Phosphorylation of cysteine string protein-alpha up-regulates the frequency of cholinergic waves via starburst amacrine cells

During the first postnatal week in rodents, cholinergic retinal waves initiate in starburst amacrine cells (SACs), propagating to retinal ganglion cells (RGCs) and visual centers, essential for visual circuit refinement. By modulating exocytosis in SACs, dynamic changes in the protein kinase A (PKA) activity can regulate the spatiotemporal patterns of cholinergic waves. Previously, cysteine string protein-alpha (CSP alpha) is found to interact with the core exocytotic machinery by PKA-mediated phosphorylation at serine 10 (S10). However, whether PKA-mediated CSP alpha phosphorylation may regulate cholinergic waves via SACs remains unknown. Here, we examined how CSP alpha phosphorylation in SACs regulates cholinergic waves. First, we identified that CSP alpha 1 is the major isoform in developing rat SACs and the inner plexiform layer during the first postnatal week. Using SAC-specific expression, we found that the CSP alpha 1-PKA-phosphodeficient mutant (CSP-S10A) decreased wave frequency, but did not alter the wave spatial correlation compared to control, wild-type CSP alpha 1 (CSP-WT), or two PKA-phosphomimetic mutants (CSP-S10D and CSP-S10E). These suggest that CSP alpha-S10 phosphodeficiency in SACs dampens the frequency of cholinergic waves. Moreover, the level of phospho-PKA substrates was significantly reduced in SACs overexpressing CSP-S10A compared to control or CSP-WT, suggesting that the dampened wave frequency is correlated with the decreased PKA activity. Further, compared to control or CSP-WT, CSP-S10A in SACs reduced the periodicity of wave-associated postsynaptic currents (PSCs) in neighboring RGCs, suggesting that these RGCs received the weakened synaptic inputs from SACs overexpressing CSP-S10A. Finally, CSP-S10A in SACs decreased the PSC amplitude and the slope to peak PSC compared to control or CSP-WT, suggesting that CSP alpha-S10 phosphodeficiency may dampen the speed of the SAC-RGC transmission. Thus, via PKA-mediated phosphorylation, CSP alpha in SACs may facilitate the SAC-RGC transmission, contributing to the robust frequency of cholinergic waves.