Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line

Abstract

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 in situ 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, in vivo Ca 2+ imaging on mechanically-stimulated zebrafish LL HC showed that ACh elicits a decrease in evoked Ca 2+ 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 2+ -dependent SK potassium channels. Significance StatementFishes 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 .

Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish

“…While our data do not directly support this idea, we speculate that reduced glutamatergic transmission in mechanically overstimulated neuromasts may be a consequence of damaged hair bundles and impaired mechanotransduction (Zhang et al, 2018). Alternatively, cholinergic efferent feedback, which has been shown to hyperpolarize lateral-line hair cells, may reduce hair-cell excitability during sustained strong current exposure to protect against excess glutamate release, excitotoxic damage, and synapse loss (Carpaneto Freixas et al, 2021). Interestingly, presynaptic ribbons were not similarly enlarged, but instead showed a modest reduction in size following mechanical injury.…”

Functional and ultrastructural analysis of reafferent mechanosensation in larval zebrafish

“…Our experiments using knockout larvae establish that the α9 cholinergic receptor subunit, which is also conserved in the hair cells of the mammalian inner ear, is pivotal for the suppression of reafferent mechanosensation in the neuromast. Recent work has determined that α9 subunits are expressed in zebrafish neuromasts, and that these can form functional homomeric acetylcholine-sensitive receptors in heterologous preparations (Carpaneto Freixas et al, 2021). Furthermore, using calcium imaging, this study showed that application of acetylcholine reduces calcium signals evoked by hair cell stimulation, and that this effect is blocked by apamin, a selective blocker of small-conductance calcium-activated potassium (SK) channels.…”

“…One originating from the cholinergic Octavolateral Efferent Nucleus (OEN) in the hindbrain and the other from the Dopaminergic Efferent to the Lateral Line (DELL) in the ventral hypothalamus (Bricaud et al, 2001; Metcalfe et al, 1985). Further, acetylcholine has been shown to inhibit hair cell activity (Carpaneto Freixas et al, 2021; Dawkins et al, 2005), making the OEN the most likely source of reafferent suppression. At the synaptic level, ideas about how acetylcholine may inhibit hair cells come from work done in the mammalian inner ear.…”

“…This inhibitory effect, however, is less related to reafferent suppression and is thought to have a protective function when animals are exposed to acoustic hyperstimulation that can induce damage due to excitotoxicity (Schneider et al, 2014, 2018; Taranda et al, 2009). Interestingly, enriched expression of the α9, but not the α10 subunits, have been observed in the hair cells of the lateral line of larval zebrafish (Carpaneto Freixas et al, 2021; Erickson and Nicolson, 2015), but whether they play a specific role in protective silencing, context-dependent modulation or treafferent suppression has not yet been determined. Nonetheless, there is now significant evidence for the role of cholinergic efferents in reafferent suppression, and their mechanism has been hypothesized to the level of individual receptor subtypes.…”

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells

“…Fish, for example, have hair cells arranged in a mechanosensory lateral line organ that is used, in part, to avoid capture. Recently it was demonstrated that these lateral line hair cells utilize α9 nAChRs at the efferent synapse to mediate ACh neurotransmission ( Carpaneto Freixas et al, 2021 ).…”