![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
*
Michèle S. Sons,2,
Jaap J. Plomp,2 and
Nils Brose1
Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Göttingen, Germany,1 Departments of Neurology and Neurophysiology, Leiden University Medical Center, Leiden, The Netherlands2
Received 13 February 2005/ Returned for modification 10 April 2005/ Accepted 26 April 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Before fusion can be initiated, synaptic vesicles must be primed into a fusion-competent state (for review, see references 16, 33, and 37). Members of the Munc13 family, mammalian homologues of Caenorhabditis elegans Unc-13 (15), play an essential role during this priming reaction (2, 3, 34, 35, 38). At the molecular level, synaptic vesicle priming is thought to depend on a conformational switch of the SNARE protein syntaxin 1 from a closed conformation, which prevents SNARE complex assembly, to an open conformation, which permits it (19). It is believed that Munc13 plays an important role during this conformational switch, since the overexpression of an open syntaxin mutant in C. elegans bypassed the strict requirement for Unc-13 (35). In C. elegans, Unc-13 is essential for vesicle priming at both cholinergic and -aminobutyric acid-ergic (GABAergic) synapses (14, 28, 35).
In mammals, the Munc13 protein family comprises three highlyhomologous members, Munc13-1, bMunc13-2/ubMunc13-2 (splice variants of the Munc13-2 gene), and Munc13-3 (16), which are differentially distributed in the brain (4) and confer differential short-term plasticity characteristics to the synapses they equip (25, 38). Transmitter release from both glutamatergic and GABAergic neurons in the hippocampus is strictly dependent on Munc13 function. In the absence of Munc13-1 and Munc13-2, these neurons show neither spontaneous nor evoked synaptic release events, yet develop normal numbers of synapses which contain an electrophysiologically normal postsynaptic AMPA (
-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid) and GABA receptor complement, but exhibit a broader active zone (45). These findings led to the conclusion that genesis and assembly of synapses between hippocampal nerve cells are largely independent of synaptic activity. Rather, synaptogenesis in the central nervous system may follow a default developmental program that is only modulated, stabilized, and refined by synaptic activity (45).
In many aspects, the neuromuscular synapse, which uses acetylcholine as a neurotransmitter, is similar to central synapses and therefore a widely used model for the study of synaptogenesis. The formation and maturation of the neuromuscular junction (NMJ) are known to rely in part on activity-dependent signals. Initially, evidence in support of this view was obtained in studies where the developmental role of synaptic transmission at the NMJ had been examined using anticholinergic or activity-blocking drugs (reviewed in references 13 and 30). More recently, genetic studies on mutant mice lacking choline acetyltransferase (ChAT), the enzyme responsible for producing acetylcholine, provided compelling evidence for the requirement of neurotransmitter release in NMJ formation (13, 30).
Based on our observations in the central nervous system (45) and the fact that Munc13-deficient mice are completely paralyzed, we expected to find a total blockade of transmitter release at the NMJ in the absence of Munc13s. We report here the unexpected finding that neuromuscular synaptic transmission is not entirely abolished in the absence of Munc13s. Nevertheless, the morphology of the NMJ shows abnormalities comparable to those seen in ChAT deletion mutant mice. We characterize the features of the neuromuscular apparatus in Munc13-deficient NMJs and discuss the role of different types of synaptic activity in regulating synaptogenesis at NMJs and the function of Munc13s at peripheral and central synapses.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Animal preparation. For spinal cord preparations, E18.5 embryos were fixed by perfusion with 4% paraformaldehyde in phosphate buffer. The spinal cord (cervical levels 3 to 5) was then dissected out under a binocular. For diaphragm preparations, E18.5 embryos were decapitated and the ribcage was quickly isolated and fixed by immersion (2 to 12 h). Subsequently, the diaphragm muscle was taken out and further processed for staining.
Histology. In toto staining of E18.5 embryos for bone and cartilage was performed by immersion fixation of the eviscerated embryo in absolute ethanol (4 days) and then acetone (3 days). After several washes in water, embryos were stained for 10 days in a solution containing 0.015% alcian blue, 0.005% alizarin red, 5% acetic acid, and 93% ethanol. After washes in water, samples were kept in 20% glycerol-1% KOH for 16 h at 37°C and then at room temperature until cleared. Samples were stored in 20% glycerol.
Western blotting. The presence of Munc13 isoforms at the NMJ was assessed by Western blotting of muscle membranes that were prepared as follows. The diaphragm muscles from 20 newborn mice were dissected out under a binocular and flash-frozen in liquid nitrogen. Diaphragms were then thawed; homogenized in buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 300 mM sucrose, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin; and centrifuged at 1,000 x g at 4°C for 10 min. The supernatant was further centrifuged at 15,000 x g at 4°C for 20 min, and the resulting pellet was resuspended in Laemmli sample buffer and analyzed in parallel with newborn mouse brain or lung homogenates (positive control) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20 µg per lane). Blots were probed with Munc13 isoform-specific rabbit polyclonal antibodies that were raised against recombinant protein fragments (Munc13-1, residues 3 to 317; bMunc13-2, residues 1 to 305; ubMunc13-2, residues 182 to 407; Munc13-3, residues 294 to 574; BAP3, residues 9 to 181; and Munc13-4, residues 889 to 1088).
Immunocytochemistry.
Fifty-micrometer-thick free-floating vibratome sections of the spinal cord were made at the cervical level and either stained for the vesicular acetylcholine transporter (vAChT) with a rabbit polyclonal antibody (1:500; Chemicon) or Nissl stained. Free-floating diaphragms from mutant and control mice were incubated with -bungarotoxin-Alexa 568 (1:2,000; Molecular Probes) or with antibodies against synapsin (rabbit polyclonal, 1:500; Synaptic Systems) or S-100 (mouse monoclonal, 1:500; DAKO), to visualize acetylcholine receptors, presynaptic terminals, and Schwann cells, respectively. Acetylcholinesterase activity was visualized histochemically by incubation of the fixed diaphragms for 30 min at 37°C in 0.5 mM 5-bromoindoxyl acetate (23). All preparations were used as whole mounts.
Imaging. Fluorescent images were acquired on a Zeiss Axiovert 200-LSM 510 confocal laser-scanning microscope, and bright-field images were obtained with a Camedia (Olympus) digital camera fixed on a Leica binocular (for embryos) or a Zeiss Axiophot upright microscope (for Nissl staining). Alternatively, fluorescent and bright-field images were acquired on an Olympus BX61 microscope with an F-view (EsiVision) digital camera coupled to image acquisition and analysis software (EsiVision).
Ultrastructural analysis. Ultrastructural investigation of the right phrenic nerve and whole-diaphragm muscle synapses was carried out on samples that had been fixed by immersion with 4% paraformaldehyde and 0.5% glutaraldehyde and classically processed for epoxy embedding with Durcupan (ACM; Fluka). Fifty-nanometer-thick sections were contrasted and observed in a LEO912AB transmission electron microscope, and digital pictures were taken with a Proscan charge-coupled device camera coupled to the EsiVision software, which was also used for quantitative analysis.
Electrophysiology.
Ex vivo electrophysiological measurement of acetylcholine release was performed at 26 to 28°C on NMJs of diaphragm nerve-muscle preparations from Munc13-1/2-DKO, Munc13-1/2/3-TKO, and control E18 embryos. Muscles were dissected and mounted in Ringer's medium (containing the following in mM: NaCl, 116; KCl, 4.5; CaCl2, 2; MgSO4, 1; NaH2PO4, 1; NaHCO3, 23; glucose, 11, pH 7.4, prebubbled with 95% O2/5% CO2). Muscle fibers were impaled at the endplate region with a 20- to 40-M
glass capillary microelectrode connected to standard recording equipment (31). Intracellular recordings of miniature endplate potentials (MEPPs), the spontaneous depolarizing events due to uniquantal acetylcholine release, were made at different NMJs within the muscle. The phrenic nerve was stimulated supramaximally via a suction electrode. The resulting muscle contraction was visually monitored, and muscle action potentials, if present, were recorded. To be able to record evoked synaptic responses (endplate potentials [EPPs]) in control preparations, muscle fibers were cut alongside the endplate region to induce depolarization to –20 to –40 mV (8). This inactivates Na+ channels so that muscle action potentials and the ensuing contractions no longer occur and the underlying EPP can be recorded. In Munc13-DKO/TKO muscles, this procedure was not necessary because depolarization to –20 to –40 mV often occurred spontaneously after impalement with the microelectrode. Munc13-DKO/TKO fibers were more fragile and thinner than controls and probably became damaged by the impalement. Also, synaptic recordings were much less disturbed by contraction of neighboring fibers, because these were much less vigorous than in control muscles. From each NMJ, 11 to 144 responses to nerve stimulation at 0.3 Hz were recorded. The mean amplitudes of EPP and MEPP recorded at each NMJ were linearly normalized to –75 mV resting membrane potential. From the grand mean values of each muscle, the number of acetylcholine vesicles released per nerve impulse (i.e., the quantal content) was calculated by dividing the mean EPP amplitude by the mean MEPP amplitude.
In intact control and Munc13-DKO/TKO muscles, MEPPs were recorded before and after application of 2.5 nM -latrotoxin (Alomone Laboratories, Jerusalem, Israel) or 0.5 M sucrose, to probe the acetylcholine vesicle pool available for immediate release. In these experiments, tetrodotoxin (1 µM; Sigma-Aldrich, Zwijndrecht, The Netherlands) was added to reduce the spontaneous contractions of fibers which can occur in embryonic muscle. All electrophysiological data are given as group mean values ± standard error, with n as the number of muscles per group and 1 to 20 NMJs sampled per muscle.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Munc13-1/2 double-deletion mutant mice (Munc13-1/2-DKO) and Munc13-1/2/3 triple-deletion mutant mice (Munc13-1/2/3-TKO) studied here showed even stronger phenotypic alterations, whereas Munc13-2/3 double-mutant mice (Munc13-2/3-DKO) were viable and fertile, indicating a dominant role of Munc13-1 in mice. Munc13-1/2-DKO and Munc13-1/2/3-TKO mice were morphologically indistinguishable from each other, and had identical phenotypes with respect to neuromuscular synaptic structure and function (see below). This could be due to the fact that NMJ axon terminals contain Munc13-1 and ubMunc13-2, but neither bMunc13-2 nor Munc13-3, as determined by Western blot analysis of muscle membrane preparations (Fig. 1A). Therefore, results obtained from Munc13-1/2-DKO and Munc13-1/2/3-TKO mice were pooled and subsequently referred to as "Munc13-1/2-DKO" for clarity. More distantly related Munc13 homologues are either faintly detectable (Munc13-4; molecular mass, 112 kDa) or not detectable (BAP3; molecular mass, 125 kDa) in muscle membrane preparations (Fig. 1A). Because Munc13-1/2-DKO mice were often born dead, all experiments were carried out on E18.5 embryos, whose central and peripheral nervous systems are developed extensively and which can be recovered alive upon hysterectomy. Munc13-2-KO and Munc13-2/3-DKO littermates were used as controls as they were indistinguishable from wild-type animals with respect to neuromuscular synaptic transmission.
|
The total paralysis we observed in Munc13-1/2-DKO embryos indicated a profound defect at the NMJ in addition to the central nervous system dysfunction seen in these mice (6, 45). To examine this in more detail, we investigated the structure and function of the NMJ using the well-characterized phrenic nerve/diaphragm muscle preparation as a model system.
Muscle morphology in Munc13-1/2 double-deficient mutants. The fragile appearance of the Munc13-1/2-DKO embryos was paralleled by an abnormally thin musculature. In E18.5 embryos, the diaphragm muscle appeared fully developed along its rostro-caudal axis, but its outermost edges indicated an impaired lateral extension of myotubes (Fig. 2A). Moreover, muscle fibers were not strictly aligned and formed intermingled bundles (Fig. 2A). Muscle cells were more loosely attached to each other, on average smaller than in diaphragms of control littermates (muscle cell area, 389 ± 25 µm2, n = 104, control, versus 136 ± 5 µm2, n = 227, Munc13-1/2-DKO), and exhibited centrally localized nuclei that had apparently not migrated to the cell periphery (Fig. 2B), indicating a maturation delay or defect of some of the myotubes. In addition, many mostly oversized blood vessels ran throughout the diaphragm (Fig. 2B), possibly due to a lack of muscle tone.
|
|
-bungarotoxin-binding acetylcholine receptors, and S-100-expressing Schwann cells were carried out. In control as well as in Munc13-1/2-DKO diaphragms, all axon terminals were juxtaposed to acetylcholine receptor clusters, and vice versa, and all terminals were ensheathed by Schwann cells (Fig. 4). However, acetylcholine receptor and synapsin stainings showed that motor endplate units in the Munc13-1/2-DKO diaphragm were only poorly aligned along the midline of the diaphragm and no longer confined to it, but rather distributed as a large array of clusters (Fig. 4). Quantitative analyses showed that the area occupied by endplates (as defined by -bungarotoxin-labeled acetylcholine receptor clusters) at the midline of the diaphragm was larger in the Munc13-1/2-DKO mice than in the control mice (4,984 ± 1,150 µm2 per 0.2 mm2, n = 5, in Munc13-1/2-DKO mice versus 2,144 ± 436 µm2 per 0.2 mm2, n = 5, in control mice, P < 0.05). In addition, the number of synapses was clearly increased in the Munc13-1/2-DKO diaphragm mouse (not shown).
|
|
Surprisingly, and in contrast to glutamatergic and GABAergic synapses in hippocampal neurons, intracellular recordings of MEPPs revealed that neurotransmitter release is not completely abolished at the NMJs of Munc13-1/2-DKO mice. MEPP amplitude (2.89 ± 0.28 mV, n = 15 muscles) was not statistically significantly different from that in control embryos (2.96 ± 0.28 mV, n = 15, P = 0.86), and MEPP frequency was more than doubled in Munc13-1/2-DKO embryos (4.42 ± 0.60 per min, n = 15), as compared to that in controls (1.80 ± 0.34 per min, n = 15, P < 0.001; Fig. 6 A). -Latrotoxin elicits massive asynchronous uniquantal acetylcholine release through exocytosis from all synaptic vesicles that are fusion competent and therefore are probably in a primed state. Application of this toxin revealed that this type of neurotransmitter release is strongly impaired at NMJs of Munc13-1/2-DKO embryos. MEPP frequency was 591 ± 137 per min (n = 8) in Munc13-/2-DKO NMJs and 1,996 ± 500 per min (n = 7, P < 0.05) in control NMJs (Fig. 6B). MEPP amplitudes were similar in Munc13-1/2-DKO embryos (4.00 ± 0.6 mV, n = 6) and controls (2.81 ± 0.30 mV, n = 7, P = 0.09). Like -latrotoxin, application of hypertonic sucrose solution, which triggers the release of fusion-competent synaptic vesicles, induced much lower MEPP frequencies in Munc13-1/2-DKO embryos (144 ± 66 per min, n = 5) as compared to controls (619 ± 100 per min, n = 7, P < 0.01) (Fig. 6C). Unexpectedly, sucrose treatment reduced MEPP amplitude to 1.33 ± 0.24 mV (n = 5) compared to 3.35 ± 0.23 mV (n = 7) in the controls. Thus, the asynchronous uniquantal acetylcholine release induced by -latrotoxin or hypertonic shock is severely reduced at Munc13-1/2-DKO NMJs.
|
Ultrastructural characteristics of NMJs in Munc13-1/2 double-deficient mutants. At the ultrastructural level, well-formed synapses were observed in both control animals and Munc13-1/2-DKO mice. Synapses in the mutants tended to contain more boutons than control synapses, which may be a correlate of the increased complexity of innervation and the larger endplate size observed in the mutants at the light microscopic level (Fig. 4). Synaptic boutons at NMJs of Munc13-1/2-DKO mice contained normal-sized small synaptic vesicles but also dense core and clathrin-coated vesicles (Fig. 7). Boutons in mutant synapses were aligned with postsynaptic densities in muscle cells and exhibited clusters of small synaptic vesicles that were occasionally observed along the plasma membrane or docked at the active zone (Fig. 7, insert). Pre- and postsynaptic membranes were continuously juxtaposed on each other and separated by a well-developed basal lamina. In the control samples, many synapses showed junctional folds reflecting a normal maturation process. In contrast, small invaginations, but no deep folds, were observed at the postsynaptic membranes of NMJs in Munc13-1/2-DKO mice (Fig. 7, insert).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We found the murine NMJ to contain Munc13-1 and ubMunc13-2, but not bMunc13-2, Munc13-3, or the more distantly related BAP3, and only trace amounts of Munc13-4. The two Munc13 isoforms expressed at the NMJ are the most closely related Munc13 variants. In contrast to other family members, they do not only share the highly conserved C-terminal region but also have highly homologous N-terminal regions which contain a C2 domain that binds the active zone components RIM1 and RIM2 (11) and a calmodulin binding site (25). Thus, Munc13-1 and ubMunc13-2 may interact with the same protein partners and have similar basic functions, and mutual compensation upon loss of one of the two isoforms is highly likely at the NMJ, as was also reported for hippocampal GABAergic synapses (45). Nevertheless, Munc13-1 and ubMunc13-2 differentially modulate short-term plasticity at hippocampal synapses (25, 38), and their coexpression at the NMJ may allow for the tuning of presynaptic molecular mechanisms over a wide range of synaptic activity rates in order to guarantee high fidelity of synaptic transmission.
Using the diaphragm NMJ as a model system, we found that upon genetic deletion of Munc13s (Munc13-1, -2, and -3) and in the absence of significant levels of the related Munc13-4 and BAP3 proteins, evoked synaptic transmission is strongly reduced while spontaneous release persists, and the NMJ system exhibits all classical developmental aberrations that are typically observed upon complete block of spontaneous and evoked synaptic transmission (13, 30). In the light of previous studies on the function of Munc13s at central synapses, two of our findings at the NMJ are very unexpected: (i) synaptic vesicle priming in the NMJ appears to be partially independent of bona fide Munc13s (Munc13-1, -2, and -3); and (ii) despite the quite large spontaneous transmitter release activity at Munc13-1/2-DKO NMJs, the innervation of the diaphragm exhibits the same developmental aberrations that are also observed in the complete absence of NMJ synaptic transmission (13, 30).
Munc13-independent synaptic vesicle priming at the NMJ. Synaptic transmission at glutamatergic and GABAergic synapses of murine hippocampal neurons is strictly dependent on the presence of Munc13-1 and -2. Munc13-3, Munc13-4, or BAP3 does not functionally replace Munc13-1 and -2 in these synapses (45). Likewise, spontaneous and evoked transmitter release at the cholinergic NMJ in C. elegans is entirely blocked in worms carrying the complete loss-of-function allele of unc-13, unc-13(s69) (35), although an Unc-13 homologue similar to BAP3 and Munc13-4 (27) is most likely present. In contrast, at murine NMJs lacking Munc13s, spontaneous transmitter release persists and some evoked transmitter release is elicitable. It is unlikely that trace amounts of Munc13-4 or BAP3 mediate the residual synaptic vesicle priming at these mutant NMJs because even robust levels of BAP3 are not sufficient to ameliorate the Munc13-deficient mutant phenotype in hippocampal synapses (45) and Munc13-4 does not bind to RIM (27). Apart from Munc13-4 and BAP3, CAPS proteins (i.e., CAPS1 and CAPS2 in mammals) have been proposed to be priming proteins (9, 27, 32, 43). However, CAPS proteins do not compensate for the loss of Munc13s from hippocampal neurons in spite of strong expression at their synaptic terminals (41, 45) and are therefore also unlikely to support synaptic vesicle priming in NMJs.
As the murine and human genomes do not contain any additional genes with homology to Munc13s, our findings indicate that some vesicle priming at the NMJ occurs in the absence of Munc13s and that therefore either non-Munc13 priming proteins must exist or vesicle priming at the NMJ can occur in part spontaneously without priming proteins. Vesicle priming independent of Munc13-1, -2, and -3 has previously been suggested to occur in chromaffin cells (3). According to the current molecular model, Munc13s mediate synaptic vesicle priming by stabilizing the open conformation of the SNARE syntaxin 1, thereby allowing the formation of SNARE dimers containing syntaxin 1 and SNAP25 or of trans SNARE complexes (16, 20, 36). Constitutive SNARE-mediated intracellular and secretory membrane fusion reactions do not require a Munc13-like priming step; Saccharomyces cerevisiae does not express homologues of Munc13-1, -2, -3, and -4 or BAP3 (27); and bona fide Munc13s first appear during evolution in organisms with a central nervous system (27), indicating that Munc13-independent spontaneous SNARE priming does occur. It is likely that synaptic vesicle exocytosis at the mouse NMJ involves in part a syntaxin variant or other SNARE complex components such as SNAP23 that are less dependent on Munc13s stabilizing the open conformation of syntaxin, with the consequence that some vesicle priming indeed occurs spontaneously.
The SNARE protein complement of murine NMJs is only partially known. NMJs lacking SNAP25 exhibit increased spontaneous transmitter release but lack evoked release (48). It is possible that spontaneous release in SNAP25 KO mutants is due to the presence of SNAP23, which can partly replace SNAP25 but has strikingly different functional features (40). Essentially, our data and the published account in the literature are best compatible with a scenario according to which Munc13-mediated vesicle priming is essential for a majority of synaptic vesicles at NMJs, while a small subpopulation of vesicles can undergo spontaneous priming. This would explain why in the absence of Munc13-1, -2, and -3 evoked transmitter release is strongly reduced while spontaneous release persists. The increase in the frequency of spontaneous release events seen in SNAP25 KO (48) and Munc13-1/2-DKO NMJs may then simply be due to the increased number of synapses formed in these mutant NMJs.
The importance of synaptic activity for NMJ formation. The phenotypic alterations seen at the Munc13-1/2-DKO NMJ are similar to those reported for the ChAT-deficient mouse NMJ, in which synaptic vesicles are no longer loaded with acetylcholine (13, 30). The same types of abnormal nerve arborizations and disorganized termination areas with more or larger synaptic endplates are also seen in SNAP25-deficient NMJs (48).
Functionally, the Munc13-1/2/3 and SNAP25 KO mice differ from ChAT KO mutants in various aspects. In the ChAT KO, transmission is blocked at the NMJ, while motor neuron cell bodies receive functionally normal synaptic inputs. In the Munc13-1/2-DKO and SNAP25 KO, synaptic transmission at spinal cord synapses is either abolished (not shown) or strongly reduced. This situation is similar to that described for Munc18-1 deletion mutant mice, which are characterized by a complete lack of spontaneous and evoked transmitter release at the NMJ as well as at central snyapses (12, 23, 46). In these mutants, motor neurons form synaptic contacts very transiently in early embryonal life and soon thereafter degenerate and retract axonal processes (23). This phenomenon, which is not seen in ChAT KO mice (13, 30) or Munc13-1/2-DKO mice, was explained by the lack of synaptic signaling onto motor neurons in Munc18-1 mutants (23). However, given the fact that in Munc13-1/2-DKO mice no motor neuron degeneration is seen despite a complete shutdown of synaptic transmission onto these neurons, this explanation may be wrong. Rather, the role of Munc18-1 may extend to the regulation of intracellular membrane trafficking events necessary for neuronal survival or of developmentally earlier and more general secretory events, whose impairment dramatically compromises neuronal survival (e.g., neurotrophin signaling). As a consequence, Munc18-1 may exert a more stringent control of exocytosis at the NMJ, abolishing not only fast synaptic transmission but also the secretion of neuroactive peptides, neurotrophic factors, or hormones. In contrast, Munc13-1/2/3 deletion still allows for the fusion of a small population of synaptic and peptidergic vesicles, and ChAT deletion still permits the release of transmitter-deficient vesicles that contain neuroactive or neurotrophic peptides. These may influence synapse formation, synapse maintenance, and neuronal survival (13, 30).
The increased number of motor neurons in the spinal cord of Munc13-DKO mice is presumably due to a cessation of their apoptosis, which occurs normally around E15 to E17 (1, 22). As mentioned above, this blockade of apoptosis is unlikely to result from a block of synaptic signaling in the spinal cord. Instead, it might be due to the local malfunction of the NMJ, thus influencing the well-described process of embryonic synapse elimination that usually leads to the consolidation of only one axon/endplate per muscle fiber (17, 18, 26, 29).
Spontaneous action potentials, which typically occur in motor neurons during development (21), are likely to be important for shaping nerve-muscle connectivity. They originate from either the spiking of premotor interneurons or the coordinated quantal neurotransmitter release from motor neurons, which is unlikely to occur in Munc13-1/2-DKO embryos. Thus, muscle action potentials and contractions driven by action potentials in motor neurons are unlikely to take place in vivo in the Munc13-DKO embryo, despite the fact that stimulation of the cut phrenic nerve can elicit action potentials and evoke some contractile response. Similarly, ChAT and SNAP25 deficiencies presumably lead to the elimination of muscle activation driven by motor neuron action potentials. Given that all three mutants exhibit the same phenotype with respect to motor neuron survival and refinement of NMJ connectivity, we conclude that the proper development of motor neurons and NMJs does not depend on a trophic action of spontaneous quantal acetylcholine release. Rather, successful and reliable action potential-driven postsynaptic depolarizations, which can even occur spontaneously during development, appear to be necessary to regulate motor neuron survival and shape mature NMJs.
It is likely that retrograde signaling from the muscle to the innervating motor neuron is involved in the effects of these action potential-driven synaptic events on motor neuron number and NMJ morphology. Interestingly, a pattern of connectivity similar to the one found in Munc13-1/2-DKO NMJs is seen in MyoD-deficient mice. Here, the abnormal branching must be due to an impaired retrograde signaling because MyoD is a muscle-specific transcription factor (47). In addition, the phenotypes of Rapsyn- and MuSK-deficient mice, which show the same aberrant motor neuron survival seen in Munc13-1/2-DKO and ChAT KO mice (7, 44), indicate a role of retrograde signaling from muscle in motor neuron survival. By analogy, it is possible that impaired transmission at the Munc13-, ChAT-, or SNAP25-deficient NMJ affects muscle electrical activity, thereby influencing the levels of myogenic regulatory factors or other signaling molecules and, in turn, retrograde signaling.
|
ACKNOWLEDGMENTS |
|---|
We thank M. Dutschmann, D. Fasshauer, J. Heeroma, A. Mansouri, and G. Meyer for helpful scientific discussion; K. Hellmann, S. Wenger, and J. Mairesse for excellent technical assistance; A. Arand and the staff of the Transgenic Animal Facility at the Max-Planck-Institute for Experimental Medicine, Göttingen, for help with mouse colonies; and J. Ficner for help with graphics.
|
FOOTNOTES |
|---|
Supplemental material for this article may be found at http://mcb.asm.org/.
F.V. and M.S.S. contributed equally to this study.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Aravamudan, B., T. Fergestad, W. S. Davis, C. K. Rodesch, and K. Broadie. 1999. Drosophila UNC-13 is essential for synaptic transmission. Nat. Neurosci. 2:965-971.[CrossRef][Medline]
3. Ashery, U., F. Varoqueaux, T. Voets, A. Betz, P. Thakur, H. Koch, E. Neher, N. Brose, and J. Rettig. 2000. Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells. EMBO J. 19:3586-3596.[CrossRef][Medline]
4. Augustin, I., A. Betz, C. Herrmann, T. Jo, and N. Brose. 1999. Differential expression of two novel Munc13 proteins in rat brain. Biochem. J. 337:363-371.
5. Augustin, I., S. Korte, M. Rickmann, H. A. Kretzschmar, T. C. Sudhof, J. W. Herms, and N. Brose. 2001. The cerebellum-specific Munc13 isoform Munc13-3 regulates cerebellar synaptic transmission and motor learning in mice. J. Neurosci. 21:10-17.
6. Augustin, I., C. Rosenmund, T. C. Sudhof, and N. Brose. 1999. Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature 400:457-461.[CrossRef][Medline]
7. Banks, G. B., T. N. Chau, S. E. Bartlett, and P. G. Noakes. 2001. Promotion of motoneuron survival and branching in rapsyn-deficient mice. J. Comp. Neurol. 429:156-165.[CrossRef][Medline]
8. Barstad, J. A., and G. Lilleheil. 1968. Transversally cut diaphragm preparation from rat. An adjuvant tool in the study of the physiology and pharmacology of the myoneural junction. Arch. Int. Pharmacodyn. Ther. 175:373-390.[Medline]
9. Berwin, B., E. Floor, and T. F. Martin. 1998. CAPS (mammalian UNC-31) protein localizes to membranes involved in dense-core vesicle exocytosis. Neuron 21:137-145.[CrossRef][Medline]
10. Betz, A., M. Okamoto, F. Benseler, and N. Brose. 1997. Direct interaction of the rat unc-13 homologue Munc13-1 with the N terminus of syntaxin. J. Biol. Chem. 272:2520-2526.
11. Betz, A., P. Thakur, H. J. Junge, U. Ashery, J. S. Rhee, V. Scheuss, C. Rosenmund, J. Rettig, and N. Brose. 2001. Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming. Neuron 30:183-196.[CrossRef][Medline]
12. Bouwman, J., A. S. Maia, P. G. Camoletto, G. Posthuma, E. W. Roubos, V. M. Oorschot, J. Klumperman, and M. Verhage. 2004. Quantification of synapse formation and maintenance in vivo in the absence of synaptic release. Neuroscience 126:115-126.[CrossRef][Medline]
13. Brandon, E. P., W. Lin, K. A. D'Amour, D. P. Pizzo, B. Dominguez, Y. Sugiura, S. Thode, C. P. Ko, L. J. Thal, F. H. Gage, and K. F. Lee. 2003. Aberrant patterning of neuromuscular synapses in choline acetyltransferase-deficient mice. J. Neurosci. 23:539-549.
14. Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77:71-94.
15. Brose, N., K. Hofmann, Y. Hata, and T. C. Sudhof. 1995. Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270:25273-25280.
16. Brose, N., C. Rosenmund, and J. Rettig. 2000. Regulation of transmitter release by Unc-13 and its homologues. Curr. Opin. Neurobiol. 10:303-311.[CrossRef][Medline]
17. Buffelli, M., R. W. Burgess, G. Feng, C. G. Lobe, J. W. Lichtman, and J. R. Sanes. 2003. Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature 424:430-434.[CrossRef][Medline]
18. Buffelli, M., G. Busetto, L. Cangiano, and A. Cangiano. 2002. Perinatal switch from synchronous to asynchronous activity of motoneurons: link with synapse elimination. Proc. Natl. Acad. Sci. USA 99:13200-13205.
19. Dulubova, I., S. Sugita, S. Hill, M. Hosaka, I. Fernandez, T. C. Sudhof, and J. Rizo. 1999. A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18:4372-4382.[CrossRef][Medline]
20. Fasshauer, D. 2003. Structural insights into the SNARE mechanism. Biochim. Biophys. Acta 1641:87-97.[Medline]
21. Hanson, M. G., and L. T. Landmesser. 2003. Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord. J. Neurosci. 23:587-600.
22. Harris, A. J., and C. D. McCaig. 1984. Motoneuron death and motor unit size during embryonic development of the rat. J. Neurosci. 4:13-24.[Abstract]
23. Heeroma, J. H., J. J. Plomp, E. W. Roubos, and M. Verhage. 2003. Development of the mouse neuromuscular junction in the absence of regulated secretion. Neuroscience 120:733-744.[CrossRef][Medline]
24. Jahn, R., T. Lang, and T. C. Sudhof. 2003. Membrane fusion. Cell 112:519-533.[CrossRef][Medline]
25. Junge, H. J., J. S. Rhee, O. Jahn, F. Varoqueaux, J. Spiess, M. N. Waxham, C. Rosenmund, and N. Brose. 2004. Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity. Cell 118:389-401.[CrossRef][Medline]
26. Kasthuri, N., and J. W. Lichtman. 2003. The role of neuronal identity in synaptic competition. Nature 424:426-430.[CrossRef][Medline]
27. Koch, H., K. Hofmann, and N. Brose. 2000. Definition of Munc13-homology-domains and characterization of a novel ubiquitously expressed Munc13 isoform. Biochem. J. 349:247-253.[CrossRef][Medline]
28. Lackner, M. R., S. J. Nurrish, and J. M. Kaplan. 1999. Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta: DAG binding to UNC-13 is required to stimulate acetylcholine release. Neuron 24:335-346.[CrossRef][Medline]
29. Lichtman, J. W., and H. Colman. 2000. Synapse elimination and indelible memory. Neuron 25:269-278.[CrossRef][Medline]
30. Misgeld, T., R. W. Burgess, R. M. Lewis, J. M. Cunningham, J. W. Lichtman, and J. R. Sanes. 2002. Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron 36:635-648.[CrossRef][Medline]
31. Plomp, J. J., G. T. van Kempen, and P. C. Molenaar. 1992. Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in alpha-bungarotoxin-treated rats. J. Physiol. 458:487-499.
32. Renden, R., B. Berwin, W. Davis, K. Ann, C. T. Chin, R. Kreber, B. Ganetzky, T. F. Martin, and K. Broadie. 2001. Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion. Neuron 31:421-437.[CrossRef][Medline]
33. Rettig, J., and E. Neher. 2002. Emerging roles of presynaptic proteins in Ca++-triggered exocytosis. Science 298:781-785.
34. Rhee, J. S., A. Betz, S. Pyott, K. Reim, F. Varoqueaux, I. Augustin, D. Hesse, T. C. Sudhof, M. Takahashi, C. Rosenmund, and N. Brose. 2002. Beta phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell 108:121-133.[CrossRef][Medline]
35. Richmond, J. E., W. S. Davis, and E. M. Jorgensen. 1999. UNC-13 is required for synaptic vesicle fusion in C. elegans. Nat. Neurosci. 2:959-964.[CrossRef][Medline]
36. Richmond, J. E., R. M. Weimer, and E. M. Jorgensen. 2001. An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature 412:338-341.[CrossRef][Medline]
37. Rosenmund, C., J. Rettig, and N. Brose. 2003. Molecular mechanisms of active zone function. Curr. Opin. Neurobiol. 13:509-519.[CrossRef][Medline]
38. Rosenmund, C., A. Sigler, I. Augustin, K. Reim, N. Brose, and J. S. Rhee. 2002. Differential control of vesicle priming and short-term plasticity by Munc13 isoforms. Neuron 33:411-424.[CrossRef][Medline]
39. Shiratsuchi, T., K. Oda, H. Nishimori, M. Suzuki, E. Takahashi, T. Tokino, and Y. Nakamura. 1998. Cloning and characterization of BAP3 (BAI-associated protein 3), a C2 domain-containing protein that interacts with BAI1. Biochem. Biophys. Res. Commun. 251:158-165.[CrossRef][Medline]
40. Sorensen, J. B., G. Nagy, F. Varoqueaux, R. B. Nehring, N. Brose, M. C. Wilson, and E. Neher. 2003. Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23. Cell 114:75-86.[CrossRef][Medline]
41. Speidel, D., F. Varoqueaux, C. Enk, M. Nojiri, R. N. Grishanin, T. F. Martin, K. Hofmann, N. Brose, and K. Reim. 2003. A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function. J. Biol. Chem. 278:52802-52809.
42. Sutton, R. B., D. Fasshauer, R. Jahn, and A. T. Brunger. 1998. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395:347-353.[CrossRef][Medline]
43. Tandon, A., S. Bannykh, J. A. Kowalchyk, A. Banerjee, T. F. Martin, and W. E. Balch. 1998. Differential regulation of exocytosis by calcium and CAPS in semi-intact synaptosomes. Neuron 21:147-154.[CrossRef][Medline]
44. Terrado, J., R. W. Burgess, T. DeChiara, G. Yancopoulos, J. R. Sanes, and A. C. Kato. 2001. Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos. J. Neurosci. 21:3144-3150.
45. Varoqueaux, F., A. Sigler, J. S. Rhee, N. Brose, C. Enk, K. Reim, and C. Rosenmund. 2002. Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming. Proc. Natl. Acad. Sci. USA 99:9037-9042.
46. Verhage, M., A. S. Maia, J. J. Plomp, A. B. Brussaard, J. H. Heeroma, H. Vermeer, R. F. Toonen, R. E. Hammer, T. K. van den Berg, M. Missler, H. J. Geuze, and T. C. Sudhof. 2000. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864-869.
47. Wang, Z. Z., C. H. Washabaugh, Y. Yao, J. M. Wang, L. Zhang, M. P. Ontell, S. C. Watkins, M. A. Rudnicki, and M. Ontell. 2003. Aberrant development of motor axons and neuromuscular synapses in MyoD-null mice. J. Neurosci. 23:5161-5169.
48. Washbourne, P., P. M. Thompson, M. Carta, E. T. Costa, J. R. Mathews, G. Lopez-Bendito, Z. Molnar, M. W. Becher, C. F. Valenzuela, L. D. Partridge, and M. C. Wilson. 2002. Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat. Neurosci. 5:19-26.[Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
