Synaptotagmin isoforms 在發育中大鼠視網膜的時空表現模式

Abstract

Synaptotagmin (Syt) constitutes a large protein family with at least seventeen isoforms, many of which serve as calcium sensors and regulate neurotransmitter releases through calcium-regulated exocytosis. In early developing rat retina, the patterned spontaneous activities (retinal waves) play an important role in regulating eye-specific segregation of retinal inputs in the dorsal lateral geniculate nucleus (dLGN). However, the underlying mechanism is still unclear. Since the retinal waves are driven by synaptic transmission, we then think Syt be involved in regulating retinal waves and/or eye-specific segregation of retinal ganglion cell (RGC) axons. In this study, we investigated the expression profiles of Syt isoforms in the developing rat retina since the lack of information about the spatiotemporal expression patterns of Syt isoforms. First, we applied the single-cell RT-qPCR (sc-RT-qPCR) to screen the expression of Syt I-IV transcripts in developing retinal ganglion cells (RGCs), the retinal output neurons projecting to central brain targets. We found that Syt III mRNA can be detected in the RGCs from the postnatal day 4 to 8 (P4-P8), which is the critical period for eye-specific segregation of the retinotopic map. In contrast, Syt I and IV mRNAs were consistently expressed in the RGCs from P2 to P9. Syt II mRNA was not detectable in the RGCs from P2 to P9, which may not be involved in RGCs development. Furthermore, by qPCR from the whole retinal lysates, we detected an increasing mRNA expression level for both Syt I and Syt III from P0 to P11. The different expression profiles of Syt III mRNA between RGCs and whole retinas suggest different roles of Syt III in output neurons (RGCs) and other types of cells (e.g. interneurons) in the developing retina. Moreover, from the P0-P11 rat retinal cDNAs, we identified that Syt III harbored S287 and R448, distinct from the previous reports (T287 and L448). Second, by immunofluorescence stainings, we found that Syt I was mainly expressed in the synaptic layers [both inner plexiform layer (IPL) and outer plexiform layer (OPL)], consistent with its role in synaptic transmission. However, Syt III showed a dot-like expression pattern in the ganglion cell layer (GCL), IPL, and neuroblast layer (NBL), implying that Syt III might be also involved in cell differentiation and maturation. Third, Syt III was largely expressed in the Brn3+ and Thy1.1+ RGCs and localized to two vesicles [small vesicles (SVs) and large dense-core vesicles (LDCVs)], early endosome/lysosome compartments and the growth cone. Finally, unlike Syt IV, we found the expression of Syt III was retinal wave activity-independent. To sum up, the unique spatiotemporal expression pattern of Syt III may provide us with some clues to investigate its possible role in the developing rat retina.