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Review
. 1998 Mar 15;18(6):2028-39.
doi: 10.1523/JNEUROSCI.18-06-02028.1998.

Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin

D L Deitcher  1 , A Ueda, B A Stewart, R W Burgess, Y Kidokoro, T L Schwarz
Affiliations
Review

Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin

D L Deitcher et al. J Neurosci. .

Abstract

Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+ influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To investigate the mechanism of transmitter release, we have examined the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion. We generated a series of mutations, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous "minis" that are seen at the synapse can be generated by a protein complex that is distinct from that required for an evoked synaptic response.

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Figures

Fig. 1.
Fig. 1.
Mapping of P-element insertions near then-syb locus. A, Lanes 1–5contain, in order, P1 genomic clones 17–42, 29–89, 39–43, 40–41, and 55–41 from polytene bands 62A and 62B. Their DNA was digested withBglII and XhoI and simultaneously probed with n-syb probe and the flanking sequence from the starter P-element. n-syb-specific bands are indicated with arrows, and the P-element flanking sequence is indicated with arrowheads. 17–42 hybridizes with both probes (asterisk). B, Map of P-elements near n-syb. The P-element upstream ofn-syb in line 34 was mobilized (arrows) to give rise to the insertions in F33 and F82. Untranslated exon sequences are indicated by open boxes, translated exons by shaded boxes, and EcoRI sites by the letter E. Top, Although the orientation of the P-element and the n-syb gene and the distance between the two have not been determined, they must fall within ∼100 kb of each other to be contained on P1 17–42. Middle, The F33 P-element inserted in exon 1 of the n-syb gene 150 bp from the initiation ATG. Bottom, The F82 P-element inserted 3–4 kb from the 3′ end of n-syb.C, Southern blot of genomic DNA from wild-type (left) and F33/TM3, Sb heterozygote (right). Lanes are digested with the indicated restriction enzymes, and the molecular weights of the bands are indicated in kilobase pairs. Both blots are probed with a 2 kbEcoRI fragment of n-syb from the 5′ untranslated region. Arrowheads indicate new bands that result from the F33 insertion. D, Southern blot of viable, revertant excision lines. Lanes 1–7 are genomic DNA from seven different F33 excisions digested withEcoRI and probed with a 2 kb EcoRIn-syb genomic fragment. The wild-type band of 2 kb is indicated, and all of the excisions are within 200–300 bp of the wild-type size.
Fig. 2.
Fig. 2.
Restriction mapping of excision alleles.A, Restriction map of the n-syb locus (top), the excision allelen-sybΔF33-8(middle), and the excision allelen-sybΔF33B (bottom). The restriction site abbreviations are the following:EcoRI, E; XhoI,X; PstI, P. Deficiencies are indicated by a dashed line, PCR primers are shown asarrows, and exons are shown as boxes(shaded boxes are coding regions, and unshaded boxes are noncoding regions). Probes used in Bare diagrammed below the wild-type n-syb and then-sybΔF33B loci. B, Genomic Southern blots of DNA from n-sybΔF33Bheterozygote. Lanes 1–4 are all digested withXhoI and are hybridized with probes 1–4, respectively. Mutant (−) and wild-type (+) bands are indicated. Inlane 1, probe 1 hybridized to both the wild-type and mutant XhoI restriction fragments. The larger size of the mutant band results from upstreamn-syb sequences and the remainder of the P-element that failed to excise fully. In lane 2, probe2 recognized the same wild-type band as in lane 1. The higher molecular weight mutant band failed to hybridize as the 3′ half of exon 1, and all of exons 2, 3, and 4 are deleted in the mutant. In lane 3, probe 3 hybridized to a wild-type band of ∼1 kb and the high molecular weight mutant band from lane 1. Probe 3 hybridized to the mutant band because the XhoI restriction site between exons 4 and 5 was deleted, but the deletion did not extend to exon 5. In lane 4, probe 4 hybridized to a single unaltered band because the probe is outside the deleted region.
Fig. 3.
Fig. 3.
Western blots of protein extracts from wild-type and n-syb mutant heterozygotes probed with anti-n-syb antiserum. The molecular weight size markers are in kilodaltons, and the band corresponding to size of then-syb protein (∼22 kDa) is indicated. Proteins extracts from either 10 heads or 10 bodies (A) or 20 heads (B) from the indicated lines were prepared as described in Material and Methods. A,n-syb protein is enriched in wild-type (wt) heads as compared with the rest of the body (wt body); the starter P-element line, line 34, has wild-type levels of n-syb. All of then-syb mutant heterozygotes have reduced levels ofn-syb protein. B,n-sybΔF33-8 produces a slightly higher molecular weight form of n-syb (indicated with anarrow) than wild type. This is most likely attributable to the use of an alternative initiation ATG in intron 1. The higher molecular weight band present in all of the lanes, running at ∼35 kDa, appears to be a protein that cross-reacts with then-syb antiserum because it does not decrease in intensity in the n-syb mutants.
Fig. 4.
Fig. 4.
Immunocytochemistry of wild-type andn-syb mutant embryos. A, Wild-type embryo fillet double-stained with n-syb antiserum (green) and Fasciclin II antibody (red). B, C, Synapses (arrows) at a wild-type NMJ stained for FasII (B) and n-syb(C). D–G, n-sybnull mutants (n-sybΔF33B) stained for FasII (D, F) and n-syb(E, G). Despite the absence of detectablen-syb, the morphology of the nerve cord and NMJ appears normal. Ventral nerve cord, VNC; axonal commissures,co; axons of the segmental nerves, SN; longitudinal muscles 6 and 7, 6, 7. Scale bar, 1 μm.
Fig. 5.
Fig. 5.
Nerve-evoked synaptic currents are absent from the neuromuscular junctions of n-syb mutants stimulated at 0.3 Hz. Evoked currents are lacking in n-syb null mutantn-sybΔF33B(A) and in the mutantn-sybF33-R (B) but are present in the parental control, line 34 (C). The external solution contained 2 mm Ca2+ for linesn-sybΔF33B andn-sybF33-R and 0.5 mmCa2+ for line 34.
Fig. 6.
Fig. 6.
Frequency of miniature synaptic currents inn-sybΔF33B,n-sybF33-R, and line 34. Error bars are SEM. Asterisks denote statistical differences from line 34 at p < 0.05 by the ANOVA test.Numbers indicate the number of cells examined. Miniature synaptic currents were recorded in the presence of 3 μmTTX in 0.5 mm Ca2+ saline.
Fig. 7.
Fig. 7.
Representative miniature synaptic currents and amplitude histograms. Shown are miniature synaptic currents forAa, n-sybΔF33B; Ba, n-sybF33-R; and Ca, line 34. Amplitude histogram for Ab isn-sybΔF33B; for Bbis n-sybF33-R; and forCb is line 34. Miniature synaptic currents were recorded in high K+ saline (20 mm) to increase their frequency and in the presence of 3 μm TTX and 0.5 mm Ca2+. The mean amplitudes wereA, 155 ± 26 pA (n = 5);B, 199 ± 16 pA (n = 7); andC, 168 ± 24 pA (n = 6), wheren is the number of cells.
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