![]() The synaptic terminal was patch clamped by using a whole-cell pipette filled with a solution containing fluorescent RBP to label synaptic ribbons (red). (A) Retinal bipolar cells were isolated from transgenic zebrafish that highly express SypHy. SypHy fusion events in response to depolarization stimuli Thick-walled borosilicate glass tubing (outer diameter: 1.5 mm, inner diameter: 0.86 mm)Ġ.5-ml Non-Stick RNase-Free microfuge tubesīubbler and lighting for zebrafish satellite housing Silicon oil-immersion objective lens (magnification 60×) Zebrafish: Transgenic zebrafish that express SypHy (synaptophysin-pHluorin fusion protein) under control of heat-shock promoter (adult, male or female) All animal studies were approved by the University of Tennessee Health Science Center (UTHSC) Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the guidelines.Ĭhemicals, peptides, and recombinant proteins (Enzymes)ģ-aminobenzoic acid ethyl ester methanesulfonate (Tricaine) The protocol is divided into the following sections, based on the temporal order (timing) of the experimental procedures. When combined with high spatiotemporal-resolution imaging and labeling of the synaptic ribbon with RBP to provide a roadmap to the specific location of fusion with respect to the CAZ, this method allows us to determine the kinetics of synaptophysin protein clearance from a single synaptic vesicle within the ribbon active zone. ![]() The sparse expression of SypHy in this line allows us to detect single fusion events in situ. Instead, we have developed the novel and unique technique described here, which is an assay based on our recently established transgenic zebrafish line that weakly expresses the exocytosis reporter, synaptophysin-pHluorin fusion protein (SypHy) ( Vaithianathan et al., 2016, 2019). However, because FM dye labels multiple vesicles and does not serve as a reporter of exocytosis, this procedure cannot be used to track the clearance of fused synaptic vesicles. The conventional procedure involves labeling of synaptic vesicles with FM dye and imaging them using total internal reflection fluorescence microscopy (TIRFM). However, monitoring the trafficking, fusion, and clearance of synaptic vesicles remains technically challenging. A well-established method for localizing synaptic ribbons is to label them with fluorescent ribeye-binding peptide (RBP) ( Vaithianathan et al., 2016, 2019 Zenisek et al., 2004). Synapses of retinal bipolar cells (BPCs) and other sensory neurons that release neurotransmitter continuously in response to graded changes in membrane potential rely on the proper function of a specialized organelle, the synaptic ribbon, which is a complex molecular structure that tethers synaptic vesicles to the cytomatrix at the active zone (CAZ) ( Matthews and Fuchs, 2010 Moser et al., 2020 Schmitz et al., 2000). An ideal approach for investigating the dynamics of nanodomain synaptic vesicles is the simultaneous labeling of synaptic vesicles and active zone-specific proteins in the living synapse that can be stimulated to release using physiological relevant stimuli and visualized by high spatiotemporal resolution imaging ( Vaithianathan et al., 2016). Due to technical constraints, the kinetics for clearance of newly fused synaptic vesicle proteins from the active zone have remained unclear. For synapses that transmit tonically, the limiting factor for sustained transmission is the availability of fusion sites to which synaptic vesicles can dock rather than the sheer number of synaptic vesicles ( Neher, 2010). Synapses of visual and auditory sensory neurons are specialized functionally and morphologically to faithfully encode a wide dynamic range of signals, while enhancing the detection of spatial or temporal changes in stimulus intensity ( Matthews and Fuchs, 2010 Moser et al., 2020).
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