Abstract
Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity.
Highlights
At chemical synapses, exocytosis of transmitter-filled synaptic vesicles is followed by compensatory endocytosis to retrieve synaptic vesicle components from the plasma membrane and to restore the cell surface area
DISTRIBUTION OF ENDOCYTOTIC PROTEINS IN THE MOUSE RETINA First, we stained vertical cryostat sections of light adapted C57BL/6JRj (BL/6) retinae for proteins involved in various modes of endocytosis, but for proteins described in the different steps of Clathrin-mediated endocytosis (CME), i.e., nucleation (AP180, Clathrin LC; Figures 1A,D), budding and scission (Endophilin, Amphiphysin, Synaptojanin, Dynamin; Figures 1B,E), and uncoating
Strong staining was detected for Clathrin LC, Endophilin1, Amphiphysin1, and Dynamin3 (Figures 1D,E), weaker staining for AP180 (Figure 1D), Synaptojanin1 (Figure 1E), and hsc70 (Figure 1F)
Summary
Exocytosis of transmitter-filled synaptic vesicles is followed by compensatory endocytosis to retrieve synaptic vesicle components from the plasma membrane and to restore the cell surface area. Multiple pathways contribute to compensatory endocytosis at chemical synapses, including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kissand-run retrieval (reviewed in Dittman and Ryan, 2009; Royle and Lagnado, 2010; Haucke et al, 2011; Saheki and de Camilli, 2012). In contrast to conventional chemical synapses, tonically active ribbon synapses support high and sustained rates of exocytosis (reviewed in Matthews and Fuchs, 2010; Mercer and Thoreson, 2011; Regus-Leidig and Brandstätter, 2012). The ultrastructural hallmark of photoreceptor synapses, the presynaptic ribbon, is an electron-dense plate-like structure which extends several hundred nm into the cytoplasm and tethers a large number of transmitter vesicles via fine filaments (Rao-Mirotznik et al, 1995; Sterling and Matthews, 2005; tom Dieck and Brandstätter, 2006). The exact function of the synaptic ribbon in photoreceptor neurotransmission is not yet fully understood, but it is widely accepted that the synaptic ribbon ensures the precise spatial and temporal control of vesicle fusion, and accommodates the high rates of synaptic transmission (Schmitz, 2009; Regus-Leidig and Brandstätter, 2012)
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