Unravelling the molecular players at the cholinergic efferent synapse of the zebrafish lateral line

Abstract

<jats:title>Abstract</jats:title><jats:p>The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness to external stimuli is modulated by descending efferent neurons. LL efferent modulation aids the animal to distinguish between external and self-generated stimuli, maintaining sensitivity to relevant cues. One of the main components of the efferent system is cholinergic, the activation of which inhibits afferent activity. Since LL hair cells (HC) share structural, functional and molecular similarities with those of the cochlea, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic cholinergic one (nAChR). However, the identity of the molecular players underlying acetylcholine (ACh)-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression data and <jats:italic>in situ</jats:italic> hybridization, we describe that α9, but not α10 subunits, are enriched in zebrafish HC. Moreover, the heterologous expression of zebrafish α9 subunits indicates that α9 homomeric receptors are functional and exhibit robust ACh-gated currents which are blocked by α-Bungarotoxin (α-Btx). In addition, <jats:italic>in vivo</jats:italic> Ca<jats:sup>2+</jats:sup> imaging on mechanically-stimulated zebrafish LL HC showed that ACh elicits a decrease in evoked Ca<jats:sup>2+</jats:sup> signals, irrespective of HC polarity. This effect was blocked by both α-Btx and apamin, indicating coupling of ACh mediated effects to SK potassium channels. Collectively, our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by the ACh-dependent activation of Ca<jats:sup>2+</jats:sup>-dependent SK potassium channels.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p>Fishes and amphibians have a mechanosensory system, the lateral line (LL), which serves to detect hydromechanical variations around the animal’s body. The LL receives descending efferent innervation from the brain that modulates its responsiveness to external stimuli. LL efferent inhibition is mediated by ACh, however the identity of the molecular players at the LL efferent synapse is unknown. Here we demonstrate that a nicotinic cholinergic receptor (nAChR) composed of α9 subunits operates at the LL efferent synapse. Activation of α9-containing (α9*) nAChRs leads to LL hair cell hyperpolarization. The inhibitory signature of this process is brought about by the subsequent activation of Ca<jats:sup>2+</jats:sup>-dependent potassium SK channels, functionally coupled to α9* nAChRs.</jats:p></jats:sec>

Using light-sheet microscopy to study spontaneous activity in the developing lateral-line system

“…Using this approach, we found that in developing neuromast organs, without any external stimuli, the efferent terminals contacting hair cells were spontaneously active (examples, day3, To further examine the role of cholinergic efferents in hair cell spontaneous activity, we took a pharmacological approach. Cholinergic efferents share a molecular mechanism that is conserved among vertebrate hair cell systems (Glowatzki and Fuchs, 2000;Hiel et al, 2000;Parks et al, 2017;Carpaneto Freixas et al, 2021). Acetylcholine released from these efferents acts on α9 or α10 nicotinic acetylcholine receptors (nAChR), which allows calcium to enter near the hair cell presynapse.…”

“…To determine if this cascade of events impacts hair cell spontaneous activity, we applied α-bungarotoxin (α-Btx) and apamin, compounds that block α9 nAChRs and SK channels respectively. Recent work has shown that these compounds are effective at disrupting efferent neurotransmission onto lateral-line hair cells (Carpaneto Freixas et al, 2021). Because cholinergic signaling is thought to impact the hair cell presynapse, for our analyses we used a membrane-localized indicator memGCaMP6s which we have used previously to specifically measure spontaneous presynaptic calcium activity (Hiutung et al, 2019).…”