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.

Figure 1.

After calcium-triggered exocytosis, plasma membrane was retrieved into large endosomes. A, The external surface of isolated bipolar terminal was decorated with cationic ferritin. The terminal fired spontaneous calcium spikes for several minutes (overlay) and was then fixed. Ferritin was internalized in numerous endosomes (circled). The bracketed endosome is shown in the inset with additional magnification (1.7×) to better demonstrate the ferritin labeling. B, Similar decoration of plasma membrane, but calcium spiking was suppressed by muscimol (overlay). The uptake of ferritin was sparse.

Figure 2.

Endocytosis was restricted to the synaptic terminal. A, Whole bipolar cell; boxed regions are magnified in B–D. B, C, The soma and axon were devoid of ferritin. Note the smooth endoplasmic reticulum (ser) associated with the plasma membrane. mt, Microtubule; mit, mitochondrion; glyc, glycogen. D, The synaptic terminal contained numerous labeled endosomes (circled), mostly larger than unlabeled synaptic vesicles.

Figure 3.

Some labeled endosomes resemble synaptic vesicles, whereas others are much larger. A, Arrowheads mark a selection of small labeled vesicles; arrows mark selected larger labeled endosomes. B, Histograms compare the single distributions of labeled and unlabeled membrane structures. The cell fired spontaneously for 11 min before fixation.

Figure 4.

Labeled synaptic vesicles return to the ribbon. A, A labeled endosome associates with a ribbon and appears to bud off a small, labeled vesicle (arrows). This section does not show the attachment of the ribbon to the plasma membrane. B, Two vesicles containing ferritin (arrows) tether to a ribbon.

Figure 8.

Weak stimulation at a conventional synapse (amacrine cell) labeled most of the reserve pool of synaptic vesicles. A, Amacrine varicosity adhered to a bipolar terminal (BT) and was decorated by ferritin, even in the synaptic cleft. The terminals were stimulated twice by brief, high-potassium depolarization and fixed 180 sec after the first stimulus. In the bipolar terminal, labeling was sparse, whereas in the amacrine varicosity (AV), labeling was present in nearly two-thirds of the vesicles. B, In the amacrine varicosity, labeled and unlabeled vesicles show the same size distribution.

Figure 5.

Endosomes forming at the plasma membrane are several times larger than a synaptic vesicle. Three sections from a series are shown, each ∼80 nm thick, with single intervening sections omitted. Section 3 shows an endosome with a neck still attached to the plasma membrane (left arrow), which is absent from sections 1 and 5, implying that the endosome must be < 160 nm in diameter. The right arrow shows a presumed earlier stage of budding.

Figure 6.

After brief depolarization (elevated potassium), ferritin-labeled endosomes were near the plasma membrane. A, This cell was exposed to high potassium (high-K) and fixed after 30 sec (fix). Labeling was sparser than after prolonged activity (Figs. 1,3) and remained near the plasma membrane. B, Labeled structures were mostly larger than synaptic vesicles. This histogram represents summed data from three synaptic terminals fixed after brief depolarization.

Figure 7.

After prolonged spiking, labeled endosomes move from the plasma membrane to distribute throughout the terminal. A, Diagram summarizes the distribution of labeled endosomes reconstructed from eight serial sections in a bipolar terminal that fired calcium spikes for 11 min before fixation. Ferritin concentrated in large endosomes (up to 1800 particles), which moved from the plasma membrane toward the cell interior. Some large, heavily labeled endosomes were associated with synaptic ribbons. B, In six experiments, ferritin increased with spiking, and endosomes gradually distributed throughout the terminal. The number of calcium bursts indicates the amount of synaptic activity, and the poststimulus incubation period represents the time from the onset of activity to fixation. The three cells on the left were stimulated by brief applications of high external potassium, and the three on the right were stimulated more extensively by spontaneous calcium action potentials during a more prolonged incubation period. Different shades of gray represent positions within the terminal, as indicated in the inset.