Ca(2+) influx and neurotransmitter release at ribbon synapses

Abstract

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.

Figure 1

Morphology of ribbon synapses. A halo of synaptic vesicles surround the electron dense synaptic ribbon. The ribbon can be plate-like, ellipsoidal, or spherical. A. Representative section of adult bullfrog amphibian papilla hair cell synapse imaged at 40000× (scale bar, 100 nm). Ribbon-attached vesicles (orange), immediately-releasable vesicles (red; vesicles docked on the membrane), ribbon anchoring sites (purple arrowheads). Modified with permission from Graydon et al., 2011 [6]. B. A mature (p14 – 17) mouse inner hair cell synapse (scale bar, 200 nm) viewed by electron microscopy. Modified from Neef et al., 2007 [3]. C. Ultrastructure of goldfish retina bipolar cell synapse (scale bar, 50 nm). The arciform density resides below the base of the synaptic ribbon. Modified from von Gersdorff et al., 1996 [84].

Figure 2

High-speed confocal Ca²⁺ imaging of presynaptic dense bodies in auditory hair cells from the turtle’s papilla. A. Fluorescence image of presynaptic dense bodies with rhodamine-tagged ctbp2-terminal binding peptide (scale bar: 1 µm). B. Regions selected for Ca²⁺ imaging. C. A depolarizing stimulus from −85 mV to −35 mV (top) on the hair cell evoked a large L-type Ca²⁺ current. D. The fluorescent signal shows a rapid initial increase with subsequent slow rise of the internal Ca²⁺ concentration. Regions far from the synapse show little initial response followed by a slow slight increase. This indicates Ca²⁺ channels are clustered at the synaptic ribbon sites. Modified from Schnee et al., 2011[39].

Figure 3

Presynaptic Ca²⁺ entry in frog hair cells. A. Image of a hair cell using differential interference contrast light microscopy. Arrowheads indicate the locations of presynaptic ribbons. B. Epifluorescence image of the same hair cell with A, which is loaded with 200 µM fluo-3. The fluorescence image shows strong fluorescence signals at the positions of ribbons. The arrow indicates the transect scanned in D. C. While the image was acquired, the hair cell was depolarized from −70 mV to −15 mV for 300 ms (upper trace). Ca²⁺ current was evoked by this stimulus (lower trace). D. Time course of Ca²⁺ entry in isolated hair cell loaded with 200 µM fluo-3. The line-scan feature of a laser-scanning confocal microscope with high temporal resolution generated a two-dimensional image. One axis is distance and the other axis is time. A transect (400 nm) across the hair cell (arrow in B) was repeatedly scanned every 2 ms for a period including the depolarization in C. Within a few milliseconds of a depolarization’s onset, fluorescence signals increased in restricted region, which was close to the presynaptic membrane, then signals spreaded gradually to the inside of the cell. The spatial scale bar in D applies to A and B. The temporal scale bar in D applies to C. E. Mobile Ca²⁺ buffer affects temporal and spatial changes in fluo-3 fluorescence. Hair cells were loaded with 200 µM fluo-3 (E1) or 200 µM fluo-3 plus 10 mM BAPTA (E2). The spread of Ca²⁺ was restricted to the presynaptic dense body by high concentration of mobile Ca²⁺ buffer (E2). A – E: isolated frog saccular hair cells (modified from Issa and Hudspeth, 1996 [40]). F. EPSCs from paired recordings from the bullfrog amphibian papilla hair cell synapse. Presynaptic hair cell dialyzed with 10 mM BAPTA was depolarized from −90 mV to −54 mV. The afferent fiber shows large amplitude EPSCs during the depolarizing pulse. Modified from Graydon et al., 2011 [6].

Figure 4

Short-term plasticity at bullfrog auditory hair cell synapses (A – C) and goldfish retina bipolar cell synapses (D – F). A. Ca²⁺ current (I_Ca) and membrane capacitance (C_m) evoked by a pair of 20 ms depolarization from −90 mV to −30 mV with 20 ms interpulse interval shows paired-pulse facilitation in the ratio of the C_m jumps. Note that I_Ca shows a slow form of calcium-dependent inactivation (inset). B. When a hair cell was depolarized from −90 mV to −30 mV for 20 ms with 5 ms interpulse interval, EPSC was recorded from the connected afferent fiber terminal. The ratio of EPSC (EPSC₂/EPSC₁) shows paired-pulse depression. C. The relationship between EPSC peak ratios and interpulse intervals. Hair cells were depolarized from −90 mV to −30 mV for 20 ms with various interpulse intervals (n = 4 – 8). The EPSC paired-pulse ratio (EPSC₂/EPSC₁) recovered exponentially with a single exponential fit from 3 to 50 ms intervals (τ = 10.9 ms; red dashed curve). The gray dashed line indicates that the ratio is 1 (EPSC₂ = EPSC₁). The ratio of Ca²⁺ charge (Q_Ca2/Q_S) was relatively constant and close to 1 (0.96 – 0.99). D. I_Ca and C_m were evoked by a pair of 25 ms pulses from −60 mV to −10 mV with 100 ms interpulse interval in dissociated goldfish retina bipolar cells. The ratio of C_m shows strong paired-pulse depression. E. A bipolar cell was depolarized by a pair of 2 ms pulses from −60 mV to −10 mV with 100 ms interpulse interval. Note that although I_Ca shows slight calcium-dependent facilitation in amplitude (inset), the ratio of C_m jumps shows strong paired-pulse depression due to vesicle pool depletion. F. Paired-pulse ratio (ΔC_m2/(ΔC_m1) and the ratio of Ca²⁺ charge (Q_Ca) with pairs of 2 ms or 25 ms pulses in dissociated bipolar cell terminals or terminals in retinal slices. Paired-pulse ratio recovered with fast time constant (τ = 1.03 s; 43 %) and slow time constant (τ = 11.8 s; 57 %). The black line is a double exponential fit. Under all conditions, the rates of recovery from depression were similar. A–C: from Cho et al., 2011 [77], D–E: Modified from Palmer et al., 2003 [109].