The Effects of Phosphorylation of Cysteine String Protein on Retinal Waves
Date Issued
2014
Date
2014
Author(s)
Chen, Ching-Feng
Abstract
During a developmental critical period, the visual system displays a robust spontaneous activity termed “retinal waves”, essential for neural circuit refinement. These waves are initiated by releasing neurotransmitters from presynaptic starburst amacrine cells (SACs) onto postsynaptic retinal ganglion cells (RGCs). Previous studies showed that the developing SACs can periodically fire action potentials and undergo Ca2+-regulated exocytosis, thus releasing excitatory neurotransmitters such as acetylcholine (ACh) and γ-amino butyric acid (GABA) and inducing periodic retinal waves. However, little is known regarding how altering the Ca2+-regulated exocytosis in SACs affects the spatial or temporal properties of retinal waves.
Cysteine string protein (CSP) was found to ensure the correct folding of fusion machinery [i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins], thus playing an important role in regulating neurotransmitter release. In addition, CSP can be phosphorylated by protein kinase A (PKA) that is highly activated during retinal waves. These results suggest that the intracellular signaling in SACs may modulate the function of CSP and thus regulate neurotransmitter release during retinal waves.
In this study, we investigated how phosphorylation of CSP affects the spatial or temporal properties of retinal waves. First, we used immunofluorescence staining to show that CSP was expressed in the inner plexiform layer (IPL) of the neonatal rat retina. Further immunostaining of dissociated retinal cells confirmed that the expression of CSP was localized to presynaptic SACs, implying that CSP may involve in regulating neurotransmitter release from SACs. The quantitative polymerase chain reaction (qPCR) experiment showed that CSPα was the dominant isoform in the developing rat retina. To investigate whether phosphorylation of CSPα in presynaptic SACs affects retinal waves, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter (pmGluR2). After overexpression of CSP or its phosphomutants in SACs, subsequent Ca2+ imaging was performed in the RGC layer to detect the spatial and temporal properties of wave-associated spontaneous Ca2+ transients. We found that the frequency of Ca2+ transients was significantly decreased by the phosphodeficient mutant (CSPα-S10A), but not by the wild-type CSPα (CSPα-WT) or the phosphomimetic mutants (CSPα-S10D and CSPα-S10E) compared to the control. In contrast, the CSPα phosphodeficient mutant had a relatively minor effect on the spatial correlation of spontaneous Ca2+ transients over distance. Whole-cell voltage-clamp recordings demonstrated that the CSPα phosphodeficient mutant reduced the frequency and the slope of wave-associated postsynaptic currents (PSCs), but not altered the electrical properties of postsynaptic RGCs, suggesting that the effects by overexpressing the CSPα phosphodeficient mutant were due to the change in the release from presynaptic SACs. Given that the CSPα phosphodeficient mutant down-regulates the frequency but does not significantly alter the spatial correlation of retinal waves, our results suggest that phosphorylation of CSPα may play an important role in regulating the temporal properties of retinal waves.
Subjects
半胱胺酸串鍊蛋白
視網膜波
星狀無軸突細胞
視網膜節細胞
鈣離子顯像技術
Type
thesis
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