M3 mAChR-KO animals were reduced or absent in irregular afferents but appearing unchanged in regular afferents. In agreement, vestibular sensory-evoked potentials, known to originate from irregular afferents, were much less enhanced by mAChR activation in M3mAChR-KO mice compared to controls. Finally, M3mAChR-KO mice display distinct behavioral phenotypes in open field activity, and thermal profiles, and balance beam and forced swim test. M3mAChRs mediate efferent-mediated slow excitation in irregular afferents, while M1mAChRs may drive the same process in regular afferents.

KCNQ2/3 regulates efferent mediated slow excitation of vestibular afferents in mammals

“…We measured VsEP waveforms at +6 dB re: 1.0 g/ms before and aser the IP administraPon of 2mM Oxo-M. Consistent with previous findings (Sinha et al, 2023), we observed an increase in P1N1 amplitude over Pme in both control and KCNQ3 KO mice following Oxo-M administraPon (Fig. 7E).…”

“…We next wanted to explore the effect of mAChR activation of on VsEP metrics in KCNQ3 KO mice. Previous research (Sinha et al, 2023) demonstrated that the activation of mAChRs using Oxotremorine-M (Oxo-M), a mAChR agonist, enhanced VsEP P1N1 amplitude and reduced P1 latency in control mice (Sinha et al, 2023). Here, we repeated this experiment in KCNQ3-KO mice.…”

“…In this study, we explored which KCNQ channel subunits might be modulated by electrical stimulation of EVS neurons and can contribute to EVS-mediated slow excitation in mammals. Previous work in turtle and rat suggested that KCNQ channels are the downstream targets of mAChR activation given their sensitivity to the KNCQ blockers, linopirdine and XE991 and lack of effect from the ERG channel blockers, E-4031 (Holt et al, 2017; Perez et al, 2009; Perez et al, 2010; Ramakrishna et al, 2021; Sinha et al, 2023). In this paper, we confirmed a similar role for KCNQ channels in our in vivo mouse preparation.…”

“…In Figure 6, UCL2077 was administered through IB route in 100% DMSO. 100% DMSO non-significantly enhances baseline (Sinha et al, 2023). Systemic administration of DMSO did not affect vestibular afferent firing when injected at volume less that 100ul (Lee et al, 2017).…”

“…Values, expressed as means ± SEM, and outcome parameters including p values are reported in the text and figures. For figure 5 and 6 control data from (Sinha et al, 2023) was reused (n=5 animals) for discharge regularity and EVS-mediated slow excitation. Additionally, four (n=4) animals were recorded simultaneously with the KCNQ3 KO mice.…”