Imaging calcium entry sites and ribbon structures in two presynaptic cells

Area of Science:

• Neuroscience
• Cell Biology
• Biophysics

Background:

• Synaptic transmission relies on calcium influx and synaptic vesicles.
• The precise localization of calcium channels relative to synaptic ribbons is crucial for understanding neurotransmitter release.
• Previous studies suggest active zones are the primary sites for calcium influx and vesicle fusion.

Purpose of the Study:

• To investigate the spatial relationship between calcium entry sites and synaptic ribbons in goldfish bipolar neurons and bullfrog saccular hair cells.
• To determine if calcium channels cluster at active zones near synaptic ribbons.
• To explore the role of active zones in exocytosis.

Main Methods:

• Cells were loaded with calcium indicators (Fluo-3 or Fluo-5F) and a calcium buffer (EGTA).
• Evanescent field microscopy was used to image the cell surface and identify calcium entry sites as fluorescent 'hot spots'.
• Immunofluorescence with an antibody against ribeye labeled synaptic ribbons.

Main Results:

• Calcium entry sites appeared as transient fluorescent hot spots, correlating with calcium channel activity.
• In both cell types, the number of calcium entry sites closely matched the number of synaptic ribbons.
• In bipolar cells, active zones, calcium entry sites, and synaptic ribbons colocalized, but vesicle fusion also occurred outside active zones.

Conclusions:

• Calcium channel clusters are located near synaptic ribbons, defining active zones in both bipolar neurons and hair cells.
• While active zones are primary sites for calcium influx and ribbon-associated exocytosis, additional vesicle fusion events can occur independently of these structures in bipolar terminals.