Calcium binding to Synaptotagmin III regulates patterned spontaneous activity and visual circuit development

Patterned spontaneous activity in developing retina, termed retinal waves, appears during the critical period of visual circuit refinement. Stage II retinal waves, critical for establishing the eye-specific segregation of retinogeniculate and retinocollicular projections, are mediated by neurotransmitter release from starburst amacrine cells (SACs) via Ca2+-dependent exocytosis, affecting neighboring SACs and retinal ganglion cells (RGCs). We previously found that the expression of synaptotagmin III (Syt III), a Ca2+ sensor protein in vesicle release, is upregulated in rat RGCs and optic nerves during P4-P6 (P4-P8 in rodents as the critical period for eye-specific segregation). Moreover, Syt III regulates the kinetics of Ca2+-dependent exocytosis through Ca2+ binding to the C2A and C2B domains. However, how and why Ca2+ binding to Syt III regulates stage II waves remain unknown. In this study, we overexpressed Syt III or Syt III-C2AB* (a mutant harboring the abolished Ca2+-binding sites) in SACs or RGCs by the cell-type specific promoters, and further performed live Ca2+ imaging to measure the subsequent changes on wave properties. First, we found that overexpressing Syt III-C2AB* in SACs decreased the wave frequency compared to Syt III. Moreover, in neonatal RGCs, Syt III dramatically increased the frequency and decreased the spatial correlation of retinal waves, but Syt III-C2AB* did not. These results suggest that Syt III in SACs or RGCs can regulate the kinetics of retinal waves through Ca2+ binding to its C2AB domains. However, Syt III in RGCs plays a relatively profound role in wave regulation compared to Syt III in SACs. Previous studies showed that RGCs are the source of glutamate release in developing retinas. To further investigate how Syt III in RGCs modulates stage-II waves, we applied ionotropic glutamate receptor antagonists and found that the frequency of wave-associated Ca2+ transients was decreased to the same level in retinas overexpressing control, Syt III, or Syt III-C2AB*. Therefore, Syt III in developing RGCs can promote glutamatergic transmission between RGCs and SACs, thus affecting the wave properties. Finally, by using in vivo electroporation, we transfected P3 rat RGCs with Syt III and found the aberrant pattern in ipsilateral retinogeniculate projections. Together, our results suggest that Syt III in developing RGCs plays an important role in regulating patterned spontaneous activity and visual circuit development during a critical development period.