High-Resolution Optical Imaging of Zebrafish Larval Ribbon Synapse Protein RIBEYE, RIM2, and CaV 1.4 by Stimulation Emission Depletion Microscopy

Abstract

The synaptic ribbon is a unique presynaptic structure with an intricate morphology in photoreceptors. Because of the resolution limit in conventional fluorescence microscopy, investigating ribbon protein locations has been challenging, especially in the early development stages of model animals. Here, we used stimulated emission depletion microscopy, a super-resolution imaging technique, to look at retina sections in 4 days post-fertilization (dpf) zebrafish. We observed that in photoreceptor cells, RIBEYE and RIM2 are expressed along the synaptic ribbon, with RIM2 consistently located inside of the horseshoe-shaped synaptic ribbon structure with RIBEYE located on the outside. The L-type calcium channel subunit, CACNA1F, exhibited small spot-like staining beneath the RIM2 and RIBEYE structures. Using morpholino antisense oligonucleotides to knock down RIBEYE expression, we observed fewer and shorter ribbons in the photoreceptor outer plexiform layers of 4 dpf fish retina as well as a reduction in RIM2 expression. The clustering of CACNA1F in these blind fish was no longer observed, but instead showed a diffuse expression in the photoreceptor terminal.

Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels

“…More recently, knockout of Ribeye in mouse was found to cause a subtle change in the size of calcium channel clusters in photoreceptors (Maxeiner et al, 2016). Notably, in contrast to the previous MO results in retina (Lv et al, 2012), calcium channels remained clustered in discrete locations in the retina. Additionally, knockout mice were found to exhibit an increase in sensitivity of mEPSC frequency to high levels of the membrane permeant analog of EGTA, EGTA-AM.…”

“…A link between Ribeye and calcium channels has previously been inferred from overexpression and knock-down studies in zebrafish. Morpholino oligonucleotide (MO) driven knock-down of ribeye leads to loss of calcium channel clusters in both retina (Lv et al, 2012) and neuromast hair cells (Sheets et al, 2012), whereas animals overexpressing Ribeye exhibit ectopic calcium channel clusters that colocalize with Ribeye protein aggregates (Sheets et al, 2012). More recently, knockout of Ribeye in mouse was found to cause a subtle change in the size of calcium channel clusters in photoreceptors (Maxeiner et al, 2016).…”

“…Ribeye can be subdivided into two domains, a ribbon-specific A-domain and a B-domain, which is nearly identical to CtBP2 (Schmitz et al, 2000). Genetic deletion of mouse Ribeye leads to a loss of synaptic ribbons in the retina, which is accompanied by a reduction in synaptic transmission from bipolar cells without observable changes in the kinetic features of release (Maxeiner et al, 2016), whereas morpholino oligonucleotide driven knockdown of Ribeye expression leads to the mislocalization of calcium channels (Sheets et al, 2011, Lv et al, 2012) and a reduction of spiking rates in afferent neurons postsynaptic to zebrafish hair cells (Sheets et al, 2011). …”

Visualizing synaptic vesicle turnover and pool refilling driven by calcium nanodomains at presynaptic active zones of ribbon synapses

“…During brief depolarization, Ca 2+ rose more rapidly and to higher levels at ribbon locations compared with nearby regions lacking a ribbon, demonstrating that Ca 2+ channels cluster near the ribbon. This finding is consistent with other types of evidence showing a high density of Ca 2+ channels near ribbon-type active zones (18)(19)(20)(21)(22)(23)(24)(25). Furthermore, even brief depolarization (3 ms) was sufficient to locally saturate the indicator dye in a diffraction-limited region near the plasma membrane.…”

“…The concentration of Ca 2+ at the ribbon active zone should rise rapidly to high levels to drive fusion of synaptic vesicles at the speed achieved during the ultrafast component. A variety of lines of evidence indicate that Ca 2+ channels cluster in the plasma membrane at ribbon locations in bipolar cell synaptic terminals (17)(18)(19)(20)(21), and Ca 2+ influx has been shown to occur preferentially at ribbons (19,22). Therefore, nanodomains of high Ca 2+ at the ribbon are likely to be involved in triggering vesicle fusion (19,(22)(23)(24)(25), a conclusion also supported by blockade of the fast component by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) but not EGTA (4).…”