Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction

Abstract

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

Fig. 1.

Quantification of synchronous and asynchronous components of release. (A) A 10-s recording of EPCs in response to 100 Hz stimulation. Regions are expanded on a fast time scale along with the motor neuron action potentials corresponding to purely synchronous (A1), to shared synchronous and asynchronous (A2), and to largely asynchronous release (A3). Asterisks indicate synchronous events. (B) Scatterplot of total integrated release (black circles) and summed amplitudes (red circles) for each 10-ms interval occurring during the first 5 s of a representative paired recording as shown in A. The horizontal line represents the maximal predicted release corresponding to amplitude of the first EPC of the train, before the onset of depression. (C) A frequency histogram of time intervals between each asynchronous event and the peak of the action potential during the 3- to 4-s window of the stimulation. The gray bar indicates the 1.5-ms window used to score events as synchronous. (D) The contributions by synchronous (red circles) and asynchronous (blue triangles) release for each second interval were obtained by integrating the charge associated with each process during sequential 10-ms intervals shown in B. The total charge contributed by the sum of the two modes is shown by the black squares.

Fig. 2.

Synchronous and asynchronous release competes for the same release machinery. (A) Recordings of the action potential and associated postsynaptic response for sample 10-ms intervals when sharing between synchronous and asynchronous was maximal. The synchronous component scored on the basis of the 1.5-ms window is indicated for each interval by an asterisk. (B) Intervals with total release corresponding to >80% of the maximal response (shown in Fig. 1B as dashed line) were used to compare the contributions by synchronous and asynchronous release during each 10-ms interval. The open symbols are the values for each of the numbered individual intervals shown in A and closed symbols represent the values for the intervals not shown in A. The distribution was best fit by linear regression to a line with a correlation coefficient of 0.81. (C) The slope values for each of 11 cell pairs are plotted against the total charge associated with each 10-s recording. The dashed line indicates a slope of −1. The average correlation coefficient of the linear fits (as shown in B) of the 11 pairs was 0.82 ± 0.05.

Fig. 3.

Effects of syt2 knockdown on release. (A) The 10-s train of EPCs in response to 100-Hz stimulation for a syt2 morpholino fish. Two regions are expanded on a fast time scale corresponding to times when, in wild-type fish, release is either largely synchronous (A1) or asynchronous (A2). Asterisks indicate EPCs considered to be synchronous. The increased frequency of synchronous events in A2 is likely due to the heightened probability that an asynchronous event will fall within the window defined for synchronous detection. (B) Comparison of the contributions of asynchronous (blue triangles) versus synchronous (red circles) to total release (black squares) over the time course of 10 s of stimulation. (C) SV2 presynaptic label (green) and postsynaptic α-bungarotoxin (α-btx) label (red) in wild type (Upper Left) and syt2 morpholino (Upper Right) fish. Znp-1 antibody labeled syt2 protein (green) colocalizes with α-btx (red) label in wild-type fish (Lower Left) but is absent in syt2 morpholino fish (Lower Right). (Scale bar, 10 μm.) (D) Example traces of spontaneous synaptic currents from wild type (Top) and syt2 morpholino (Bottom) fish. A bar graph of spontaneous event frequency for wild-type fish (392 ± 263; n = 6 recordings), syt2 morpholino fish (18 ± 14; n = 10 recordings), and syt7 morpholino fish (17 ± 21; n = 6 recordings). ***P < 0.001.

Fig. 4.

Zebrafish syt7 gene and transcripts. (A) The location of exons on the gene (Upper) and transcript (Lower) are shown for zebrafish syt7. Exons are numbered on the basis of the mammalian gene (25, 26). The relative sizes of individual exons are indicated, along with the percentage identity to rat syt7 on the amino acid level (color indicators). The domains, predicted on the basis of homology to the mammalian gene are: transmembrane (black), calcium-binding C2 domains (dark gray), and alternatively spliced (light gray) regions. (B) RT-PCR analysis on wild-type and syt7 morpholino fish. The exon structures of the RT-PCR products and morpholino position are shown schematically. As a control the α-subunit of muscle acetycholine receptors was amplified from the same sample. (C) Syt7 antibody label (green) colocalizes with α-btx labeling (red) in wild type (Upper) but is absent at α-btx-labeled sites in syt7 morpholino fish (Lower). (Scale bar, 10 μm.)

Fig. 5.

Effects of syt7 knockdown on release. (A) A 10-s recording of EPCs in response to 100-Hz stimulation for a syt7 morpholino fish. Two regions are expanded on a fast time scale corresponding to times where, in wild-type fish, release is either largely synchronous (A1) or asynchronous (A2). (B) Comparison of the contributions of asynchronous (blue triangles) versus synchronous (red circles) to total release (black squares) over the time course of 10-s stimulation.

Fig. 6.

Quantitative overall comparisons of the effects of syt2 and syt7 morpholinos on synchronous and asynchronous release components. (A) Scatterplot of the charge associated with synchronous (red circles) and asynchronous (blue triangles) for the individual recordings from wild type, syt2 morpholino, and syt7 morpholino fish. The black lines connect the charges associated with asynchronous and synchronous contributions in each individual recording. (B) The total release over 10 s for syt2 morpholino (n = 6 cells), syt7 morpholino (n = 11 cells), and wild-type (n = 11 cells) fish were comparable (F = 1.36; P = 0.27). (C) Comparison of the contribution of summed release during the initial second of stimulation, to the total summed release measured over the entire 10-s period (F = 9.96; P < 0.0001). For pairwise comparisons, **P < 0.02; ***P < 0.0005. (D) Comparison of the fraction of release that occurred synchronously over the 10 s of stimulation (F = 66.4; P < 1 × 10⁻¹¹). (E) Comparison of the fraction of the release that occurred synchronously during the 3- to 4-s window of stimulation (F = 74.9; P < 5 × 10⁻¹¹). The values have not been corrected for contamination by asynchronous or spontaneous events. This will overestimate the contribution by synchronous release, particularly for syt2 knockdown where asynchronous and spontaneous release are elevated. For pairwise comparisons in D and E, ****P < 1 × 10⁻¹⁰.