Increased synaptic microtubules and altered synapse development in Drosophila sec8 mutants

Background Sec8 is highly expressed in mammalian nervous systems and has been proposed to play a role in several aspects of neural development and function, including neurite outgrowth, calcium-dependent neurotransmitter secretion, trafficking of ionotropic glutamate receptors and regulation of neuronal microtubule assembly. However, these models have never been tested in vivo. Nervous system development and function have not been described after mutation of sec8 in any organism. Results We identified lethal sec8 mutants in an unbiased forward genetic screen for mutations causing defects in development of glutamatergic Drosophila neuromuscular junctions (NMJs). The Drosophila NMJ is genetically malleable and accessible throughout development to electrophysiology and immunocytochemistry, making it ideal for examination of the sec8 mutant synaptic phenotype. We developed antibodies to Drosophila Sec8 and showed that Sec8 is abundant at the NMJ. In our sec8 null mutants, in which the sec8 gene is specifically deleted, Sec8 immunoreactivity at the NMJ is eliminated but immunoblots reveal substantial maternal contribution in the rest of the animal. Contrary to the hypothesis that Sec8 is required for neurite outgrowth or synaptic terminal growth, immunocytochemical examination revealed that sec8 mutant NMJs developed more branches and presynaptic terminals during larval development, compared to controls. Synaptic electrophysiology showed no evidence that Sec8 is required for basal neurotransmission, though glutamate receptor trafficking was mildly disrupted in sec8 mutants. The most dramatic NMJ phenotype in sec8 mutants was an increase in synaptic microtubule density, which was approximately doubled compared to controls. Conclusion Sec8 is abundant in the Drosophila NMJ. Sec8 is required in vivo for regulation of synaptic microtubule formation, and (probably secondarily) regulation of synaptic growth and glutamate receptor trafficking. We did not find any evidence that Sec8 is required for basal neurotransmission.

Drosophila CG2095 / sec8 Figure 1 Drosophila CG2095 / sec8. A: Amino acid alignment of human (Accession number NP_068579), rat (Accession number Q62824), fly (Accession number AY905551), nematode (Accession number NP_492732) and yeast (Accession number NP_015380) Sec8. The amino acid sequence of Drosophila CG2095 is 33% identical to rat and human Sec8. Drosophila amino acids 440-460 (underlined) represent the peptide sequence used to generate Sec8 antibodies. B: Genomic map of Drosophila CG2095 (sec8). Grey bars represent exons. sec8P1 is a lethal transposon insertion into the first exon. sec8∆1 is a deletion created by imprecise excision of the transposon. The sec8∆1 deletion removes almost the entire sec8 gene, with the exception of the first 37 bp of exon 1 and last 73 bp of the final exon 9.

Background
The Sec6/8 complex, or 'exocyst', is an octomeric protein complex thought to comprise the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. This complex has been most extensively studied in yeast, where it is required for membrane trafficking and secretion [1,2]. In multicellular organisms, Sec proteins are expressed at particularly high levels in the nervous system [3,4]. The functional role of Sec proteins in neurons, however, including whether they always function as a canonical octomeric complex, remains unclear.
A mouse sec8 knockout has been created, but dies early in embryonic development [16], precluding significant functional or developmental analyses. In contrast, the Drosophila sec8 knockouts described here survive through embryogenesis, and hypomorphs survive throughout larval development into pupation. This provides the first opportunity to describe a sec8 mutant phenotype at any synapse. The Drosophila NMJ is a particularly good model synapse for this purpose, since it is glutamatergic and accessible to powerful microscopic and electrophysiological techniques throughout development. Here, we provide the first description of sec8 mutant synaptic phenotypes. We focused our analysis on (1) growth of presynaptic terminals, (2) basal neurotransmission, (3) ionotropic glutamate receptor trafficking and (4) microtubule density, since Sec8 has specifically been implicated in each of these processes.
We found that sec8 mutant NMJs show no obvious defect in growth of presynaptic arborizations or neurotransmitter secretion, but do show mild defects in glutamate receptor trafficking and relatively dramatic alterations in synaptic microtubule density. We hypothesize that Sec8's most important role in vivo is spatially-restricted inhibition of microtubule stability, and that disruption of microtubule regulation may underlie most, if not all, sec8 mutant phenotypes.

Identification and generation of sec8 mutants
We identified the transposon insertion mutant P{SUPor-P}CG2095 KG02723 in simultaneous unbiased forward screens for Drosophila mutants with alterations in NMJ presynaptic growth and glutamate receptor cluster formation [5,6]. We selected this mutant for further study on the basis of a dramatic semi-penetrant presynaptic overgrowth phenotype and reproducible loss of glutamate receptor immunoreactivity (see results, below). The KG02723 P-element insertion was mapped by the Berkeley Drosophila Genome Project using inverse PCR to the first exon of predicted gene CG2095 [17]. We confirmed the KG02723 P-element insertion site in CG2095 by sitespecific PCR primers.
We cloned and sequenced full-length CG2095 cDNA from the wild-type Drosophila strain Oregon R (Accession number AY905551). A BLAST search using this Oregon R CG2095 amino acid sequence showed 33% identity to rat and human Sec8 (Fig. 1A). Conversely, BLAST searches using mammalian Sec8 against the translated Drosophila genome showed close matches only to CG2095. Thus, CG2095 represents the sole Drosophila sec8 homolog. Hereafter, we refer to CG2095 as sec8.
Because the P{SUPor-P}CG2095 KG02723 P element insertion mutant represents a mutation in the Drosophila sec8 gene, we refer to this allele as 'sec8P1'. We verified that the P-element insertion was responsible for the sec8P1 phenotype (see below) by complementation analysis and remobilization of the P-element [18] to create precise excisions ('revertants'). These revertants are phenotypically indistinguishable from other control (w 1118 ) animals. Furthermore, sec8P1 mutants retain their phenotype in trans to a deficiency, Df(3R)Tpl10, that completely eliminates the CG2095 gene. These results show that the sec8P1 phenotype is specifically due to the P-element insertion in sec8.
Many transposon insertion mutants, including the sec8P1 mutants, represent hypomorphic alleles. Therefore, we generated deletions of the sec8 gene via imprecise excision of the KG02723 P-element in sec8P1, using standard methods [18]. Among the excision mutants isolated was a CG2095-specific deletion that we refer to as 'sec8∆1'. To confirm that sec8∆1 only disrupts the sec8 gene and provide an accurate molecular description of this mutation, we sequenced the sec8 genomic region in sec8∆1 mutants. Sequencing showed that the sec8∆1 deletion begins before the ATG, 37 bp into the first 5' exon, and ends 73 bp prior to the 3' end of the last exon. Neighboring genes are completely intact. sec8∆1 therefore represents a zygotic null. sec8∆1 homozygous mutants hatch at the same time as controls (approximately 24 h after egg laying, AEL), but do not undergo their first molt and die as first instar larvae (~ 46 h AEL). The molecular nature of the sec8P1 and sec8∆1 mutations is summarized in Fig. 1B.
Sec8 is found in the Drosophila NMJ Figure 2 Sec8 is found in the Drosophila NMJ. A: Third instar larval NMJs on ventral longitudinal muscles 6 and 7, stained using antibodies against Sec8 (green), GluRIIA (red), and the neuronal membrane marker anti-HRP (blue). All results in this study are derived from experiments on 6/7 NMJs in segments A3-A4. Lower row shows NMJ terminals at higher magnification. Scale bars = 5 µm. B: Confocal fluorescent images showing intersegmental nerve branch b (ISNb) innervating ventral longitudinal muscles in first instar larvae (24-28 h AEL). NMJs were visualized using the neuronal membrane marker anti-HRP (magenta). Sec8 was visualized using anti-Sec8 antibodies (green). Scale bar: 10 µm. Insets show individual synaptic boutons at higher magnification (approximately 2 µm × 2 µm). C: Quantification of total animal Sec8 protein, measured using protein blots from first instar control and mutant larvae, probed with anti-Sec8 antibody.
Homozygous sec8P1 and sec8∆1 mutants both hatched at approximately normal time (22)(23)(24) h AEL) and appeared grossly normal by light microscopy (appropriate size, segmentation, mouthooks, etc.). There was no obvious difference in mutant animal size, behavior or viability immediately after hatching. However, homozygous sec8∆1 mutants did not undergo their first molt and did not live past first instar larval stage (~ 48 h AEL). Homozygous sec8P1 mutants appeared to molt and pupate normally, and therefore lived through larval development, but did not eclose and therefore died as pupae. In view of the viability and the fact that sec8 is almost completely deleted in sec8∆1 mutants, we conclude that sec8P1 represents a hypomorphic allele, while sec8∆1 represents a zygotic null allele.

Drosophila Sec8 is abundant in embryonic and larval neuromuscular junctions (NMJs)
The Drosophila embryonic/larval neuromuscular junction (NMJ) is a well-established model glutamatergic synapse that can be used to examine the effects of Sec8 loss on synaptic development and function. To test whether Sec8 is expressed at the NMJ, we raised rabbit polyclonal antibodies against Sec8 using a synthetic peptide composed of Sec8 amino acids 440-460, and affinity purified these antibodies using the original peptide epitope. Immunoreactivity of this antibody was widely distributed throughout many tissues and was particularly enriched in synapses, including the NMJ ( Fig. 2A). Because the pre and postsynaptic membranes are separated by only approximately 15 nm [19,20], it is impossible to distinguish definitively by light microscopy whether Sec8 immunoreactivity directly at the NMJ is pre or postsynaptic. Nevertheless, several indications suggest that Sec8 is both pre and postsynaptic. First, there is clearly Sec8 immunoreactivity throughout the muscle cell outside the area delimited by presynaptic HRP staining; therefore, sec8 must be expressed in the postsynaptic muscle cell. Postsynaptic localization of Sec8 is also suggested by the fact that Sec8 immunoreactivity overlaps immunoreactivity for the postsynaptic glutamate receptor subunit GluRIIA. Three-dimensional reconstructions and rotations (not shown) show Sec8 immunoreactivity extending above the interior face of the muscle, consistent with presynaptic localization. We did not observe any Sec8 immunoreactivity in segmental nerve axons, suggesting that presynaptic Sec8, if present, must be localized specifically at the terminals, but there is little or no immunoreactivity in the centers of presynaptic boutons. Thus, presynaptic Sec8 must be localized specifically near the terminal membrane. We obtained essentially identical results using a recently described and independently generated antibody [21] (data not shown).
Sec8 antibody immunoreactivity in the embryonic/first instar (L1) neuromusculature was dramatically reduced in homozygous sec8P1 mutants, and was almost undetectable in sec8∆1 mutants (Fig. 2B). On immunoblots using whole-animal extracts, our Sec8 antibody recognized a single 111 kDa band (not shown). The intensity of this band was reduced, on average, to approximately one-half normal in both sec8P1 and sec8∆1 L1 mutants (Fig. 2C). This suggests that despite the deletion of the sec8 gene in sec8∆1 mutants, and almost complete elimination of Sec8 in the NMJ of homozygous mutants, there is substantial maternal contribution of Sec8 in other tissues, as previously shown for other sec proteins [7,8,[21][22][23].
We conclude from our immunohistochemical results that Sec8 is probably expressed both pre and postsynaptically at the Drosophila NMJ, and that sec8 mutant NMJs have almost no remaining Sec8 but substantial maternal Sec8 persists in other parts of the sec8∆1 mutant animals.

sec8 mutants show increased NMJ growth during larval development
Sec proteins have been implicated in the membrane addition required for neurite outgrowth [7][8][9][10]. Formation of Drosophila larval NMJs involves extensive neurite outgrowth and presynaptic arborization. Examination of motor nerve terminals in the ventral body wall neuromusculature of Drosophila sec8 mutants therefore provides an excellent opportunity to test whether Sec8 plays a role in nerve terminal growth in vivo. Approximately halfway through Drosophila embryonic development (12 h After Egg Laying, AEL), motor neuron growth cones exit the CNS and travel along the developing segmental nerve toward their body wall muscle targets. Approximately 13 h AEL, development of ventral NMJs begins when motor neuron growth cone filopodia contact the target muscles. Over the next several hours, filopodia-ringed growth cones in contact with appropriate targets collapse to form nascent presynaptic processes of indistinct shape, termed 'prevaricosities'. By 17-22 h AEL, parts of the prevaricosities begin to constrict and/or swell such that presynaptic terminals (known as synaptic boutons) are formed [24][25][26][27][28][29][30]. These presynaptic boutons are functional very early; electrophysiological analysis shows that NMJ transmission occurs within minutes of contact between motor nerve axons and muscles [31,32]. At the time of hatching (22-24 h AEL), presynaptic boutons are still relatively indistinct, morphologically, but nonetheless are clearly highly functional since neuromuscular transmission is required for hatching and subsequent crawling of the newly hatched first instar (L1) larva. During larval development (24-120 h AEL), the presynaptic arborization grows dramatically in order to accommodate rapidly growing larval muscles. This growth involves additions in NMJ length, branches and bouton number (from about NMJ morphology in sec8 mutants becomes abnormal during larval growth In addition, long slender neurites (normally not present) extend across the muscle to the right (distally) of the NMJ center. Abnormal NMJ growth such as this was never observed in control animals (N>200). D: Quantification of the number of presynaptic boutons (left) and the number of branches (right) at the 6/7 NMJ in first instar (24-48 h AEL) larvae. E: Quantification of the number of presynaptic boutons (left) and the number of branches (right) at the 6/7 NMJ in third instar (110-120 AEL) larvae. sec8∆1 mutants die before third instar (skull and crossbones) and thus third instar morphology could not be assayed in that genotype.
Fewer synaptic glutamate receptors are electrophysiologically detectable in sec8 mutants Figure 4 Fewer synaptic glutamate receptors are electrophysiologically detectable in sec8 mutants. A: Portions of representative patch clamp recordings from control and sec8∆1 mutant first instar larvae (22-24 h AEL), showing spontaneous excitatory junction currents (sEJCs) from the muscle 6 NMJ. B: Glutamate-gated currents triggered by pressure ejection of 1 mM glutamate on to patch-clamped muscle 6, from control and sec8∆1 mutants. Amplitudes of glutamate-gated currents are quantified in C. C: Quantification of glutamate-gated current amplitudes from control and sec8∆1 mutants. D: sEJCs from control and sec8∆1 mutant first instar larvae, showing delayed single channel closings observable during the falling phase of the currents. Lines have been added to the figure to indicate the baseline and single receptor current amplitude. Single-channel glutamate receptor amplitudes were measured from delayed closings such as these; quantification of these amplitudes is shown in E. E: Quantification of single glutamate receptor current amplitudes from control and sec8∆1 mutants. F: Portions of representative two-electrode voltage clamp recordings from control and sec8P1 third instar larvae (110-120 h AEL), showing spontaneous excitatory junction currents (sEJCs) in the muscle 6 NMJ. On the right are shown portions of recordings at expanded time scale, which sacrifices an overall impression of sEJC amplitude differences but better demonstrates the resolution with which larval sEJCs were detected. G: Cumulative frequency histogram of sEJC amplitudes in third instar animals. Homozygous sec8P1 mutant animals had fewer large events, compared with controls. 8-10 in at hatching to 50-100 in the third instar 6/7 NMJ). Boutons also become more distinct during NMJ development, such that the third instar NMJ typically appears as 'beads on a string' [28][29][30]33].

sec8 mutants show robust neurotransmission but a slight decrease in the number of postsynaptic glutamate receptors
Sec8 has been suggested to play a role in calcium-dependent neurotransmitter secretion [11]. To determine whether transmission in sec8 mutants is functionally abnormal, we used voltage clamp synaptic electrophysiology on NMJs. First we examined sec8∆1 mutants, since Sec8 was almost completely eliminated from the NMJ in these animals (Fig. 2B). Whole-cell patch clamp recordings from homozygous sec8∆1 mutants showed that sec8∆1 mutant NMJs were capable of robust endogenous synaptic transmission (Fig. 4A). This result is consistent with the fact that newly hatched sec8∆1 mutant larvae appeared to crawl and feed efficiently. The frequency of spontaneous synaptic currents (aka 'spontaneous excitatory junction currents', or 'sEJCs') was normal in sec8∆1 mutants (Control = 13.30 ± 1.25 Hz, n = 11; sec8∆1 = 12.34 ± 2.78 Hz, n = 10, p = 0.750). These data suggest no major disruption of presynaptic function after loss of Sec8. However, sEJC amplitude in sec8∆1 mutant first instar NMJs was mildly but significantly reduced (K-S statistic = 0.379, p < 0.0001). Reduced sEJC amplitude suggests loss or mislocalization of postsynaptic glutamate receptors.
To test glutamate receptor function directly, we pressure ejected 1 mM glutamate on to patch-clamped (-60 mV) postsynaptic muscle. Pressure ejection of glutamate provides a means of measuring postsynaptic receptor function independent of presynaptic glutamate release. Drosophila NMJ receptors have low affinity for glutamate compared to mammalian glutamate receptors [34]; 1 mM is barely saturating. Currents triggered by pressure ejection of glutamate were significantly reduced in sec8∆1 mutants compared to controls ( Fig. 4B-C; Control = 1858 ± 222.4 pA, n = 13; sec8∆1 = 1181 ± 105.5 pA, n = 11, p = 0.016). This reduction in glutamate-gated current amplitude could be due to a decrease in the number of functional postsynaptic glutamate receptors, or a change in the biophysical properties of individual receptors. In embryonic/ L1 Drosophila, these two possibilities can be distinguished by measuring the single channel current size of synaptic receptors directly. The input resistance of embryonic/L1 Drosophila muscle cells, coupled with the large conductance of insect muscle glutamate receptors, allows wholecell patch clamp mode discrimination of delayed single channel closing on the falling phase of some sEJCs [35][36][37][38]. The amplitude of these delayed synaptic receptor closings was not significantly different between sec8∆1 mutants and controls ( Fig. 4D-E; control = 12.02 ± 1.15 pA, n = 11; sec8∆1 = 10.70 ± 0.94 pA, n = 10, p = 0.392), suggesting that the decrease in glutamate-gated current size measured in sec8 mutants is due to a loss of functional postsynaptic receptors, rather than a change in receptor properties. These latter experiments could not be repeated in sec8P1 mutant third instar larvae because synaptic receptors are not accessible for rapid agonist application because presynaptic terminals tend to be embedded deep within a subsynaptic reticulum, SSR. Decreased muscle input resistance in L3 animals also makes it impossible to discriminate single channel current amplitudes. However, two-electrode voltage clamp recordings revealed that, as in sec8∆1 mutant L1 animals, sEJC amplitude was significantly reduced in sec8P1 mutant third instar larvae ( Fewer glutamate receptors are immunocytochemically detectable in sec8 mutants. A: Confocal images showing NMJs on ventral longitudinal muscles 15, 6/7, 13 and 12 in first instar larvae (24-28 h AEL). NMJs were visualized using the neuronal membrane marker anti-HRP (magenta). Postsynaptic glutamate receptors were visualized using antibodies raised against the glutamate receptor subunit GluRIIA (green). Scale bar: 10 µm. Quantification of GluRIIA cluster sizes is shown in B. B: Top: Average GluRIIA cluster size was significantly reduced in sec8 mutant first instar larvae. Middle: Frequency histogram of GluRIIA cluster sizes. Bottom: Average GluRIIA cluster size was significantly reduced in sec8P1 mutant third instar larvae, and this phenotype is rescued by a precise excision of the P-element ('sec8rev5'). Similar results were obtained using antibodies against GluRIIB and GluRIIC (see text). C: Confocal images showing ventral longitudinal NMJs in first instar larvae, as in A, stained using antibodies against cysteine string protein (CSP; top panels), discs-large (DLG; middle panels), and synaptotagmin (SYT; bottom panels). Scale bar: 10 µm. D: Quantification of staining in C. sec8 mutants showed no significant difference in cysteine string protein (CSP, top), discs-large (DLG, middle) or synaptotagmin (SYT, bottom) immunofluorescence compared with controls.
loss of postsynaptic glutamate receptors in both mutant alleles throughout larval development.
To confirm the electrophysiological results and determine whether loss of receptors in sec8 mutants might be due to loss of a specific receptor subtype, we performed immunocytochemical experiments on the same synapses examined electrophysiologically (muscle 6 NMJs). Drosophila NMJs contain two spatially, pharmacologically and biophysically distinct subtypes of postsynaptic glutamate receptor [38][39][40][41][42], referred to as 'A-type' and 'B-type' receptors. A and B-type receptors appear to be molecularly identical heterotetramers that differ by one subunit: Atype receptors contain the subunit GluRIIA, but not GluRIIB, and B-type receptors contain the subunit GluRIIB, but not GluRIIA. Both receptor subtypes also contain the subunits GluRIIC (aka GluRIII), GluRIID and GluRIIE [39][40][41].
We also quantified the immunoreactivity of the presynaptic proteins cysteine string protein (CSP) and synaptotagmin (SYT), as well as the MAGUK protein discs-large (DLG), which is primarily localized postsynaptically. These antigens do not form distinct immunoreactive clusters. Therefore, we quantified CSP, SYT and DLG immunoreactivity by measuring average fluorescence intensity in the NMJ minus average non-NMJ muscle background intensity over an identical area of neighboring muscle membrane, normalized to control genotype (e.g.: immunoreactivity = (f NMJ -f muscle ) mutant / (f NMJ -f muscle ) control ). This approach avoids use of fluorescence intensity from a different wavelength as a control, which would be inappropriate because fluorescence of different fluorophores and detection of different emission spectra can vary independently from that of the 'target' fluorophore. Immunoreactivity for CSP, DLG, and SYT was normal in both sec8P1 and sec8∆1 (  [43], and DLG is required for proper localization of GluRIIB [38,44], normal immunoreactivity for CSP, SYT and DLG is consistent with relatively normal neurotransmission and GluRIIB clustering that we observed.
Taken together, our electrophysiological and immunocytochemical results suggest that sec8 mutant NMJs are capable of relatively normal neurotransmission, except for a slight loss of postsynaptic glutamate receptors.

The loss of glutamate receptors in sec8 mutant NMJs is due to mislocalization of glutamate receptor protein
Although the loss of postsynaptic glutamate receptors that we observed in sec8 mutants was slight, we felt this phenotype deserved further investigation since Sec8 has been implicated in glutamate receptor trafficking, but Sec8dependent trafficking of non-NMDA receptors has not been demonstrated [12][13][14]. Drosophila NMJ glutamate receptors are most similar in amino acid sequence to mammalian non-NMDA kainate receptors [40,41]. The Drosophila genome also encodes NMDA receptor subu-nits, which are expressed in the CNS but not in muscle [45,46].

Drosophila Sec8 inhibits microtubule formation in vivo
Previous work has demonstrated that exocyst proteins associate biochemically with microtubule proteins [10], and that this association destabilizes microtubules [15]. To test whether Sec8 regulates microtubules at the Drosophila NMJ, we examined pre and postsynaptic NMJ microtubules using an antibody against acetylated tubu-Glutamate receptor subunit mRNA and protein is not reduced in sec8 mutants Figure 6 Glutamate receptor subunit mRNA and protein is not reduced in sec8 mutants. A: Relative mRNA levels for glutamate receptor subunits GluRIIA (left), GluRIIB (middle), and GluRIIC (right), as measured by quantitative real-time RT-PCR in control and sec8 mutant first instar larvae. B: Example immunoblot from first instar larvae (24-28 h AEL) probed with a anti-GluRIIB antibody. The arrow points to the GluRIIB band. GluRIIA immunoblots have been previously described [71]. GluRIIA and GluRIIB bands are eliminated in Df(2L)SP22 mutants, in which GluRIIA and GluRIIB genes are deleted (not shown). Loading control was total protein; to ensure that the same amount of protein was loaded for both control and mutant, total extracted protein quantity was measured using a Bradford assay and then the same amount of protein was loaded on to the gel for each mutant (either 20 µg for GluRIIB blots or 50 µg for GluRIIA blots). Loading was also checked with Coomassie staining (not shown). C: Quantification of total GluRIIA (left) and GluRIIB (right) protein, as measured from immunoblots such as the one shown in B.
Microtubule density is inversely proportional to Sec8 expression Figure 7 Microtubule density is inversely proportional to Sec8 expression. A: Confocal images showing a portion of a NMJ on ventral longitudinal muscles 6 & 7 in third instar larvae (110-120 h AEL), visualized using antibodies against tubulin (green) and the essential glutamate receptor subunit GluRIIC (red). Upper left half of the image shows confocal projection; lower right half of the image shows 3D isosurface reconstruction of the same data. This shows the close association between pre and postsynaptic microtubules and the NMJ. B: Microtubules in postsynaptic larval muscles. Muscle nuclei ('N') are surrounded by microtubule networks (labeled with anti-acetylated tubulin antibody, magenta) that often extend (arrows) toward the neuromuscular junction (labeled with anti-HRP, green). This suggests that muscle microtubules may be critical for delivery of postsynaptic molecules to the NMJ. C: Quantification of synaptic microtubule density. To measure synaptic microtubule density, immunofluorescence from only the non-axonal area delimited by HRP staining (magenta) was measured. D: Quantification of extrasynaptic microtubule density. To measure extrasynaptic microtubule density, immunofluorescence from the muscle area not delimited by HRP staining was measured. E: Confocal images showing intersegmental nerve branch b (ISNb) innervating ventral longitudinal muscles in first instar larvae (24-28 h AEL). Neuronal tissue was visualized using anti-HRP antibody (magenta). Microtubules were visualized using an anti-acetylated tubulin antibody, which recognizes only stably polymerized alpha and beta tubulin (green). Individual NMJs are identified (arrows, labels). In 24B Gal4;UAS-sec8 animals, sec8 cDNA is overexpressed in postsynaptic muscle cells, resulting in small but not statistically significant reductions in microtubule immunofluorescence. In sec8 mutants, microtubule immunofluorescence is increased. This increase is highest at the synapse, where Sec8 is normally localized. Qualitatively identical results were obtained using another antibody that recognizes all tubulin (not shown). Scale bar: 10 µm.

Overexpression of tubulin phenocopies the loss of glutamate receptors seen in sec8 mutants
Our data suggest that Drosophila Sec8 plays a role in glutamate receptor localization and microtubule regulation. These two phenotypes could represent separate functions for Sec8, or could be due to the same root cause. Mammalian glutamate receptors are trafficked to the synapse via the microtubule network [55][56][57]. Therefore, it is reasonable to surmise that the defects in postsynaptic glutamate receptor localization measured in sec8 mutants might be due to misregulation of microtubules. To address this, we mimicked the increase in microtubules observed in sec8 mutants by overexpressing alpha tubulin. Overexpression of an alpha tubulin transgene in postsynaptic muscles using the gal4-UAS system doubles microtubule density (Images not shown; Control MT density = 1.00 ± 0.17, N = 11; UAS-α tub/B24 gal4 tubulin = 2.21 ± 0.24 a.u., n = 12, p = 0.001), presumably due to a shift in the dynamic equilibrium between disassembled and assembled tubulin subunits. This shift toward an increase in microtubules caused a significant decrease in the size and number of postsynaptic glutamate receptor clusters, similar to that observed in sec8 mutants ( Fig. 8; Control GluRIIA cluster size = 0.45 ± 0.02 µm 2 , n = 115 clusters in 8 animals; UASα tub/B24 gal4 = 0.28 ± 0.01 µm 2 , n = 104 clusters in 9 animals, p < 0.0001; Number of control clusters = 0.68 ± 0.06 clusters/µm 2 , n = 13 NMJs; UAS-α tub/B24 gal4 = 0.47 ± 0.05 clusters/µm 2 , n = 14 NMJs, p = 0.014). These results suggest that the loss of postsynaptic glutamate receptors observed in sec8 mutants may be secondary to disruption of microtubule networks.

Discussion
Ours is the first detailed examination of any multicellular Sec8 mutant. Sec proteins are thought to play a role in almost every cell type, but are particularly highly enriched in the brain. Therefore, most interest in Sec protein function is within the context of neuronal development and function. Sec8, alone or as part of the exocyst complex, has been implicated in several aspects of neural development and function including: (1) neurite outgrowth [7][8][9][10], (2) calcium-dependent neurotransmitter secretion [11], (3) trafficking of ionotropic glutamate receptors [12][13][14] and (4) regulation of neuronal microtubule assembly [10,15]. We explored each of these areas of potential function in Drosophila sec8 mutants. Surprisingly, we did not see any evidence that Sec8 is required for neurite extension or nerve terminal growth, at least in motor neurons. Instead, sec8 mutant NMJ motor arborizations were morphologically normal at time of hatching, but then developed more branches and more boutons during larval development, compared to controls. This suggests that Sec8 suppresses neurite growth. The sec8 mutant NMJ overgrowth phenotype is unlikely to be an artifact for several reasons: (1) Although our immunoblots showed that 50% of total late embryonic/L1 Sec8 was left after deletion of the gene, Sec8 in the NMJ at the start of larval development was already almost undetectable.
(2) Regardless of whether the amount of Sec8 is reduced to 50% or 0% (the difference between a functional hypomorph or a null), there was no detectable impairment of presynaptic terminal growth. Instead, NMJ growth was enhanced in the sec8 mutants. (3) The NMJ growth enhancement occurred entirely during larval development -when maternal sec8 was fading to its lowest levels. This sec8 mutant NMJ overgrowth phenotype is dramatically different from the phenotype observed in sec5 mutants [7]. Drosophila sec5 mutants showed a slight increase in NMJ growth during L1 development (c.f. Murthy et al., Fig 3B), but then progressive deterioration in the ability to form new motor nerve terminals through later larval development [7]. This suggests, along with other evidence (see below), that metazoan Sec proteins may not always function together as part of a canonical octomeric complex.
Because Sec proteins are required for membrane secretion in yeast, neuronal Sec proteins have long been implicated in neurotransmission, which relies on synaptic vesicle membrane insertion and cycling. However, we did not see any evidence that Sec8 is critical for neurotransmitter secretion, consistent with recently published descriptions of Drosophila sec5 and sec15 mutants [7,21], and sec10 RNAi expression in the fly NMJ [58]. Our result is unlikely to be an artifact of incomplete removal of Sec8 from the synapse, because even though sec8 zygotic null mutants showed substantial maternal contribution of Sec8 protein (~ 50% normal levels overall), immunocytochemistry and confocal microscopy showed that Sec8 was almost completely absent from mutant early larval NMJs. Thus, we were able to examine synaptic function following severe genetic reductions in synaptic Sec8. No mutant evidence to date suggests a role for Sec proteins in neurotransmission. Our data add Sec8 to the list of tested Sec proteins, and strengthen this conclusion.
Mammalian Sec8 has been shown to interact with NMDAtype glutamate receptors and the PDZ-domain protein SAP102 [12]. This interaction appears critical for delivery of NMDA receptors to synaptic membrane in cultured cells [12]. Sec8 also interacts with PSD-95 and SAP-97 [13,14], which are two proteins thought to be important for delivery of non-NMDA (AMPA & kainate) glutamate receptors to postsynaptic membranes. Thus, Sec8 has been implicated in trafficking of all types of glutamate receptors. We observed mislocalization of postsynaptic glutamate receptors in Drosophila sec8 mutants, consistent with a role for Sec8 in glutamate receptor trafficking. However, the mechanism by which Drosophila Sec8 regulates glutamate receptor trafficking may not be the same as the mechanism proposed for mammalian neurons. As mentioned above, mammalian glutamate receptor trafficking by Sec8 is thought to occur via interactions with the PDZ-domain proteins SAP102, PSD-95 or SAP-97. DLG is the sole Drosophila member of the mammalian PSD-95/SAP97/SAP102 protein family [59][60][61]. In Drosophila DLG mutants, only B-type receptor trafficking/ localization is disrupted; trafficking and localization of Atype receptors appear normal [38]. But in the sec8 mutants described here, trafficking of both A-and B-type receptors was affected. Therefore, our evidence suggests that loss of Sec8 does not selectively disrupt a DLG-dependent glutamate receptor trafficking pathway, which would be expected to lead to loss of only B-type receptors.
Exocyst proteins, including Sec8, have recently been shown to associate with microtubules in vitro and in cultured cells, and overexpression of sec proteins inhibits microtubule polymerization [10,15]. Overexpression of Drosophila sec8 cDNA in vivo also caused a noticeable but statistically insignificant reduction in microtubule immunoreactivity, and sec8 loss of function mutants showed dramatically increased microtubule immunoreactivity in NMJs. These results suggest that Sec8, probably in conjunction with other sec proteins, locally inhibits synaptic microtubule network assembly and/or stability.
The misregulation of microtubules in sec8 mutants could be responsible for the glutamate receptor mislocalization we observed, since glutamate receptors, like other transmembrane proteins, are thought to be transported to synapses via microtubule networks [55][56][57]. Consistent with the idea that the receptor trafficking defects are secondary to microtubule misregulation, postsynaptic overexpression of tubulin increased muscle microtubule density and phenocopied the loss of synaptic glutamate receptors measured in sec8∆ 1 mutants. Misregulation of microtubules in sec8mutants might also explain the presynaptic growth defects that we observed in sec8 mutants. Presynaptic growth in the Drosophila NMJ is known to be dependent on microtubule stability in both pre and postsynaptic cells [49][50][51][52][53][54]. Misregulation of microtubules by either pre or postsynaptic Sec8 might therefore be expected to trigger defects in presynaptic growth.
Regulation of microtubules by Sec8 might also explain why expression of a sec8 transgene in sec8 mutant neurons (elav C155 gal4/UAS-sec8; sec8) fails to rescue the synaptic overgrowth (Data not shown), and why overexpression of a sec8 transgene in sec8 mutant muscle (UAS-sec8; 24B gal4 or UAS-sec8; H94 gal4) fails to reverse sec8 mutant receptor trafficking phenotypes (Data not shown). Traditionally, these results might be taken as genetic evidence that our phenotypes may be due to 'background' mutations. But nervous-system-specific expression sec8 (elav C155 gal4/UAS-sec8; sec8) rescues the mutant viability, sec8P1 mutants retain their phenotype in trans to a deficiency (Df(3R)Tpl10) that removes the sec8 gene region, and precise excision of the P-element in sec8P1 mutants completely reverses all synaptic phenotypes (see results). These results all argue that: (1) The sec8 mutant flies die owing to problems in the nervous system but not the NMJ defects, and (2) all NMJ phenotypes are indeed due to specific disruption of sec8.
Considered together, our results suggest that a primary role for Sec8 at synapses may be spatially-restricted regulation of microtubule networks. But we cannot conclude that this is a primary function for other Sec proteins. A growing body of evidence suggests that metazoan exocyst proteins may not always function as a canonical heterooctomeric complex; functionally distinct subcomplexes appear to exist in different cell types or even within the same cell [21,23,[62][63][64]. There are, for example, important differences between the sec8 mutant phenotypes described here and the phenotypes previously described for Drosophila Sec5 mutants with regard to NMJ formation [7,22]. Similarly, Drosophila expressing sec10 RNAi in the neuromusculature showed no NMJ changes, and Sec10 was not detected in the NMJ [58]. More recently, a study of Drosophila Sec15 mutants found more differences between sec mutant phenotypes and argued explicitly for independent Sec protein roles [21]. Thus, several lines of evidence, including expression patterns and phenotypes, suggest that Sec proteins function independently in at least some tissues and/or at different times. Nevertheless, all Drosophila sec mutants show at least partial defects in membrane trafficking, membrane protein distribution and/or cell polarization -processes that rely, at least to some extent, on microtubules. Thus, microtubule regulation may be a common thread tying together Sec protein function in vivo. One might imagine that different subsets of differentially-regulated Sec proteins transiently associate in specific cellular compartments to prune the dynamic microtubule network. This pruning and shaping might ensure that membrane packages are delivered past, or are dropped off at, appropriate places. We think that it is important for future work on Sec proteins to consider this possibility.

Conclusion
Sec8 is abundant in the Drosophila NMJ. Sec8 is required in vivo for regulation of synaptic microtubule formation, and (probably secondarily) regulation of synaptic growth and glutamate receptor trafficking. We did not find any evidence that Sec8 is required for basal neurotransmission.

Genetics
To identify genes required for glutamatergic synapse development, we screened GT1 and SuPor-P transposon insertions from the Berkeley Drosophila Gene Disruption Project [17]. Specifically, we first identified homozygous mutants using the FlyBase Insertions Query Form http:// flybase.bio.indiana.edu/. Approximately 10% (220 of 2185 stocks) of the insertion lines available in March 2003 contained lethal inserts. We examined dechorionated embryos from each of the 220 stocks to ensure that homozygous mutants developed to at least late stage embryogenesis (16-17 h after egg laying, AEL), which is when NMJs begin to form. Of the 220 mutants, 202 developed into morphologically mature embryos (condensed CNS, clear segmentation, trachea, mouth hooks, etc.). These 202 mutants were rebalanced using a chromosomespecific GFP balancer for unambiguous identification of homozygous P-element mutants and then screened immunocytochemically using anti-HRP and anti-GluRIIA antibodies to visualize presynaptic nerve terminals and postsynaptic glutamate receptors, respectively. The remaining 18 stocks were discarded from future consideration. P{SUPor-P}CG2095 KG02723 mutants (herein referred to as 'sec8P1') were identified as a member of the subgroup of mutants with defects in both presynaptic morphology and postsynaptic glutamate receptor expression. A more complete description of this screen, and the mutants identified therein, can be found elsewhere [5,6]. sec8∆1 mutants were created by imprecise excision of P{SUPor-P}CG2095 KG02723 from sec8P1, using standard methods for P-element mobilization [18]. Four hundred and forty-five independent mutant lines of flies were created; approximately 8% of these (35 of 445) were homozygous lethal. The lethal lines were then screened using a PCR-based assay with three pairs of primers. One pair of primers was designed to amplify a portion of sec8 near the P-element insertion. A second set of primers was designed to amplify a portion of the gene CG2082, located immediately 5' to CG2095, and the third set of primers was designed to amplify a portion of gene CG2091, located immediately 3' to sec8. Mutants in which the first (sec8 specific) primer pair did not produce a product but the second and third primer sets did suggested that sec8 was specifically deleted but the neighboring genes were left intact. Eight mutants of this type were identified. Lethal lines containing partial deletions of sec8 were further characterized using several sets of PCR primers. Both forward and reverse primers were designed to every 1 kb of genomic DNA beginning 500 bp upstream and downstream of sec8. Using these primers, one deletion mutant (sec8∆1) was isolated in which the first forward primer (positioned approximately 500 bp 5' of sec8) and the last reverse primer (positioned approximately 500 bp 3' of sec8) produced a product of approximately 1 kb. This PCR product was sequenced to determine the exact location and size of this deletion (sec8∆1).
Precise excisions of P{SUPor-P}CG2095 KG02723 ('revertants') were produced by the same P-element mobilization that produced sec8∆1. Revertants were initially identified based on eye color and reversion of the lethal insertion phenotype, then confirmed by PCR. Measurements from revertants, in which the sec8 P-element was precisely excised, are indicated in the text and figures. For all experiments, homozygous mutant larvae were identified by use of an appropriate GFP-tagged balancer chromosome. 'Control' genotypes in all experiments are w 1118 . Animals were age-matched for all experiments using timed laying plates and morphological staging of latestage embryos.

Molecular biology
Wild-type CG2095 (sec8) cDNA was cloned by RT-PCR amplification of embryonic RNA. Total RNA was isolated using Trizol extraction and reverse transcribed using cDNA-specific primers and cloned. The 3256 bp sec8 cDNA was ligated into the pUAST vector using the KpnI and XbaI sites, and then sequenced for confirmation. Our results are consistent with the predicted gene structure shown in FlyBase, although we noted several polymorphisms. We used our cDNA clone sequence to predict the CG2095 amino acid sequence. Our predicted Oregon R CG2095 amino acid sequence contains three differences compared to the predicted CG2095 translation product as annotated in FlyBase: Oregon R CG2095 encodes an M at amino acid 119, a D at amino acid 328, and a Y at amino acid 669.
Fly transformation was performed by DNA microinjection of embryos using standard methods (Genetic Services, Inc, Cambridge MA). Ten independent UAS-sec8 transgenic strains were created.
Quantitative real-time RT-PCR of Drosophila glutamate receptor subunits was performed as previously described [41]. Briefly, total RNA was isolated from 22-24 h AEL embryos using Trizol extraction and reverse transcribed using sequence specific primers. Real-time RT-PCR was then performed using subunit cDNA-specific primers in an Opticon 2 real-time cycler (MJ Research, Waltham MA), using SYBR green (Molecular Probes, Eugene, OR) for fluorescent measurement of amplicon quantity. Nonspecific fluorescence due to primers was eliminated by measuring fluorescence after a short 'holding step' at a temperature sufficient to melt primer dimers but not desired product (MJ Research Technical Note # 004). As a loading control, actin 5C RNA levels were also measured for every extraction using actin 5C-specific primers, and GluRII RNA levels normalized using this measurement. Each measurement represents the actin 5C c(t) divided by a GluRII c(t), where both measurements were made from the same RNA isolation and RT reaction.

Immunocytochemistry
To generate Sec8 antibodies, rabbit polyclonal antisera was raised against a synthesized peptide composed of Sec8 amino acids 440-460 (GTSNNSDAFKEHRRNAS-DASV). The antiserum was affinity purified and used at 1:500. For staining and microscopy, animals were manually dissected and fixed for 30-60 min in either Bouin's fixative (when Sec8 or GluRII antibodies were used), or 4% paraformaldehyde (for all other staining). Note that the Sec8 antibody recently described in [21] as not showing immunoreactivity at the fly NMJ, actually does so (and shows staining very similar to our antibody) when preparations are fixed in Bouin's fixative. Mouse monoclonal anti-GluRIIA ('8B4D2', Iowa Developmental Studies Hybridoma Bank, Iowa City, IA) was used at 1:100. Rabbit polyclonal anti-GluRIIB and anti-GluRIIC [39] were gifts from Dr. Aaron DiAntonio (Washington University, St. Louis, MO) and used at 1:2000 and 1:3000, respectively. Fluorescently conjugated anti-HRP (Jackson Immunore-search Labs, West Grove, PA) was used at 1:100. Mouse monoclonal anti-CSP was used at 1:200 [65]. Mouse monoclonal anti-Discs Large (DLG) ('4F3', Iowa Developmental Studies Hybridoma Bank, Iowa City, IA) was used at 1:1000. Mouse monoclonal anti-synaptotagmin (SYT) was used at 1:500 [66]. Mouse monoclonal antiacetylated tubulin (Sigma) was used at 1:1000. Goat antirabbit or goat anti-mouse fluorescent (FITC or TRITC) secondary antibodies (Jackson Immunoresearch Labs, West Grove, PA) were used at 1:400. Confocal images were obtained using an Olympus FV500 laser-scanning confocal microscope. All images represent maximum intensity projections of confocal Z-stacks. Image analysis and quantification was performed on maximum intensity projections of Z-stacks using ImageJ software. Threedimensional surface reconstructions were generated from confocal Z-stacks using Amira 3.1 (Mercury Computer Systems, Chelmsford, MA).
Quantification of glutamate receptor cluster size was performed as previously described [37,38,42,67]. Every cluster on a 6/7 NMJ from each animal was measured. Drosophila NMJ glutamate receptor clusters form over the course of hours and then stabilize to form distinct clusters with relatively invariant diameter [37,67]. These diameters stay relatively constant, even throughout larval development [30]. Because changes in the number of postsynaptic receptors within each cluster are linearly proportional to changes in cluster area; cluster area is a quantitative and sensitive optical measure of receptor number in the Drosophila NMJ [30,37,67]. Cluster area was quantified and measured from maximum intensity Z-projections of confocal image stacks, using automated edge finding and area measurement in ImageJ software (NIH, Bethesda MD). This method agrees well with manual measurements [37], is highly reproducible [38,42,67], and involves no experimenter bias for cluster selection because the software selects and measures every bright spot regardless of shape or size. An alternative measurement of glutamate receptor abundance is fluorescence intensity. Although fluorescence intensity measurements gave qualitatively identical results for this study (data not shown), we do not prefer them for embryonic/L1 receptor abundance measurements because intensity measurements assume changes in protein density within individual PSDs (for which we have no evidence) or underestimate differences in receptor abundance due to fluorescence measurement from pixels between individual GluR clusters. Because immunoreactivity for microtubules and the synaptic proteins DLG, CSP and SYT did not show distinct clusters, we quantified their abundance using fluorescence intensity. However, all of these measurements are relative to 'background' fluorescence (measured from non-muscle areas for microtubules, or nonsynaptic areas for DLG, CSP and SYT) in the same image and filter channel [68]. We feel that this is an important wavelength-matched control for variation due to possible differences in staining, mounting of the preparation, or microscope settings.

Immunoblots
After protein isolation for immunoblotting, protein abundance was quantified using Bradford assays, and the same amount of protein was loaded per lane on 6% SDS-polyacrylamide gels. The amount loaded was 20 µg for GluRIIB blots, 50 µg for GluRIIA blots and 20 µg for Sec8 blots. After electrophoresis, protein was transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked with 5% milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl and 0.1% Tween 20) for 1-2 h at room temperature. Anti-Sec8 was used at 1:500 and anti-GluRIIA ('8B4D2' concentrate, Iowa Developmental Studies Hybridoma Bank, Iowa City, IA) was used at 1:200. GluRIIB antibodies usable for immunoblots have not previously been described. To generate Anti-GluRIIB antibodies for use in immunoblots, rabbit polyclonal antisera was raised against synthetic peptides containing C-terminal sequence of GluRIIB: RQSRDSTSTGYSSLEQITSAS-SAKKKK. For immunoblotting, GluRIIB antibodies were used at 1:400. Note that these GluRIIB antibodies are not the same as used for in situ staining; as described above, the GluRIIB antibodies used for in situ staining are described in Marrus et al. [39]. After incubating at 4°C overnight, the membranes were washed with TBS-T. Antimouse HRP-conjugated secondary antibodies were used at 1:5000 for GluRIIA blots and anti-rabbit AP-conjugated secondaries were used at 1:1000 for GluRIIB and Sec8 blots. Protein bands on blots were visualized using enhanced chemiluminescence (GluRIIA) or via a colorimetric reaction (GluRIIB and Sec8). Both GluRIIA and GluRIIB bands are completely eliminated in appropriate mutants (Df(2L) [AD9] and Df(2L) [SP22]) [40] and Liebl, Ng, Sheng, Karr and Featherstone, unpublished).

Electrophysiology
All electrophysiology was performed on the ventral body wall muscle 6 at 19°C. For electrophysiology on first instar (L1) larvae we used a whole-cell patch clamp technique. Third instar larval muscles are too large for whole cell patch clamp. Therefore, we used two-electrode voltage clamp technique for third instar (L3) larval recordings. Both techniques were performed as previously described [36,38,68]. The sEJCs analyzed in this study, in either first or third instar larvae, are unlikely to include events resulting from endogenous action potentials, based on two pieces of evidence: (1) Endogenous action potential evoked activity is much larger, typically patterned, and thus easily recognizable, and (2) Drosophila sEJCs recorded in 0 mM calcium with 5 µm TTX occur with less frequency but are not larger, in both embryos/first instars and third instar larvae [ [68] and unpublished observations]. Therefore, although we do not methodologically exclude any particular type of synaptic event and therefore properly use the conservative term 'sEJC' to describe what we are measuring, all of the events shown and analyzed in this study are likely to represent only true spontaneous monovesicular 'mEJCs', or 'minis'.
For patch clamp electrophysiology on L1 (22-24 h AEL) larvae, animals were manually dissected by gluing (Histoacryl Blue, Braun Germany) them to sylgard-coated coverslips under standard Drosophila saline (135 mM NaCl, 5 mM KCl, 4 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM TES, 72 mM sucrose). A slit was then made along the dorsal midline using a glass capillary pulled to a sharp point, and the body walls glued flat to the coverslip. After dissection, the exposed muscle sheath was enzymatically removed by 30-90s exposure to 1 mg/ml collagenase (type IV, Sigma). Muscle 6 was then whole-cell patch clamped (-60 mV) in standard Drosophila saline (135 mM NaCl, 5 mM KCl, 4 mM MgCl, 1.8 mM CaCl, 5 mM TES, 72 mM sucrose) using standard patch-clamp techniques. Pressure ejection of 1 mM glutamate dissolved in extracellular saline utilized a Picospritzer III (General Valve/Parker Hannifin, Fairfield NJ). Pressure ejection pipettes were approximately the same size and tip diameter as unpolished patch pipettes. Glutamate leak was minimized by continuous back-pressure; no evidence for glutamate leak and subsequent desensitization of receptors was ever observed, and would be identical between genotypes. Data were acquired using an Axopatch 1 D amplifier and a PC running pClamp9 software (Axon Instruments, Union City, CA). Data were subsequently analyzed using Clampfit9 (Axon Instruments).
For two-electrode electrophysiology on third instar (L3) larvae (110-120 hr AEL), Dissections were made as with first instar larvae, except that collagenase was not used. Larval dissections and recordings were also performed under standard Drosophila saline, as described above. Standard two-electrode voltage clamp techniques were used (holding potential -60 mV), as previously described [36]. Electrodes for TEVC were filled with 3 M KCl. Data was acquired using an Axon GeneClamp 500B and a PC running Axoscope software. Data were subsequently analyzed using Clampfit9 (Axon Instruments) In all cases (data from both L1 and L3 animals) sEJCs were detected and analyzed using Clampfit9's template-matching method [69,70], which identifies synaptic events based on shape matching to a data-based ideal template. In Drosophila embryos and L1 larvae, small sEJCs as recorded and analyzed for this study represent a mixture of spontaneous calcium-dependent and calcium-independent monovesicular events. Changing the calcium