Ultrafast Exocytosis Elicited by Calcium Current in Synaptic Terminals of Retinal Bipolar Neurons

Abstract

Using high resolution capacitance measurements, we have characterized an ultrafast component of transmitter release in ribbon-type synaptic terminals of retinal bipolar neurons. During depolarization, capacitance increases to a plateau of approximately 30 fF with a time constant of approximately 1.5 ms. When not limited by activation kinetics of calcium current, the small pool is depleted even faster, with a time constant of 0.5 ms. After the ultrafast pool is depleted, capacitance rises with a slower time constant of approximately 300 ms. EGTA (5 mM) depresses the slower capacitance rise but leaves the ultrafast phase intact. BAPTA (5 mM) depresses both components of exocytosis. With paired-pulse stimulation, the ultrafast pool recovers from depletion with a time constant of approximately 4 s. The ultrafast component may represent fusion of docked vesicles at the base of the synaptic ribbon, while the slower component represents more distal vesicles on the ribbon.

Synaptic transmission at retinal ribbon synapses

“…Several additional pieces of evidence support this supposition. First, the fusion of this physiologically defined vesicle pool is not prevented by the addition of millimolar exogenous calcium buffer EGTA to the presynaptic cytosol, consistent with a vesicle location very near to the sites of calcium entry (Mennerick and Matthews, 1996;Sakaba et al, 1997). Secondly, there is an excellent correlation between the total number of vesicles docked at the base of a bipolar cell's synaptic ribbons and in physical contact with the plasma membrane (≈1000-1400, for a small and large terminal respectively) and the number of vesicles that comprise the rapidly releasing pool of vesicles as determined by capacitance measurements (≈1100) ; see also Neves and Lagnado, 1999).…”

“…In addition to the releasable pool, a smaller, faster pool of synaptic vesicles has been identified in bipolar cells (Mennerick and Matthews, 1996;Sakaba et al, 1997;Neves and Lagnado, 1999). This pool, referred to as the ultrafast or rapidly releasable pool, was discovered by examining the exocytotic response to a relatively brief (<50 ms) activation of voltage-gated calcium channels (Mennerick and Matthews, 1996).…”

“…This pool, referred to as the ultrafast or rapidly releasable pool, was discovered by examining the exocytotic response to a relatively brief (<50 ms) activation of voltage-gated calcium channels (Mennerick and Matthews, 1996). Vesicles in this pool fuse with rates similar to the maximal rates achieved in flash-photolysis experiments, in which calcium was rapidly and globally elevated by uncaging calcium (Heidelberger et al, 1994).…”

“…Secondly, there is an excellent correlation between the total number of vesicles docked at the base of a bipolar cell's synaptic ribbons and in physical contact with the plasma membrane (≈1000-1400, for a small and large terminal respectively) and the number of vesicles that comprise the rapidly releasing pool of vesicles as determined by capacitance measurements (≈1100) ; see also Neves and Lagnado, 1999). It is also noteworthy that the rate of fusion of vesicles in this pool is so rapid, that it is hindered by the activation kinetics of the presynaptic calcium channels (Mennerick and Matthews, 1996). When the calcium channels are bypassed, as in a flash photolysis of caged-calcium experiment, the brief delays that are observed between the rise in calcium and the onset of exocytosis (<1 ms for Ca 2+ >200 μM) can be attributed solely to the calcium-binding properties of the calcium sensor for exocytosis (Heidelberger et al, 1994).…”

“…From the calcium dependence of the rate of exocytosis derived from flash experiments on photoreceptors, an average vesicle in this putative pool might experience a calcium concentration in the 20-40 μM range . Although vesicles in this pool must be quite close to the calcium channels to experience such calcium concentrations, these vesicles still may not be as closely situated to the presynaptic calcium channels as their bipolar cell counterparts, which release with a time constant of 1.6 ms (Mennerick and Matthews, 1996) and are thought to sample ≈325 μM calcium (Heidelberger, 2001). This greater distance and the consequently lower calcium requirements in the rod could be related to the arciform density, which we suggest acts as a diffusion barrier to entering calcium and/ or to prevent vesicles from docking beneath the synaptic ribbon.…”