R E S EA R C H A R T I C L E

Presynaptic Architecture of the Larval Zebrafish Neuromuscular Junction Frederik Helmprobst, Miriam Frank, and Christian Stigloher* Division of Electron Microscopy, Biocenter, University of W€urzburg, 97074 W€urzburg, Germany

ABSTRACT This article shows the ultrastructural architecture of larval zebrafish (Danio rerio) neuromuscular junctions in three dimensions. We compare classical electron microscopy fixation techniques with high-pressure freezing followed by freeze substitution (HPF/FS) in combination with electron tomography. Furthermore, we compare the structure of neuromuscular junctions in 4and 8-dpf zebrafish larvae with HPF/FS because this allows for close-to-native ultrastructural preservation. We discovered that synaptic vesicles of 4-dpf zebrafish larvae are larger than those of 8-dpf larvae. Furthermore, we describe two types of dense-core vesicles and quantify a filamentous network of small filaments

interconnecting synaptic vesicles as well as tethers connecting synaptic vesicles to the presynaptic cell membrane. In the center of active zones, we found elaborate electron-dense projections physically connecting vesicles of the synaptic vesicle pool to the presynaptic membrane. Overall this study establishes the basis for systematic comparisons of synaptic architecture at high resolution in three dimensions of an intact vertebrate in a close-to-native state. Furthermore, we provide quantitative information that builds the basis for diverse systems biology approaches in neuroscience, from comparative anatomy to cellular simulations. J. Comp. Neurol. 523:1984–1997, 2015. C 2015 Wiley Periodicals, Inc. V

INDEXING TERMS: electron tomography; electron microscopy; high-pressure freezing; freeze substitution; synapse

Chemical synapses are highly complex cellular specializations that allow precise and reliable signaling between nerve cells. Neuromuscular junctions (NMJs) constitute special types of synapses that mediate communication between motor neurons and muscle cells. A plethora of specialized proteins sustains precise signaling and therefore synaptic function. For purified preparations of synaptic vesicles alone, which resemble a core component of the synaptic signaling machinery, more than 80 integral membrane proteins have been described (Takamori et al., 2006). Among these is the SNARE complex fusion machinery component synaptobrevin/VAMP1 and the Ca21 sensor synaptotagmin that triggers fast synaptic vesicle exocytosis by repelling complexin from the SNARE complex (Tang et al., 2006). Syntaxin, on the other hand, is an integral membrane protein located in the presynaptic membrane and together with soluble components SNAP-25 and Munc-18 builds up the functional SNARE complex (Hata et al., 1993). In addition to the SNARE complex components, a second protein complex involved in synaptic vesicle fusion has been identified that is thought to facilitate and regulate synaptic signaling (for review see S€udhof, 2012). The components of this complex are enriched within a specialized zone of C 2015 Wiley Periodicals, Inc. V

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the presynaptic membrane where most membrane docked synaptic vesicles are localized and synaptic vesicle fusion is happening at a high rate, the so-called presynaptic active zone (Couteaux and Pecot-Dechavassine, 1970). Identified core components of this second presynaptic complex are RIM, RIM-BP, a-liprins, Munc13, and ELKS (for review see S€udhof, 2012). Notably, RIMs, the Rab3-interacting molecules, play a central role as organizers of active zones by bridging between the synaptic vesicle component Rab3 (Takamori et al., 2006) and the voltage-gated Ca21 channels that generate the trigger for synaptic vesicle fusion by releasing extracellular Ca21 into the presynaptic space, which makes RIMs core components mediating synaptic vesicle docking and priming (Koushika et al., 2001; Schoch et al., 2002; Deng et al.,

Additional Supporting Information may be found in the online version of this article. Grant sponsor: Universit€atsbund W€ urzburg; Grant number: AZ14-48. *Correspondemce to: Christian Stigloher, Division of Electron Microscopy, Biocenter, University of W€ urzburg, Am Hubland, 97074 W€ urzburg, Germany. E-mail: [email protected] Received October 31, 2014; Revised February 13, 2015; Accepted March 4, 2015. DOI 10.1002/cne.23775 Published online April 9, 2015 in Wiley Online (wileyonlinelibrary.com)

The Journal of Comparative Neurology | Research in Systems Neuroscience 523:1984–1997 (2015)

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Zebrafish neuromuscular junction architecture

2011; Kaeser et al., 2011). Although many of the components of the synaptic vesicle fusion machinery and the active zone complex members have been identified, a systematic quantification of the ultrastructural architecture of synapses in a near-native state with high 3D resolution techniques is an important basis for the comprehensive understanding of neural signal transduction. Quantitative data and 3D models of synapses allow for a better interpretation of electrophysiological recordings and form the basis for cellular simulations (Freche et al., 2011; Taflia and Holcman, 2011; Schneggenburger et al., 2012). Despite this, precise synaptic architectural studies are still missing for many model systems. The teleost zebrafish (Danio rerio) has become an important vertebrate model organism for the analysis of motoneuronal development (Westerfield et al., 1986, 1990; Westerfield and Eisen, 1988; Drapeau et al., 2001; Zeller et al., 2002) and function studied by electrophysiology (Wang et al., 2008; Moreno and Ribera, 2009, 2010). Furthermore, the zebrafish is an emerging model for understanding motoneuron disorders such as amyotrophic lateral sclerosis and spinal muscular atrophy (Kabashi et al., 2011; Babin et al., 2014; Patten et al., 2014). NMJs of the zebrafish have been analyzed by classical electron microscopy techniques (Waterman, 1969; Westerfield et al., 1990; Drapeau et al., 2001; Bruses, 2011; Denker et al., 2011). However, a systematic analysis of their 3D architecture with high resolution as provided by electron tomography is missing up to now. We focused our study on two time points, 4 and 8 days postfertilization (dpf), because they represent two stages with different swimming behaviors (Buss and Drapeau, 2001; Drapeau et al., 2002; M€uller and van Leeuwen, 2004; Ingebretson and Masino, 2013). These stages are relevant for electrophysiological studies, Ca21 imaging analysis (Thiele et al., 2014), and behavior as well as neural development (Bally-Cuif and Vernier, 2010) and yet are small enough to be high-pressure frozen as intact animals without dissection (Nixon et al., 2009; Schieber et al., 2010). This allows us to study a nervous system in its physiological state at high resolution with minimal preparation artifacts. Here we show the ultrastructure of NMJs from 4- and 8-dpf zebrafish larvae using high-pressure freezing followed by freeze substitution (HPF/FS) and compare this with samples classically fixed with aldehydes. In accordance with previous studies, HPF/FS preparations ensured better ultrastructural preservation, closer to the native state (Rostaing et al., 2004; Weimer, 2006; Nixon et al., 2009; Schieber et al., 2010; Stigloher et al., 2011). Therefore, we focused exclusively on this technique in subsequent sample preparations.

We systematically quantify the components of the synapse and provide 3D models based on electron tomographic reconstructions. Although the overall architectural features are consistent, we observed a reduction of the mean synaptic vesicle size when comparing synaptic vesicle pools between these two stages. Furthermore, we observed two distinct populations of dense-core vesicles. Additionally, we describe several types of filaments connecting synaptic vesicles in zebrafish NMJs resembling filamentous structures initially described in other model systems. First, we found filaments interconnecting synaptic vesicles resembling structures that were first described 3 decades ago in mammalian neurons (Landis et al., 1988; Hirokawa et al., 1989). More recently, these connecting filaments were imaged by electron tomography in mammals (Siksou et al., 2007), lamprey (Gustafsson et al., 2002), Drosophila (Jiao et al., 2010), and Caenorhabditis elegans (Stigloher et al., 2011). Notably, filaments that closely resemble the type of filaments we describe here have been thoroughly analyzed without freeze substitution by direct electron tomography in the vitrified state in rodent synaptosomes and were named connectors (FernandezBusnadiego et al., 2010). Second, we found filamentous structures connecting synaptic vesicles to the presynaptic membrane that resemble so-called tethers that were also imaged in the aforementioned study (Fernandez-Busnadiego et al., 2010). Third, at the center of active zones, more voluminous electron-dense structures were found that contact synaptic vesicles and connect them to the presynaptic membrane. Similar electron-dense projections have been described with electron tomography in a large diversity at many types of synapses in classically fixed preparations such as in amphibians (Harlow et al., 2001), locusts (Leitinger et al., 2012), and mammals (Nagwaney et al., 2009). Furthermore, these presynaptic dense projections were analyzed in vitrified and freezesubstituted synapses in several model systems such as Drosophila (Fouquet et al., 2009; Jiao et al., 2010), C. elegans (Stigloher et al., 2011; Kittelmann et al., 2013a; Watanabe et al., 2013a), and mammalian hippocampal neurons (Siksou et al., 2007). With quantitative information especially on the vesicle pool and 3D reconstructions of synaptic architecture, our study provides essential parameters for cellular simulations and comparisons with other synapse types in other systems.

MATERIALS AND METHODS Animals Wild-type zebrafish (Danio rerio) larvae of the AB strain (RRID: ZIRC_ZL1) were used and kept on a 14 hours light/10 hours dark cycle at 28.5 C in 30%

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Danieau’s solution (Westerfield, 2000). The animals used in this study were staged according to days postfertilization (Kimmel et al., 1995). All experiments were performed according to the animal welfare regulations of the District Government of Lower Franconia.

Chemical fixation For chemical fixation, zebrafish larvae were anesthetized with tricaine as described previously (Westerfield, 2000) and fixed for 1 hour with 2.5% glutaraldehyde, 0.05 M KCl, and 2.5 mM MgCl2 in 0.05 M cacodylate buffer (pH 7.2). After fixation, larvae were washed with 50 mM cacodylate buffer (pH 7.2) and then fixed with 2% OsO4 in 50 mM cacodylate buffer for 1.5 hours on ice. Larvae were washed with H2O and contrasted with 0.5% uranyl acetate in H2O overnight at 4 C. After a further washing step, zebrafish larvae were stepwise dehydrated through an ethanol series and embedded in epon. Epon-infiltrated samples were polymerized at 60 C for 72 hours.

High-pressure freezing and freeze substitution HPF/FS was performed as described previously for morphological analysis of C. elegans and D. rerio (Weimer, 2006; Nixon et al., 2009; Stigloher et al., 2011), with modifications. 4- and 8-dpf zebrafish larvae were anesthetized with tricaine and transferred into 0.7% low-melt agarose (FMC BioProducts, Rockland, ME) in 30% Danieau’s solution (30 C) and loaded into the freezing chamber [Specimen Carriers Type A (200 lm) for 4-dpf larvae, Type B (300 lm) for 8-dpf larvae and B (0 lm) as lid for both; Leica Microsystems GmbH, Wetzlar, Germany]. With the EM HPM 100 (Leica Microsystems GmbH), larvae were high-pressure frozen at a freezing speed >20,000 K/second and a pressure >2,100 bar. The vitrified zebrafish/agarose pellets were transferred immediately in liquid nitrogen to an EM AFS2 freeze substitution system (Leica Microsystems GmbH). Freeze substitution was conducted as described previously (Stigloher et al., 2011), except that epon was used for embedding and that the infiltrated samples were polymerized at 60 C for 72 hours.

Sectioning and EM preparations Serial sections (semithick sections with a thickness of 150 or 250 nm) were cut through the somites of embedded zebrafish larvae with a Leica EM UC7 and a histodiamond knife (Diatome, Biel, Switzerland). The sections were transferred to pioloform-coated slotted or meshed grids (Plano GmbH, Wetzlar, Germany). The sections were stained and contrasted with 2.5% uranyl

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acetate in ethanol for 15 minutes and Reynolds lead citrate for 10 minutes. A carbon coat of approximately 5 nm thickness was added to the tomography samples with a MED 010 (Balzers Union AG, Balzers, Liechtenstein). For fiducial-aided alignment, 12-nm ProtA-Aubeads (Dianova GmbH, Hamburg, Germany) were bound nonspecifically to both sides of the tomography samples by incubating in an undiluted solution for 5 minutes, followed by a single washing step. HPF/FS samples were checked for ice crystal formation by surveying the preservation of the muscle cells and the nuclei in which ice formation is most apparent and causes clearly discernible artifacts. In case of ice formation, samples were excluded from further analysis.

EM and electron tomography A 200 kV JEM-2100 (JEOL, Munich, Germany) transmission electron microscope equipped with a TemCam F416 4k 3 4k camera (Tietz Video and Imaging Processing Systems, Gauting, Germany) was used for image and tilt series acquisition from 315,000 to 340,000 magnification. We collected tilt series from 28 NMJs from two 4-dpf and 30 NMJs from two 8-dpf high-pressure frozen zebrafish larvae. To show the extent of a whole synapse, we generated three consecutive tomograms from one NMJ of a 4-dpf zebrafish larva. Additionally, four tilt series from NMJs from two chemically fixed 4-dpf zebrafish larvae were collected. The tilt series were recorded from at least 265 to 165 with 1 increments with SerialEM (Mastronarde, 2005). For better resolution, two tilt series were collected from one position with a rotation of the grid by 90 between the two tilt series. The tilt series were aligned and reconstructed with weighted back-projection algorithms, and consecutive tomograms were joined with eTomo, which is part of the IMOD software package (Kremer et al., 1996; IMOD RRID: nif-0000–31686).

Modeling and measurements A complete 3D annotation was performed from five tomograms per stage (4- and 8-dpf larvae) by using 3dmod, which is included in the IMOD software package (Kremer et al., 1996). The organelles of the NMJ were annotated with the “closed” object function by tracing contours around the organelles. For vesicle annotation (excluding multivesicular bodies, which were annotated with “closed” objects because of their irregular shape), the maximum vesicle radius (from the center to the outer membrane) was taken, and the vesicle was annotated with the “scattered” object function in 3dmod thereby creating a sphere. Synaptic clear-core vesicles in direct contact with the presynaptic membrane without a visible filament in between were

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defined as docked vesicles and annotated in a different color to distinguish them from the synaptic vesicles in the vesicle pool. A surface model of objects was generated by meshing contours. Filaments connecting clear-core vesicles with each other or with the membrane were annotated on three different tomogram slices with “open” objects. The cylindrical volume of synapses (the volume of the annotated cell membrane), filament lengths, number of vesicles, and their radii were determined with the program “imodinfo” of the IMOD software package. All measurements were performed with the measurement tool of 3dmod. The size of the synaptic cleft was determined by measuring the thickness of the cleft at 10 positions in the tomogram. The thickness of the cleft was calculated as the mean of these 10 measurements. The thickness of the dense projections in the zdimension was determined with the “open” object function to measure the distance between two points on either end of the structure.

Statistical analysis For statistical analyses the Kolmogorov-Smirnov test (KS test; http://www.physics.csbsju.edu/stats/KS test.html) and the Mann-Whitney U test (U test; http:// elegans.som.vcu.edu/~leon/stats/utest.html) were used. For our analyses, we defined results as highly significant at P < 0.001 and significantly different at P < 0.05.

(Waterman, 1969; Westerfield et al., 1990). We analyzed tomograms from semithick sections (Fig. 1) from chemically fixed (Fig. 1A-D) and high-pressure-frozen (Fig.1E-H) NMJs of 4-dpf zebrafish larvae. In both approaches, larval NMJs appear overall to be similar in semithick sections except that mitochondria are less visible (Fig. 1B,F), but in single tomogram slices (Fig. 1C,D,G,H) the differences between these techniques in ultrastructural preservation are clearly observable. Membranes appear smoother in samples generated by HPF/FS. Furthermore, we noted a difference in the appearance of mitochondria, which are more electron dense in the HPF/FS samples compared with chemically fixed tissues. The filamentous network between vesicles and between vesicles and membranes, which has been described in former studies for different types of synapses from several organisms as well as synaptosome preparations (Landis et al., 1988; Hirokawa et al., 1989; Gustafsson et al., 2002; Siksou et al., 2007; Fernandez-Busnadiego et al., 2010), is clearly visible in larval zebrafish NMJs but seems to be collapsed in chemically fixed tissues. The cytoplasm exhibits a more homogeneous distribution in HPF/FS samples, whereas structureless areas were visible more often in the cytoplasm of chemical fixed samples and mitochondria appeared swollen (Fig. 1C). Because of the better ultrastructural preservation in general and of filamentous structures, mitochondria, and membranous structures in cryoimmobilized and freeze-substituted zebrafish NMJs in particular, we further focused on these techniques to analyze systematically the architecture of zebrafish NMJs.

RESULTS Electron tomography of chemically fixed and HPF/FS samples

Synaptic architecture

HPF/FS provides excellent tissue preservation for studying the fine architecture of synapses (Rostaing et al., 2004; Siksou et al., 2007) because fixation artifacts resulting from slow chemical fixation are reduced by a fast physical immobilization during vitrification. This technique has been established also for larval stages of the zebrafish D. rerio (Nixon et al., 2009; Schieber et al., 2010). We decided first to compare HPF/FS with classically fixed preparations of larval zebrafish NMJs because previous studies of these structures were based solely on chemical fixation (Waterman, 1969; Westerfield et al., 1990; Drapeau et al., 2001; Bruses, 2011; Denker et al., 2011). Additionally, electron tomography can be applied to both of these preparations to obtain a high resolution in 3D. Here we use the combination of these techniques to study the ultrastructure of NMJs of zebrafish larvae. These specialized synapses can be readily identified by using previously published morphological descriptions as reference

We acquired tomograms from 4-dpf and 8-dpf zebrafish larval NMJs and annotated the vesicle pool and all cell organelles as described above to obtain a quantifiable overview of the synaptic architecture in 3D. Figure 2 shows serial tomograms from a 4-dpf zebrafish larva (Fig. 2A,B), which were annotated (Fig. 2C,D) with IMOD, resulting in 3D models (Fig. 2E,F). Within the synaptic cell membrane (light green), vesicles [synaptic vesicles (white), docked synaptic vesicles (red), two types of dense cores (brown and yellow), and multivesicular bodies (turquois) with internal vesicles (purple)], endoplasmic reticulum (light blue), mitochondria (orange), endosomal structures (dark brown), and frequently also microtubules (dark blue) were generally observed (Figs. 2, 3). The electron-dense synaptic cleft with the basal lamina between the NMJ and the muscle was identified as described previously (Waterman, 1969; Westerfield et al., 1990). The mean size of the synaptic clefts was not significantly different in 4-dpf

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Figure 1. Differences in ultrastructural preservation in chemically fixed and cryoimmobilized and freeze substituted samples. The overview shows electron micrographs of a chemically fixed (A) and an HPF/FS-treated (E) synapse (S) of a 250-nm-thick section from a 4-dpf zebrafish larva. Synapses are surrounded by muscle cells (M) with an electron-dense synaptic cleft (white arrows) in between (B,C,F,G). C and G show a tomogram slice per synapse. Mitochondria (asterisks) and synaptic vesicles (black arrows) are annotated. A higher magnification image of the boxed region in C and G is shown in D and H, respectively. Scale bars 5 1 mm A,E; 500 nm in B,F; 200 nm in C,G; 100 nm in D,H.

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Figure 2. Three-dimensional model of an NMJ of a 4-dpf zebrafish larva. Three serial tomograms (600 nm overall thickness) were joined to show the extent of an NMJ. A and B show single slices of the tomograms. The tomograms were annotated in 3dmod (C,D) to highlight cellular structures in the synapse. The synapse is next to a muscle cell (M) with a synaptic cleft (SC) between and surrounded by a membrane (light green). In the synapse, synaptic vesicles (white), mitochondria (orange), endoplasmic reticulum (light blue), two types of dense-core vesicles (brown and yellow), and large multivesicular bodies (turquois) with internal vesicles (purple) were found. Docked synaptic vesicles at the presynaptic membrane are annotated in red. At the active zone we observed electron-dense structures (green), which are surrounded by many docked vesicles. E and F show the whole 3D model of the synapse. The magenta planes in E show the borders, where the three tomograms were joined. Scale bars 5 500 nm.

(64.9 6 14.0 nm, n 5 16 synaptic clefts from 15 NMJs from two individuals, mean 6 SD) and 8-dpf (60.7 6 10.5 nm, n 5 18 synaptic clefts from 18 NMJs

from two individuals, mean 6 SD) zebrafish larvae. Notably, the synaptic cleft size also does not differ significantly (U test) in comparing aldehyde fixation with

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Figure 3. Differences in size of the synaptic vesicles in NMJs from 4-dpf and 8-dpf zebrafish larvae. Tomograms of NMJs from 4-dpf (A) and 8-dpf (E) zebrafish larvae were taken and the structures were annotated (B,F). In the synapse, synaptic vesicles (white), docked vesicles (red) at the cell membrane (light green), mitochondria (orange), endoplasmic reticulum (light blue), endosomal structures (dark brown), microtubules (dark blue), two types of dense-core vesicles (brown and yellow), and large multivesicular bodies (turquois) were found. 3D models are shown either with all structures (C,G) or with only the vesicle pools of the synapses visible (D,H). The measurements of the sizes of the synaptic vesicles pools at 4 and 8 dpf (I) shows that in 4-dpf zebrafish larvae the radius of synaptic vesicles (23.9 6 2.5 nm, n 5 1,095 vesicles, mean 6 SD) is highly significantly larger than that in 8-dpf zebrafish larvae (22.2 6 2.7 nm, n 5 1,020 vesicles, mean 6 SD). Scale bars 5 200 nm.

high-pressure-frozen samples; in classically fixed 4-dpf zebrafish larvae the measured cleft sizes were 63.3 6 8.8 nm (n 5 4 synaptic clefts from four NMJs from two individuals, mean 6 SD). Within the presynaptic active zone electron-dense structures (green) contacting docked and undocked synaptic vesicles were observed (Fig. 2B,D). The fine architecture of similar presynaptic specializations was

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described via electron tomography analysis in synapses of other animals such as rodents, frog, lamprey, fly, and worm (Harlow et al., 2001; Gustafsson et al., 2002; Zhai and Bellen, 2004; Weimer, 2006; Siksou et al., 2007; Nagwaney et al., 2009; Jiao et al., 2010). The most abundant vesicles are synaptic vesicles, followed by dense-core vesicles and, rarely, clathrin-coated vesicles. More specifically, we detected two types of

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Zebrafish neuromuscular junction architecture

dense-core vesicles, which differ morphologically and in size (Figs. 2, 4). Moreover, we observed so-called multivesicular bodies in the NMJs of 4-dpf as well as in 8-dpf zebrafish larvae. The density of the multivesicular bodies was 3.2 6 1.1 vesicles/mm3 (n 5 28 single and double tilt tomograms, mean 6 SEM) in NMJs of 4-dpf zebrafish and 3.7 6 1.3 vesicles/mm3 (n 5 29 single and double tilt tomograms, mean 6 SEM) in NMJs of 8-dpf zebrafish.

The sizes of synaptic vesicles differ between 4- and 8-dpf NMJs We took tomograms of 4-dpf (Fig. 3A) and 8-dpf (Fig. 3E) zebrafish NMJs and created 3D models from the

tomograms (Fig. 3C,D,G,H). With these models, we measured radii from synaptic vesicles (Fig. 3I), from the center to the outer membrane of the vesicle, in 4- and 8-dpf zebrafish larvae (five double tilt tomograms from two separate individuals per age) and observed a significant difference in the vesicle size. Synaptic vesicles in the 4-dpf zebrafish larvae had an average radius of 23.9 6 2.5 nm (n 5 1,095 vesicles, mean 6 SD), whereas synaptic vesicles of 8-dpf NMJs were significantly smaller (P < 0.001, U test) with a radius of 22.2 6 2.7 nm (n 5 1,020 vesicles, mean 6 SD). The density of synaptic vesicles is not significantly different at these two ages. In the 4-dpf larvae the average vesicle density of the NMJs is 2,429.6 6 297.0 vesicles/ mm3 (n 5 13 single and double tilt tomograms, mean6 SEM) and in 8-dpf samples 2,601.4 6 485.9 vesicles/mm3 (n 5 10 single and double tilt tomograms, mean 6 SEM).

Two kinds of dense-core vesicles In the vesicle pools of the zebrafish NMJs two kinds of dense-core vesicles were observed, which differ in size and appearance (Fig. 4). One type of dense-core vesicles (Fig. 4A-C) shows an electron-dense core

Figure 4. Two types of dense-core vesicles differ in size and appearance. Three different tomogram slices through the middle of type 1 dense-core vesicles (DCV) are shown (A-C). Type 1 DCVs have an electron-dense core surrounded by an electronclear structure between the membrane and the core. In D-F three different tomogram slices through the middle of type 2 DCVs with a homogeneous dense structure are shown. Type 1 DCV have a radius (G) of 38.4 6 6.0 nm (n 5 50 vesicles from 26 single and double tilt tomograms, mean 6 SD) in 4-dpf zebrafish larvae and a mean radius of 34.3 6 5.8 nm (n 5 28 vesicles from 16 single and double tilt tomograms, mean 6 SD) in 8-dpf zebrafish larvae, differing significantly [**P < 0.001 (4 dpf) and *P < 0.05 (8 dpf), KS test] from the radius of type 2 DCV of 31.6 6 5.1 nm (n 5 43 vesicles from 18 single and double tilt tomograms, mean 6 SD) in 4-dpf zebrafish larvae and 29.0 6 5.3 nm (n 5 59 vesicles from 24 single and double tilt tomograms, mean 6 SD) in 8-dpf samples. The density (H) of type 1 dense-core vesicles is significantly higher in NMJs of 4-dpf zebrafish, 17.7 6 2.5 vesicles/mm3 (n 5 28 single and double tilt tomograms, mean 6 SEM), than in NMJs of 8-dpf zebrafish, 10.9 6 3.3 vesicles/mm3 (n 5 29 single and double tilt tomograms, mean 6 SEM). The density (H) of type 2 DCVs is 11.0 6 2.1 vesicles/mm3 (n 5 28 single and double tilt tomograms, mean 6 SEM) in NMJs of 4-dpf zebrafish larvae and 21.9 6 4.4 vesicles/mm3 (n 5 29 single and double tilt tomograms, mean 6 SEM) in NMJs of 8-dpf zebrafish larvae. The density of multivesicular bodies does not differ significantly in 4-dpf (3.2 6 1.1 vesicles/mm3, n 5 28 single and double tilt tomograms, mean 6 SEM) and 8-dpf (3.7 6 1.3 vesicles/mm3, n 5 29 single and double tilt tomograms, mean 6 SEM) larvae. Scale bars 5 50 nm.

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surrounded by an electron-clear structure between the membrane and the core (type 1). These vesicles have a mean radius of 38.4 6 6.0 nm (n 5 50 vesicles from 26 single and double tilt tomograms, mean 6 SD) in 4-dpf zebrafish larvae and a mean radius of 34.3 6 5.8 nm (n 5 28 vesicles from 16 single and double tilt tomograms, mean 6 SD) in 8-dpf zebrafish larvae (Fig. 4G). They differ significantly (P < 0.001 in 4-dpf and P < 0.05 in 8-dpf larvae, KS test) from the smaller, completely electron-dense vesicles (type 2; Fig. 4 D-F) with a mean radius of 31.6 6 5.1 nm (n 5 43 vesicles from 18 single and double tilt tomograms, mean 6 SD) in 4-dpf zebrafish larvae and 29.0 6 5.3 nm (n 5 59 vesicles from 24 single and double tilt tomograms, mean 6 SD) in 8-dpf samples. The density (Fig. 4H) of type 1 dense-core vesicles is 17.7 6 2.5 vesicles/mm3 (n 5 28 single and double tilt tomograms, mean 6 SEM) in NMJs of 4-dpf zebrafish, which is significantly higher (P < 0.05, KS test) than in NMJs of 8-dpf zebrafish with a density of 10.9 6 3.3 vesicles/mm3 (n 5 29 single and double tilt tomograms, mean 6 SEM). The type 2 dense-core vesicles have a density of 11.0 6 2.1 vesicles/mm3 (n 5 28 single and double tilt tomograms, mean 6 SEM) in NMJs of 4-dpf zebrafish larvae and 21.9 6 4.4 vesicles/mm3 (n 5 29 single and double tilt tomograms, mean 6 SEM) in NMJs of 8-dpf zebrafish larvae and did not differ significantly.

The filamentous network The synaptic vesicles are interconnected (“connectors,” Fig. 5A,B) and connected to the membrane (“tethers,” Fig. 5C,D) by small filamentous structures described previously in other model systems (Landis et al., 1988; Hirokawa et al., 1989; Siksou et al., 2007; Fernandez-Busnadiego et al., 2010; Stigloher et al., 2011). Synaptic vesicles directly touching the presynaptic membrane without filament between were defined as docked vesicles (Fig. 5E,F). We measured the connector and tether length in 4- and 8dpf zebrafish larvae from five double tilt tomograms per age (two individuals per age). The connector’s length (Fig. 5G) did not differ significantly, with 27.4 6 23.1 nm (n 5 193 connectors, mean 6 SD) in the 4-dpf compared with 27.5 6 21.1 nm (n 5 194 connectors, mean 6 SD) in the 8dpf larvae. The length of tethers at 4 dpf is 51.4 6 25.0 nm (n 5 42 tethers, mean 6 SD), which also did not differ significantly from the 45.7 6 21.1 nm (n 5 57 tethers, mean 6 SD) tethers at 8 dpf.

presynaptic membrane (Fig. 6). Most of these “dense projections” were also in close contact with docked vesicles. In a few NMJs clusters of dense projections were located close to each other, often with docked vesicles in between. For further analysis, we used only those dense projections that could be clearly isolated from surrounding ones. Although the individual shapes show a high level of complexity and diversity in morphological appearance, they typically share a wide base at the presynaptic membrane and a number of branch-like filaments reaching into the presynaptic space, often connected to a synaptic vesicle. Figure 6 shows typical dense projections in 4-dpf (Fig. 6A-F,J) and 8-dpf (Fig. 6G-I) zebrafish. As is evident from the differences between Figure 6A-C and D-F, the number of vesicles connected to the structures shows a high variability. We analyzed seven dense projections from five different synapses from two 4-dpf and five dense projections from four different synapses from two 8dpf zebrafish larvae. The average number of synaptic vesicles connected to a dense projection in 4-dpf zebrafish was 3.0 6 1.8 (n 5 7 dense projections, mean 6 SD) and in 8-dpf zebrafish was 4.4 6 1.7 (n 5 5 dense projections, mean 6 SD), and the average number of docked vesicles connected to a dense projection was 2.6 6 1.6 (n 5 7 dense projections, mean 6 SD) in 4-dpf zebrafish and 0.8 6 0.8 (n 5 5 dense projections, mean 6 SD) in 8-dpf zebrafish. Notably, in a single case we observed a contact between a dense projection and a type 1 dense-core vesicle. For both age groups we measured the width of the base of the structures, the thickness (z-dimension), the length (maximum distance between the center of the base of the structure and its longest filament), and the volume. For 4-dpf zebrafish we measured an average width of 57.5 6 24.6 nm, a thickness of 50.2 6 12.8 nm, a length of 93.0 6 12.8 nm, and a volume of 66.4 6 36.8 3 103 nm3 (n 5 7 dense projections, mean 6 SD). The average width in 8-dpf zebrafish was 68.6 6 22.8 nm, thickness 61.5 6 28.3 nm, length 106.9 6 23.8 nm, and volume 95.2 6 79.4 3103 nm3 (n 5 5 dense projections, mean 6 SD). All results are summarized in Table 1. Within the age groups the measurements showed a high variability, and no significant differences between 4-dpf and 8-dpf zebrafish were observed.

DISCUSSION Electron-dense structures link synaptic vesicles to the presynaptic membrane For the majority of NMJs we observed electrondense structures connecting synaptic vesicles to the

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This article presents a thorough analysis of the ultrastructural architecture of NMJs of larvae of the model organism D. rerio (zebrafish). To gain access to a closeto-native state, we used cryoimmobilization by HPF/FS.

The Journal of Comparative Neurology | Research in Systems Neuroscience

Zebrafish neuromuscular junction architecture

Figure 5. The filamentous network between synaptic vesicles and between the membranes and vesicles. Single tomogram slices are shown, in which synaptic vesicles are interconnected by filaments (connectors; A,B), and connected to the membrane via small filaments (tethers; C,D). Example of a docked synaptic vesicle located directly at the cell membrane without a visible filament between is shown in E and F. In B, D, and F the synaptic vesicles are annotated in white, the connectors in yellow, the tethers in blue, the cell membrane in light green, and the docked vesicles in red. B1, D1, and F1 show 3D models of the annotated structures. The connectors between synaptic vesicles were measured in 4-dpf and 8-dpf zebrafish larvae (G). A magenta/green version of this figure is available as Supporting Information Figure 1. Scale bars 5 100 nm in A-F; 50 nm in B1-F1.

This approach allowed us to cryoimmobilize zebrafish larvae within milliseconds as intact animals and to study a vertebrate synapse without dissection (Nixon et al., 2009). Applying electron tomography allowed us to model and quantify synapses systematically in three dimensions.

Differences between the two fixation techniques We compared the ultrastructural preservation of NMJs in HPF/FS samples with the preservation of classical glutaraldehyde-fixed samples and observed clear differences between these two approaches. First, mitochondria

The Journal of Comparative Neurology | Research in Systems Neuroscience

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appear more electron dense in high-pressure-frozen samples. Second, the cytoplasm has a more homogeneous distribution in cryofixed samples. Third, the membranes appear smoother because volume changes are minimized. These results show, in line with previous studies in other models, that HPF protocols prevent dehydration artifacts and give a better neuronal tissue preservation (Rostaing et al., 2004, 2006). The vesicle size did not differ between classical and HPF/FS samples.

Differences between 4- and 8-dpf zebrafish NMJs We measured a mean size of the synaptic cleft in HPF/FS fish larvae of 4- and 8-dpf of approximately 60 nm, but in classically fixed fish (Drapeau et al., 2001) it was shown that the synaptic cleft in 3-dpf zebrafish larvae is approximately 120 nm. This difference most likely is due to the different age of the zebrafish larvae because the synaptic cleft size did not differ in classically fixed vs. HPF/FS-treated 4-dpf zebrafish or with the different measurement techniques. Although the synapses at those two ages are morphologically comparable overall, the vesicle size of NMJs in 8-dpf HPF/FS zebrafish larvae is decreased compared with 4-dpf larvae. A similar effect was observed during maturation in rats, in which the vesicle size decreased and the number of vesicles increased proportionately (Markus et al., 1987). These results indicate a dependence of vesicle size on the maturation stage of NMJs and might be related to the functional transition of these synapses to control the different swimming behavior in zebrafish (Buss and Drapeau, 2001; Drapeau et al., 2002; M€ uller and van Leeuwen, 2004; Ingebretson and Masino, 2013).

Dense-core vesicles Although synaptic vesicles differed only in size, dense-core vesicles appeared in two morphologically distinct populations. Dense-core vesicles of type 1 resemble the characteristics of 80 nm PiccoloBassoon transport vesicles as observed in cultured mammalian hippocampal neurons (Zhai et al., 2001; Shapira et al., 2003). It will be an important next step to analyze whether these vesicles show immunoreactivity for active zone components and/or neuropeptides and how far both populations of dense-core vesicles can be discerned from their content. In contrast to previous reports regarding mammalian neurons (Vaughn, 1989), we did not observe special electron-dense spicule-like structures emanating systematically from dense-core vesicles.

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Figure 6. Dense projections link synaptic vesicles to the presynaptic membrane. Shown are tomograms of dense projections in 4-dpf (A,D) and 8-dpf (G) zebrafish. They were segmented (B,E,H) and rendered into 3D models (C,F,I). Light green, presynaptic membrane; dark green, dense projection; white, synaptic vesicles; red, docked vesicles. All segmentations and 3D models show only the vesicles directly connected to dense projections. A-C and DF: Typical dense projections in 4-dpf larvae. G-I: Dense projections found in 8-dpf larvae. J: Same tomogram as in D but showing all dense projections found. For a better overview, all vesicles are shown as transparent (J). A magenta/green version of this figure is available as Supporting Information Figure 2. Scale bars 5 50 nm in in A,B,D-H,J; 100 nm in C,I.

Filamentous network By applying electron tomography we observed and quantified a filamentous network interconnecting vesicles and connecting the vesicles to the membrane. With a mean length of approximately 30 nm, the vesicleinterconnecting filaments in zebrafish are comparable to the filaments that were first described in frog and mouse (Landis et al., 1988; Hirokawa et al., 1989). Because of the size range of 30-60 nm and the distribution measured in these studies, it was assumed that a component of these filaments could be synapsin (Landis et al., 1988; Hirokawa et al., 1989). Later these vesicle-interconnecting filaments were also described with different electron

The Journal of Comparative Neurology | Research in Systems Neuroscience

Zebrafish neuromuscular junction architecture

TABLE 1. Characterization of Dense Projections1

4 dpf

Mean SD 8 dpf

Mean SD

Number of synaptic vesicles

Number of docked vesicles

Width (nm)

Thickness (nm)

Length (nm)

Volume (103 nm3)

6 3 4 1 4 2 1 3.0 1.8 7 3 5 3 4 4.4 1.7

3 5 3 1 0 3 3 2.6 1.6 2 1 0 0 1 0.8 0.8

51.4 53.3 107.3 40.1 42.4 70.5 37.4 57.5 24.6 95.2 84.5 40.5 50.6 72.1 68.6 22.8

53.6 54.6 65.5 31.2 61.2 50.7 35.0 50.2 12.8 102.1 43.7 44.8 36.6 80.3 61.5 28.3

101.5 107.4 90.8 80.9 105.7 91.4 73.4 93.0 12.8 114.8 81.0 84.6 117.2 137.3 106.9 23.8

128.2 75.1 94.1 27.6 67.2 43.3 29.0 66.4 36.8 233.4 52.9 42.2 56.2 91.5 95.2 79.4

1

Variability in the number of synaptic and docked vesicles at the dense projections from 4-dpf (n 5 7 dense projections from five different synapses from two individuals) and 8-dpf (n 5 5 dense projections from four different synapses from two individuals) zebrafish neuromuscular junctions along with the width, the thickness in the z-dimension, the length, and the volume of the dense projections.

microscopy fixation and imaging techniques in other synapses (Gustafsson et al., 2002; Siksou et al., 2007; Fernandez-Busnadiego et al., 2010; Stigloher et al., 2011). Notably, these filaments were still visible in triple synapsin knockout mice (Siksou et al., 2007), indicating that there might be more than one protein involved in vesicle-interconnecting filaments. The tethers connecting vesicles to the cell membrane that we described in zebrafish NMJs show a wide range in length. This type of filament was previously described by using electron tomography in rodents (Siksou et al., 2007), and direct cryoelectron tomography studies in rodent synaptosomes also showed a wide range of lengths between 5 nm and 120 nm. This led to the hypothesis that different types of tethers exist (Fernandez-Busnadiego et al., 2010).

Dense projections A third type of filamentous structures that has been described at synapses are presynaptic dense projections emanating from the presynaptic membrane. As previously reported for several other types of synapses from different models, we also could observe dense projections in the center of active zones. The application of electron tomography allowed us to describe these elaborate structures in 3D. Comparable to our previous observations of dense projections from NMJs in C. elegans (Stigloher et al., 2011), dense projections in zebrafish larvae consist of a central, dense structure with fine filamentous arms emanating from the central core. In contrast to the more compact C. elegans dense projections, in zebrafish larvae these

structures show a more tree-like shape. In their treelike appearance zebrafish larval NMJs are more reminiscent of dense projections that have been described for Drosophila, called T-bars because of their apparent shape in classically fixed samples (for review see Wichmann and Sigrist, 2010). Interestingly, the T-like shape is less prominent in HPF/FS samples (Fouquet et al., 2009). In contrast to the highly organized arraylike structural organization of frog NMJs (Harlow et al., 2001), zebrafish larval NMJs appear not to be as regularly organized. Because our analysis was focused on larval stages, we cannot judge whether the regularity of the dense projections might arise later during development and therefore might be due to the maturation stage or to the special neuroanatomical and physiological demands related to the principally different types of muscles. Immunoelectron microscopy studies of the molecular components of dense projection structures in locusts (Leitinger et al., 2012) and Drosophila (Fouquet et al., 2009) reveal the ELKS/CAST homolog Bruchpilot as one central component of this structure. Furthermore, similar studies in C. elegans identified UNC-10/RIM, SYD-2/liprin, and UNC-13 (Yeh et al., 2005; Weimer et al., 2006) as dense projection components, and a genetic disruption of UNC-10/RIM or SYD-2/liprin leads to a delocalization of docked vesicles away from dense projections (Stigloher et al., 2011), indicating the physiological significance of dense projections as synaptic organizers mediating synaptic efficiency by linking synaptic vesicles to presynaptic voltage-gated Ca21 channels (Harlow et al., 2001; Fouquet et al., 2009).

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Next, it will be interesting to test the activity dynamics of the filamentous network of zebrafish NMJs using optogenetic tools, which will allow control of the activation state, and to describe activity-dependent structural changes. The usefulness of the combination of optogenetics with cryopreservation for EM analysis has been recently elegantly shown in C. elegans (Kittelmann et al., 2013b; Watanabe et al., 2013a) and mammalian hippocampal synapses (Watanabe et al., 2013b), but a precise analysis of the activity-dependent changes in the filamentous network interconnecting synaptic vesicles is still missing.

ACKNOWLEDGMENTS We thank M. Schartl for the generous use of the fish facility. Furthermore, we are very thankful to G. Krohne, A. Wehman, D. Liedtke, S. Markert, R. Kittel, C. Wichmann, J. Reingruber, D. Holcman and C. Lillesaar for supportive comments on the manuscript and fruitful discussions throughout the project. We further thank C. Gehrig and D. Bunsen for excellent technical support.

CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest.

ROLE OF AUTHORS All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: FH, CS. Acquisition of data: FH, MF. Analysis and interpretation of data: FH, MF, CS. Drafting of the manuscript: FH, MF, CS. Critical revision of the manuscript for important intellectual content: FH, MF, CS. Statistical analysis: FH, MF, CS. Study supervision: CS.

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