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SHORT REPORT

Unconventional molecular regulation of synaptic vesiclereplenishment in cochlear inner hair cells

Christian Vogl1,*, Benjamin H. Cooper2, Jakob Neef1, Sonja M. Wojcik2, Kerstin Reim2, Ellen Reisinger3,Nils Brose2,4,5, Jeong-Seop Rhee6, Tobias Moser1,4,5,* and Carolin Wichmann1,4,7,*

ABSTRACT

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient

vesicle replenishment to indefatigably encode sound. In neurons,

neuroendocrine and immune cells, vesicle replenishment depends

on proteins of the mammalian uncoordinated 13 (Munc13, also

known as Unc13) and Ca2+-dependent activator proteins for

secretion (CAPS) families, which prime vesicles for exocytosis.

Here, we tested whether Munc13 and CAPS proteins also regulate

exocytosis in mouse IHCs by combining immunohistochemistry with

auditory systems physiology and IHC patch-clamp recordings of

exocytosis in mice lacking Munc13 and CAPS isoforms.

Surprisingly, we did not detect Munc13 or CAPS proteins at IHC

presynaptic active zones and found normal IHC exocytosis as well

as auditory brainstem responses (ABRs) in Munc13 and CAPS

deletion mutants. Instead, we show that otoferlin, a C2-domain

protein that is crucial for vesicular fusion and replenishment in IHCs,

clusters at the plasma membrane of the presynaptic active zone.

Electron tomography of otoferlin-deficient IHC synapses revealed a

reduction of short tethers holding vesicles at the active zone, which

might be a structural correlate of impaired vesicle priming in

otoferlin-deficient IHCs. We conclude that IHCs use an

unconventional priming machinery that involves otoferlin.

KEY WORDS: Ribbon synapse, Priming, Tether, Munc13, CAPS,

Otoferlin

INTRODUCTIONThe mechanisms that establish fusion competence of synaptic

vesicles are classically defined as tethering, docking and priming.

In this framework, vesicles are first loosely tethered to the

presynaptic active zone membrane, then closely attach to the

membrane upon docking and finally undergo further maturation

steps to gain full fusion competence. Recent high-resolution

ultrastructural work has indicated that such clear distinctions

of morphological and functional preparatory steps in vesicle

fusion might have been too simple. Instead, protein tethers of

different lengths and numbers have been proposed to establish

vesicular fusion competence (Fernandez-Busnadiego et al., 2010;

Fernandez-Busnadiego et al., 2013; Siksou et al., 2009). In

neurons, neuroendocrine, immune and airway epithelial cells, this

process employs priming factors belonging to the mammalian

uncoordinated 13 (Munc13, also known as Unc13) and Ca2+-

dependent activator proteins for secretion (CAPS) families

(Dudenhoffer-Pfeifer et al., 2013; Imig et al., 2014; Speidel

et al., 2005; Zhu et al., 2008). The Munc13 protein family

includes the neuronal isoforms Munc13-1, Munc13-2, Munc13-3

(also known as Unc13a, Unc13b and Unc13c, respectively)

and brain-specific angiogenesis inhibitor I-associated protein 3

(Baiap3), as well as the non-neuronal Munc13-4 isoform

(Unc13d), whereas CAPS1 and CAPS2 (also known as CADPS

and CADPS2, respectively) constitute the CAPS protein family

(Ann et al., 1997; Augustin et al., 2001; Betz et al., 2001; Brose

et al., 1995; Koch et al., 2000; Shiratsuchi et al., 1998; Speidel

et al., 2003). Munc13s and CAPSs are evolutionarily conserved

(i.e. UNC-13 and UNC-31 in C. elegans, and dUnc13 and dCaps

in Drosophila; Aravamudan et al., 1999; Renden et al., 2001;

Richmond et al., 1999), and genetic deletion causes dramatic

defects, ranging from severe reduction to complete loss of the

readily releasable pool of synaptic vesicles (RRP) and total arrest

of spontaneous and evoked neurotransmission in several cell

types (Augustin et al., 1999; Jockusch et al., 2007; Liu et al.,

2010; Varoqueaux et al., 2002).

Replenishment of the RRP is likely rate-limiting for tonic

neurotransmitter release at ribbon synapses. Governed by

receptor potentials, each inner hair cell (IHC) active zone

transmits acoustic information through graded release of up to

hundreds of vesicles per second. For this challenging task, IHC

synapses must employ mechanisms of vesicle replenishment

that involve otoferlin, a multi-C2 domain protein that is crucial

for exocytosis in cochlear IHCs and vestibular hair cells (Roux

et al., 2006; Dulon et al., 2009; Pangrsic et al., 2012). Otoferlin

is required for hearing (Roux et al., 2006; Yasunaga et al.,

1999) and thought to act as a priming factor and vesicular

Ca2+-sensor for release in IHCs (Johnson and Chapman, 2010;

Pangrsic et al., 2010; Roux et al., 2006). However, which other

proteins contribute to establishing vesicular fusion competence

in IHCs remains to be determined. Here, we combined

functional and morphological approaches to investigate the

roles of Munc13-like priming factors in IHCs. Our data indicate

that the conventional Munc13- and CAPS-dependent priming

machinery of central nervous system (CNS) synapses does not

operate in exocytosis at IHC ribbon synapses.

1Institute for Auditory Neuroscience and InnerEarLab, Department ofOtolaryngology, University Medical Center Gottingen, 37099 Gottingen,Germany. 2Department of Molecular Neurobiology, Max Planck Institute ofExperimental Medicine, 37075 Gottingen, Germany. 3Molecular Biology ofCochlear Neurotransmission Group, InnerEarLab, Department of Otolaryngology,University Medical Center Gottingen, 37075 Gottingen, Germany. 4CollaborativeResearch Center 889, University of Gottingen, 37099 Gottingen, Germany.5Center for Nanoscale Microscopy and Molecular Physiology of the Brain,University of Gottingen, 37073 Gottingen, Germany. 6Neurophysiology Group,Department of Molecular Neurobiology, Max Planck Institute of ExperimentalMedicine, 37075 Gottingen, Germany. 7Molecular Architecture of SynapsesGroup, Institute for Auditory Neuroscience and InnerEarLab, University MedicalCenter Gottingen, 37099 Gottingen, Germany.

*Authors for correspondence (christian.vogl@med.uni-goettingen.de;tmoser@gwdg.de; carolin.wichmann@med.uni-goettingen.de)

Received 31 August 2014; Accepted 16 December 2014

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 638–644 doi:10.1242/jcs.162099

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RESULTS AND DISCUSSIONHearing is unaffected in mouse mutants lacking Munc13 orCAPS priming factorsTo assess the impact of genetic disruption of Munc13 and CAPSproteins on auditory function, we recorded auditory brainstemresponses (ABRs) evoked by short tone bursts and clicks in

knockout (KO) mice for Munc13-1, -2, -3, -4 and Baiap3 as well asCAPS1 and CAPS2. Given that genetic deletion of Munc13-1 andCAPS1 results in perinatal lethality, we recorded ABRs from mice

heterozygous for these genes. We did not observe alterations ofABR thresholds nor changes in amplitudes or latencies of the ABRwave I, reporting the compound action potential of the spiral

ganglion, in any of the mutants when compared to wild-type (WT)littermates (Fig. 1; supplementary material Fig. S1). Moreover, werecorded distortion product otoacoustic emissions to evaluate outer

hair cell function, but did not detect a statistically significantchange for any of the mutant mouse strains suggesting intactcochlear amplification (data not shown). Therefore, disruption ofMunc13 and CAPS does not seem to affect sound encoding in the

cochlea. However, we note that testing the effect of completedeletion of Munc13-1 and CAPS1 in IHCs will require futureexperiments on conditional knockout mice, as the heterozygous

state tested here might provide protein copy numbers that stillsupport normal functionality (Augustin et al., 1999).

Loss of Munc13 or CAPS priming factors does not alter Ca2+

currents and exocytosis of IHCsTo clarify the contributions of the main Munc13 and CAPS

isoforms to IHCs presynaptic function, we analyzed presynapticCa2+ currents and exocytosis in the respective deletion mutantmice. We used an organotypic culture approach to investigate theeffect of genetic deletion of both CAPS1 and CAPS2 (hereafter

CAPS1/2-DKO) or both Munc13-1 and Munc-13-2 (hereafterMunc13-1/2-DKO) on Ca2+-driven exocytosis in IHCs in vitro

(Fig. 2). Cultured organs of Corti appear to mature analogously to

the in vivo situation (Sobkowicz et al., 1982) and are suitable forpatch-clamp recordings of presynaptic function (Nouvian et al.,2011; Reisinger et al., 2011). After a week in culture, the overall

organ of Corti morphology was preserved and IHCs abundantlyexpressed otoferlin (Fig. 2A). When comparing IHC Ca2+

currents from CAPS1/2-DKO and Munc13-1/2-DKO with datafrom WT and otoferlin-knockout (Otof-KO) mice, we did not

detect differences in voltage-dependence, amplitude (Fig. 2B,maximal amplitudes WT, 292625 pA; Otof-KO, 291631 pA,CAPS1/2-DKO: 306630 pA, Munc13-1/2-DKO: 286620 pA;

mean6s.e.m., P.0.05 between all groups) or kinetics (Fig. 2C).Exocytosis was monitored as changes in membrane capacitance(DCm) in response to the maximal Ca2+ influx elicited by

depolarization of varying durations (Fig. 2D–G). Although theDCm values were indistinguishable between WT, CAPS1/2-DKOand Munc13-1/2-DKO IHCs, those of Otof-KO exhibited

dramatically reduced exocytosis, consistent with previous reportsusing acute preparations (Beurg et al., 2010; Roux et al., 2006).Given that exocytosis in IHCs acquires otoferlin-dependencearound P4 in vivo (Beurg et al., 2010), our findings indicate a

functional maturation of IHCs in organotypic culture and furthervalidate this approach for studying IHCs of perinatally lethalmutant mice. Detailed analysis of DCm values for stimuli of 2–

50 ms duration by exponential fitting revealed comparable RRPsize and depletion kinetics in CAPS1/2-DKO and Munc13-1/2-DKO IHCs (Fig. 2H; WT, 26.864.2 fF; CAPS1/2-DKO,

20.965.4 fF; Munc13-1/2-DKO, 31.965.5 fF; P.0.05 between

all groups), whereas the strongly reduced exocytosis of Otof-KOprohibited such analysis. Moreover, we tested presynaptic IHCfunction in mice lacking Munc13-4 and Baiap3, but did not detect

any changes in Ca2+ currents or exocytosis (supplementarymaterial Fig. S1B). We conclude that Munc13-like primingfactors are dispensable for IHC presynaptic function.

Cochlear IHCs apparently lack Munc13-like priming factorsPrevious reports have indicated that Munc13-1 protein is absentfrom chicken cochlear extracts (Uthaiah and Hudspeth, 2010),

Fig. 1. Hearing thresholds remain unaffected in Munc13 and CAPSdeletion mutants. (A) Domain overview of Munc13-like protein isoformshighlighting the conservation of the Munc13 homology domain (MHD;adapted from Koch et al., 2000). C1, C1 domain; C2, C2 domain. ComparableABR thresholds in (B) Munc13-1+/2 and Munc13-42/2, (C) Munc13-22/2,(D) Munc13-32/2 and (E) CAPS1+/2 CAPS22/2 mice and age-matched WTanimals. ABR waveforms (I–V) in response to 80 dB click stimuli are shownfor (F) CAPS1+/2 CAPS22/2 and (G) Munc13-1+/2 mice respectively. nvalues are shown on the figures.

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however, the expression patterns of the remaining Munc13 andCAPS isoforms in the cochlea remain to be established.Therefore, we performed immunostainings with extensively

tested antibodies for all Munc13 and CAPS isoforms (Cooperet al., 2012) on acutely dissected organs of Corti fromhearing P16–P17 WT mice (Fig. 3). Consistent with ourelectrophysiological data arguing against a functional role of

these proteins in presynaptic IHC function, we did not detectspecific Munc13 or CAPS immunofluorescence within IHCs.Rather, immunoreactivities of all tested proteins, including

Munc13-1 but not Munc13-2 were restricted to presynapticterminals of efferent olivocochlear neurons, as evident from

colocalization with the neuronal synaptic vesicle marker synapsin(isoforms 1 and 2), which served as internal positive controls inthese experiments. We did not find a specific immunolabeling for

Munc13-4 in organs of Corti (i.e. a labeling that was absent fromknockout tissue) with currently available antibodies. Although wecannot exclude that there is Munc13-4 expression in IHCs, weconclude, based on our analysis of presynaptic function

(supplementary material Fig. S1B), that this isoform plays aminor – if any – role in vesicular release from IHC active zones.In the present study, we could establish that IHCs seem to operate

without Munc13s or CAPS proteins, a finding that is in line withthe notion that their interacting partners neuronal soluble

Fig. 2. Ca2+ currents, RRP size and sustained exocytosisare conserved in CAPS1/2-DKO and Munc13-1/2-DKO butexocytosis is strongly impaired in Otof-KO animals.(A) Maximum projection of a confocal z-stack taken from a WTorganotypic culture after 7 days in vitro (DIV) immunostained forotoferlin (green) and F-actin (red); filled arrowheads indicateIHCs; clear arrowheads outer hair cells, OHC. (B) Whole-cellCa2+ current–voltage relationships from cultured Munc13-likepriming factor mutants and Otof-KO IHCs at DIV7–8. Voltage-dependence and Ca2+ current amplitudes are retained across allgenotypes. (C) Mean6s.e.m. sample traces of a 50 msdepolarization to the maximum Ca2+ current potential for eachgenotype. (D) Exocytic DCm in response to varying stepdepolarizations and corresponding Ca2+ current integrals showcomparable exocytosis in WT, CAPS1/2-DKO and Munc13-1/2-DKO IHCs. In Otof-KO, exocytosis is dramatically reduced.(E) Mean maximum Ca2+ current and corresponding DCm of therespective groups for a 50 ms depolarization. (F) DCm plotted asa function of Ca2+ current charge shows comparable releaseefficiency between Munc13-like mutants and WT IHCs. (G) InitialDCm were fitted with an exponential function to estimate RRPsizes as quantified in H. All genotypes, apart from Otof-KO,showed comparable RRP amplitudes. *P,0.05; ***P,0.001 forOtof-KO versus WT. n values are shown on the figures.

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N-ethylmaleimide-sensitive-factor attachment receptors (SNAREs)

appear to be absent from IHCs (Nouvian et al., 2011). Based on ourfindings, we propose that the priming machinery of IHCs ismolecularly distinct from conventional neuronal active zones and

likely involves the synaptic ribbon and/or bassoon (Frank et al.,2010; Snellman et al., 2011) and otoferlin (Pangrsic et al., 2010).

Otoferlin is enriched in IHC active zone membranes andregulates vesicular tether formationGiven that IHC exocytosis seems to operate without the classical

priming factors, we aimed to further investigate the mechanismby which otoferlin facilitates vesicle replenishment. Our confocaldata revealed an enrichment of otoferlin at the active zonemembrane (Fig. 4A), a localization analogous to Munc13 and

CAPS priming factors at neuronal active zones. In theseexperiments, we employed an antibody that recognizes anintraluminal C-terminal epitope of otoferlin and observed

otoferlin clustering in the active zone membrane at the base ofthe ribbon that could also be found with immunogold labelings(data not shown). Otoferlin enrichment at the release site might

indicate an involvement in vesicle tethering, which has beenimplicated as an ultrastructural correlate of vesicular fusioncompetence (Fernandez-Busnadiego et al., 2010; Frank et al.,

2010). Therefore, we used electron tomography of IHC synapsesto investigate vesicular tethers at active zones of WT and

otoferlin-deficient IHCs, instantaneously frozen following

stimulation with high K+ to analyze ongoing synaptic activity(Fig. 4B–G). We focused our analysis on filamentous tethersconnecting membrane-proximal vesicles to the presynaptic density

and active zone membrane, as this population has been proposed torepresent readily-releasable vesicles (Fernandez-Busnadiego et al.,2010; Frank et al., 2010). Interestingly, we failed to detect tethers

,5 nm, thought to comprise assembled SNARE complexes(Fernandez-Busnadiego et al., 2010), consistent with a previousreport suggesting that IHC ribbons operate without neuronal

SNAREs (Nouvian et al., 2011). As previously described (Rouxet al., 2006), there was no difference in the number of membrane-proximal and overall ribbon-associated vesicles (data not shown).However, we detected a statistically significant reduction, of

roughly two-thirds, in the fraction of tethers shorter than 10 nm,resulting in significantly increased average tether lengths at Otof-KO active zones (Fig. 4E–H). It is tempting to speculate that the

loss of short tethers is directly related to the exocytosis deficit,analogous to the reduction of short tethers in synaptosomesfollowing treatment with clostridial neurotoxins or hypertonic

sucrose (Fernandez-Busnadiego et al., 2010). Whether otoferlin isa tether constituent and, if so, homophilic interactions of otoferlinmolecules localized to vesicular and plasma membranes are

involved, remains to be investigated in future studies. Additionally,it will be crucial to identify and characterize interaction partners of

Fig. 3. IHCs apparently lack Munc13-like priming factors. Single confocal planes of P16–P17 WT organs of Corti immunostained for (A) Munc13-1(B) Munc13-3, (C) CAPS1 and (D) CAPS2. Calbindin (calb.) was used as IHC marker and synapsin (isoforms 1 and 2) to counterstain olivocochlear efferentpresynaptic terminals. Individual channels are presented alongside for clarity. The inset in D shows a schematic illustration of a typical IHC (blue) with afferent(white) and efferent (red) neuronal innervation showing a single synaptic complex for simplicity.

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otoferlin that might contribute to establishing vesicular fusioncompetence.

In summary, our study provides evidence for an

unconventional molecular priming machinery at cochlear IHCribbon synapses. Using deletion mutants for Munc13 and CAPSpriming factors, we show for the first time that (1) hearing is

intact in the absence of various Munc13-like priming factors, (2)IHC presynaptic exocytosis is normal in Munc13-1/2-DKO andCAPS1/2-DKO mice whose release at glutamatergic CNS

synapses is strongly impaired or completely abolished, and (3)Munc13 and CAPS proteins appear to be absent from IHC

presynaptic active zones. Instead (4), otoferlin appears to berequired to form short tethers (,10 nm) between vesicles andactive zone membranes, providing a candidate mechanism for the

highly efficient replenishment of synaptic vesicles in IHCs.

MATERIALS AND METHODSAnimals, organotypic culture and ABRsAll animal experiments conformed to national animal care guidelines and

were approved by the animal welfare office of Lower Saxony. Wild-type

C57Bl/6 (WT), CAPS1/2-DKO (Jockusch et al., 2007; Speidel et al.,

2005), Munc13-1/2-DKO (Augustin et al., 1999; Varoqueaux et al.,

Fig. 4. Otoferlin is enriched in IHC active zone membranes andplays a role in vesicular tethering. (A) Immunostaining for otoferlin andthe synaptic ribbon marker CtBP2, showing apparent apposition of thetwo proteins at the active zone (arrowheads). Asterisks indicate IHCnuclei. The boxed area is presented as a confocal z-stack of the indicatedribbon, illustrating the clustering of otoferlin in a plane that is segregatedfrom those containing the CtBP2-labeled ribbon. (B) Representativetomographic sections of (Bi) WT and (Bii) Otof-KO ribbon synapses afterstimulation with high K+ to evoke membrane turnover with correspondingrendered models. Blue, active zone membrane; magenta, presynapticdensity; yellow, membrane-associated synaptic vesicles (SVs).(C) Representative images of vesicles connected to the plasmamembrane (PM) through a filamentous tether (clear arrowhead). Despitethe comparable numbers of tethered synaptic vesicles in both genotypes(D), the reduction of short tethers leads to a highly significant shifttowards longer tethers in Otof-KO samples (E), and an increased meantether length (F). (G) Normalized histograms (5 nm binned) and(H) cumulative probability density plots of tether lengths raw data.*P,0.05; ***P,0.001 for Otof-KO versus WT. n values are shown onthe figures.

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2002) and otoferlin-KO (Otof-KO; Reisinger et al., 2011) mice of either

sex were used to prepare organotypic cultures of the organ of Corti as

described previously (Nouvian et al., 2011; Reisinger et al., 2011).

Briefly, organs of Corti were dissected from embryonic day 18–19

mutant or WT mice of either sex in HEPES-buffered HBSS

supplemented with 250 ng/ml fungizone and 10 mg/ml Penicillin G

(Sigma-Aldrich), then placed on CellTakTM-coated coverslips (BD

Biosciences) and incubated in DMEM/F12 with 5% FBS for 7–8 days

prior to electrophysiological characterization.

Munc13-1+/2 (Augustin et al., 1999), Munc13-2-KO (Varoqueaux et al.,

2002), Munc13-3-KO (Augustin et al., 2001) and CAPS1+/2-CAPS2-KO

(Jockusch et al., 2007) as well as Munc13-4-KO (Jinx mice; Jackson

Laboratories) and Baiap3-KO (Wojcik et al., 2013) were used alongside

littermates or age-matched (3–10 weeks old) C57Bl/6 WT mice for testing

ABR thresholds as described previously (Neef et al., 2009).

Patch-clamp of IHCsPerforated-patch recordings were performed on IHCs from either acutely

dissected P14 mice (Munc13-4-KO; Baiap3-KO) or IHCs from cultured

apical organs of Corti (Munc13-1/2-DKO; CAPS1/2-DKO; Otof-KO)

using an extracellular solution composed of (in mM): NaCl (103), KCl

(2.8), MgCl2 (1), HEPES (10), TEA-Cl (35), D-glucose (11.2) and CaCl2(2 for acute preparations, 10 for cultures); ,300 mOsm/l, pH 7.2 and a

Cs-based intracellular solution containing (in mM): Cs-gluconate (130),

TEA-Cl (10), 4-amino-pyridine (10), MgCl2 (0.05) and HEPES (10);

,290 mOsm/l, pH 7.1 freshly supplemented with amphotericin B

(250 mg/ml). We employed an elevated [Ca2+] in order to enhance Ca2+

currents and RRP release kinetics. All experiments were done at 22–25 C

using an EPC10 amplifier with PatchMaster software (HEKA electronics).

Capacitance measurements were performed using the Lindau-Neher

technique (Lindau and Neher, 1988; Moser and Beutner, 2000). Currents

were leak-subtracted with a p/10 protocol (Armstrong and Bezanilla,

1974); IHCs with leak currents exceeding 250 pA at the holding potential

of 287 mV were discarded from analysis. Liquid-junction potentials were

calculated with the Igor Pro LJP calculator and corrected offline.

Immunohistochemistry and confocal microscopyImmunohistochemistry was performed on acutely dissected apical organs

of Corti whole-mount preparations as described previously (Neef et al.,

2009). Specimens were imaged using Leica SP2 or SP5 laser-scanning

confocal microscopes with a 1.4 NA 636 or 1006 oil-immersion

objective, respectively. Images were thresholded for background

subtraction and deconvolved using ImageJ (Schneider et al., 2012).

AntibodiesThe following primary antibodies (supplementary material Fig. S1A)

were used for immunolabellings: polyclonal rabbit anti-Munc13-1,

anti-bMunc13-2 (brain-specific Munc13-2 isoform), anti-ubMunc13-2

(ubiquitous Munc13-2 isoform), anti-Munc13-3 antibodies (Cooper

et al., 2012; Varoqueaux et al., 2005), anti-CAPS1, anti-CAPS2

(Synaptic Systems), guinea pig antibody against both synapsin 1 and 2

(Synaptic Systems), mouse anti-calbindin D28k (Swant) and mouse

or rabbit anti-otoferlin antibodies (Abcam/Synaptic Systems), detected

by species-specific secondary anti-IgG antibodies conjugated to

AlexaFluorH 488, 568 and 647 (Life technologies). Phalloidin–

AlexaFluorH568 (Life technologies) was used to label actin-

containing stereocilia.

Electron microscopy and tomographic reconstructionFor high-pressure freezing, explanted apical-turn organs of Corti from

three WT and two Otof-KO mice (all P14) were placed in 200-mm

aluminium type A specimen carriers filled with stimulation solution

(Pangrsic et al., 2010). Type B lids were dipped in hexadecene (Sigma-

Aldrich) and the specimens were frozen with a HPM100 (Leica).

Freeze substitution was performed in an AFS2 (Leica) as described

previously (Rostaing et al., 2006). For electron-tomography, 250-nm

sections (Ultracut E ultramicrotome; Leica) were collected on formvar-

coated 100 copper mesh grids and post-stained with 4% uranyl-acetate

and Reynold’s lead-citrate. Subsequently, 10-nm gold particles were

applied to the grid. Single or double tilt series were acquired at a JEOL

JEM 2100 transmission electron microscope at 200 kV at tilt angles

ranging from 262–255˚ to +55–+62˚ with 1˚ increments using

Serial-EM software. Tomograms were generated using the IMOD

package etomo and rendered using 3dmod (http://bio3d.colorado.edu/

imod/). Tether lengths were analyzed in 3D from tomograms using

ImageJ by determination of starting (x1y1z1) and ending (x2y2z2)

coordinates in virtual sections and calculating the lengths with the

following formula: ![(x22x1)2+(y22y1)2+(z22z1)2]. Synaptic vesicles

connected to the presynaptic density and active zone membrane were

considered in the analysis.

Statistical analysisData are presented as mean6s.e.m. One-way ANOVA with post-hoc

Tukey were performed on normally distributed data of multiple groups

(assessed by Kolmogorov–Smirnov test); P#0.05 was accepted as

statistically significant. For comparison of fractional data (i.e. tether

length), a x2-test was used.

AcknowledgementsWe thank Jens Rettig for providing Munc13-4-KO mice. We thank ChristianRudiger, Stefan Thom, Sandra Gerke and Christiane Senger-Freitag for experttechnical assistance. Moreover, we would like to express our gratitude to NicolaStrenzke and Tina Pangrsic-Vilfan for supporting the study and critically readingthe manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsC.V., C.W. and T.M. designed the study. C.V. performed electrophysiology. C.V.and B.H.C. performed immunohistochemistry, C.W. performed EM tomography.E.R., S.M.W., K.R. and N.B. performed mouse mutagenesis and provideddiscussion and input into the manuscript preparation. C.V., J.N. and C.W.analyzed the data. C.V., C.W. and T.M. prepared the manuscript.

FundingThis work was supported by an intramural grant of the University Medical CenterGoettingen to C.V. and grants of the German Research Foundation (DFG)through the Collaborative Research Center SFB 889 [projects A2 to T.M, A4 toE.R., A7 to C.W. and B1 to J.R. and N.B.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.162099/-/DC1

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SHORT REPORT Journal of Cell Science (2015) 128, 638–644 doi:10.1242/jcs.162099

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