Zebrafish hair cells and ribbons synapses. (A) Schematic depicts a larval zebrafish. Pink patches outline the location of hair cells in the inner ear required for hearing and balance, as well as hair cells in the lateral-line system. Green patches represent the location of the anterior and posterior lateral-line ganglia. The cell bodies of neurons in these ganglia project to and innervate hair cells in the lateral line. (B) An overview of the anatomy of a single patch of hair cells in the lateral line, referred to as a neuromast. Hair cells (pink) are surrounded by supporting cells (internal, blue and peripheral, orange) and innervated by both afferent (green) and efferent neurons (yellow). Mechanosensory hair bundles (purple) at the apex of hair cells project out into the water to detect local water flow. (C) Diagram of a single hair cell. Hair cells are activated when hair bundles are deflected, for example by local water flow. This apical deflection opens mechanosensitive channels allowing in cations including potassium and calcium. This apical activity depolarizes the hair cell, resulting in presynaptic calcium influx and release of glutamate onto the afferent neuron. Inset: magnified view of a hair-cell ribbon synapse. Shown are key evolutionarily conserved synaptic proteins discussed in this review. In hair cells, a presynaptic density called a ribbon (red) helps to recruit synaptic vesicles (white circles) to the synapse near clusters of calcium channels (Ca_V1.3). The ribbon is made up primarily of the protein Ribeye. Slc17A8 (Vglut3) and DMXL2 (Rbc3a) colocalize in or near synaptic vesicles. Synaptojanin and Otoferlin are also critical for ribbon-synapse function although their precise localization has not been definitively shown.

Morphological examination of hair-cell synapses in zebrafish. (A) Classically, transmission electron microscopy (TEM) has been used to visualize hair-cell synapses. Shown is a micrograph of a hair-cell synapse from a zebrafish lateral-line hair cell. In this micrograph, the presynaptic ribbon is a dark spherical density. Surrounding the presynaptic ribbon are synaptic vesicles (SV). Beneath the presynaptic ribbon along the plasma membrane is the postsynaptic density (PSD). (B) The neurod:GFP transgene (green) can be used to label the afferent neurons innervating lateral line (shown in A,B), as well as afferents that innervate inner-ear hair cells. Afferent fibers can be labeled with the commercial antibody HNK-1/Zn12 (pink). (C)Neurod:GFP (green) can be co-labeled with a Synaptophysin antibody (pink) to label both afferent fibers and all efferent synapses respectively. (D) Efferent synapses, which can also be labeled with a Vamp2 antibody (pink), can be further sub-classified by a co-label such a tyrosine hydroxylase (TH, green; white overlap indicates dopaminergic synapses). (E) Pre- and post-synaptic densities can be labeled with CtBP (pink) and pan-MAGUK (green) antibodies respectively. (F) Synaptic vesicles, labeled with cysteine string protein (CSP, pink) are enriched at the basolateral membrane of hair cells near synaptic ribbons labeled with Ribeye antibody (green). Scale bar = 100 nm in (A) and 5 μm in (E) (for B–E) and (F).

Functional analysis of hair-cell synapses in the zebrafish lateral line. (A) In the lateral line, rapid uptake of the vital dye FM 1-43 (cyan), shown in this example, indicates mechanotransduction is intact in these hair cells. (B–D) Extracellular recordings from the afferent cell bodies in the posterior lateral-line ganglia can be used as a read-out of synapse function. During these afferent recordings, evoked spikes can be detected when innervated hair cells are stimulated along their axis of sensitivity (B,C). Step stimuli (B, anterior step shown) are useful to quantify spike number and the timing of the first spike. Sine stimuli (C, anterior-posterior sine stimulus shown) are useful to quantify the precision of spike timing within the waveform. Note that each afferent only responds to one direction of stimuli (for example the anterior but not posterior phase of the sine wave in C). Even in the absence of stimuli there is spontaneous spiking in that can be used as a read-out of synaptic function (D). (E,E’) A transgenic line expressing a membrane-localized calcium indicator GCaMP6s (cyan) can be used to detect mechanotransduction-dependent calcium influx in apical hair bundles (E) and calcium influx at synaptic ribbons (E’) when apical and basal planes are imaged respectively. (F) Transgenic fish expressing SypHy, an indicator of vesicle fusion can be used to detect presynaptic vesicle fusion at hair-cell synapses. (G) GCaMP6s can also be used to detect postsynaptic calcium activities in the afferent process beneath hair cells. All of the transgenic approaches outlined in (E–G) can be used in combination with a transgenic line that marks synaptic ribbons via a Ribeye b-mCherry fusion protein. The scale bar in (A,G) = 5 μm.