Endocytosis and Vesicle Recycling at a Ribbon Synapse

Abstract

At ribbon synapses, where exocytosis is regulated by graded depolarization, vesicles can fuse very rapidly with the plasma membrane (complete discharge of the releasable pool in approximately 200 msec). Vesicles are also retrieved very rapidly (time constant of approximately 1 sec), leading us to wonder whether their retrieval uses an unusual mechanism. To study this, we exposed isolated bipolar neurons from goldfish retina to cationized ferritin. This electron-dense marker uniformly decorated the cell membrane and was carried into the cell during membrane retrieval. Endocytosis was activity-dependent and restricted to the synaptic terminal. The labeling pattern was consistent with direct retrieval from the plasma membrane of large, uncoated endosomes 60-200 nm in diameter. Even after extensive synaptic activity lasting several minutes, most of the ferritin remained in large endosomes and was present in only approximately 10% of the small vesicles that constitute the reserve pool. By contrast, after brief stimulation at a conventional terminal, ferritin did not reside in endosomes but was present in approximately 63% of the small vesicles. We suggest that the bipolar ribbon synapse sustains its rapid exocytosis by retrieving membrane in larger "bites" than the clathrin-dependent mechanism thought to dominate at conventional synapses. The resulting large endosomes bud off small vesicles, which reenter the reserve pool and finally the releasable pool.

Synaptic transmission at retinal ribbon synapses

“…Indeed, both evagination with increasing stimulation intensity and the placement of the next vesicle at the crest have been reported in lateral line ribbon synapses (Fields and Ellisman, 1985). Moreover, the relatively large, tubular endocytic figures observed after strong depolarization could represent the retrieval of several vesicles' worth of membrane (Paillart et al, 2003;Royle and Lagnado, 2003), such as might occur from retrieving several vesicles following compound fusion (although other interpretations are possible). Alternatively, but not exclusively, the global calcium stimulus provided by calcium uncaging might trigger the fusion of numerous vesicles outside the active zone.…”

“…The significance of these species differences is unclear because, on the one hand, the immunolabeling studies in nonmammalian vertebrates were performed using antibodies to mammalian isoforms and, on the other hand, little is known about the calcium dependence of release at mammalian retinal ribbon synapses. A straightforward interpretation of isoform expression and the calcium sensitivity of release may be further complicated by the putative role of synaptotagmin in endocytosis and the different modes of endocytosis that a cell may preferentially utilize (Llinas et al, 2004;Holt et al, 2003;Paillart et al, 2003).…”

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

“…In this way, functional vesicle pools that were previously labelled with FM-dye can be directly identified in electron micrographs. This approach has been used extensively and highly successfully in cultured neurons 7,10,24,28,30,[32][33][34] , a number of large, peripheral terminals [26][27][28][29]31,[35][36][37] , and large central release sites such as calyx of Held 25 revealing important information about organizational principles of vesicle pools. However, only recently 38 has this method been successfully applied to small central synapses in native brain tissue, where neurons are retained in relevant circuits with defined cytoarchitecture.…”

Bulk endocytosis at neuronal synapses

“…Interestingly, when preparations were rested after 15 min stimulation, the cisternae disappeared and SVs reappeared, suggesting the SVs could have derived from the cisternae [1]. Since this initial seminal study, a considerable number of studies have demonstrated that ADBE also occurs in rat hippocampal [6,17] and cerebellar granule [18] neurons, lamprey reticulospinal synapses [5], mouse calyx of Held [19] and auditory inner hair cells [20], snake [21] and Caenorhabditis elegans motor terminals [22], and goldfish bipolar neurons [8].…”