Munc13-independent vesicle priming at mouse photoreceptor ribbon synapses

Abstract

Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.

Figure 1.

Specificity of Munc13 isoform-specific antisera. A, Schematic representation of protein fragments (framed in green) used to raise specific antibodies against the canonical Munc13 isoforms/splice variants. C1, C1 domain; C2, C2 domain; MHD, Munc13 homology domain (Koch et al., 2000); MUN, Mun domain (Li et al., 2011). B–D, Western blot detection of Munc13-1, ubMunc13-2, bMunc13-2, and Munc13-3 using our own and commercial (panMunc13 TL; BD Biosciences) antibodies in homogenates from newborn brain (B), and adult retina (C, D) or olfactory bulb (D) homogenates of the indicated genotypes. The specificity of each antibody is indicated by the absence of corresponding immunoreactive band in extracts from the respective deletion-mutant mice. D, Immunoblot labeling with an anti-ubMunc13-2 serum revealed that ubMunc13-2 is strongly expressed in the olfactory bulb and that an unidentified protein is selectively expressed in Munc13-2-deficient but not in wild-type mice (right). A similar fragment was observed in Munc13-2-deficient retina extracts (left). E, In homogenates from Munc13-2 KO and Munc13-1/2 DKO hippocampal neurons, the ubMunc13-2 antibody recognizes a similar fragment. D, E, The asterisks indicate the 70–75 kDa fragments recognized by the ubMunc13-2 antibody in samples from Munc13-2-deficient mice. F, Single-plane confocal images taken upon immunocytochemical detection of Munc13 isoforms in vertical sections of adult mouse retina of the indicated genotypes. A weak, residual signal was observed for ubMunc13-2 at both the OPL and IPL of the Munc13-2 deletion-mutant retina. This may be consistent with the presence of a protein fragment observed in the Munc13-2 KO by Western blot (D). In contrast, the punctate labelings observed with the antisera directed against bMunc13-2 and Munc13-3 were no longer present in the corresponding KO retina. Note the occurrence of a nonspecific nuclear labeling obtained upon application of the antiserum directed against bMunc13-2. ONL, INL, Outer and inner nuclear layers; OPL, IPL, outer and inner plexiform layers; GCL, ganglion cell layer. Scale bars, 20 μm.

Figure 2.

Munc13 isoforms are differentially localized in the mouse retina. Single-plane confocal images of Munc13 labelings. A, Munc13-1, ub- and bMunc13-2, and Munc13-3 exhibited distinct punctate labeling patterns at the plexiform layers of the retina suggestive of a differential synaptic localization. B, C, ubMunc13-2 distribution extensively overlapped with that of the ribbon protein CtBP2 and of the vesicular glutamate transporter VGluT1, revealing its presence at ribbon synapses of the OPL (B) and the IPL (C). D, No such association was observed with bMunc13-2. E, A polyclonal antibody raised in guinea pig against bMunc13-2 yielded strictly the same labeling pattern as the rabbit polyclonal antibody used otherwise (left). Upon double labeling for bMunc13-2 and ubMunc13-2, no overlap was observed (middle and right panels). ONL, INL, Outer and inner nuclear layers; OPL, IPL, outer and inner plexiform layers; GCL, ganglion cell layer. Scale bars: A, E, 10 μm; B–D, 2 μm.

Figure 3.

Analysis of ubMunc13-2 transcripts in Munc13-2 KO retina. A, Domain structure of mouse ubMunc13-2 with relevant amino acid residue numbers. The bars above the domain structure represent the antigens used to raise the antibodies used in the present study [52, light gray, residues 182–408; panMunc13 TL (BD Biosciences), dark gray, residues 544–757], the MUN domain required for priming function in neurons as defined by Basu et al. (2005) (light pink, residues 782–1421), and the minimal Munc13 priming domain required in chromaffin cells as defined by Stevens et al. (2005) (dark pink, residues 1017–1601). The exons (not drawn to scale) encoding ubMunc13-2 are shown below the domain structure. The stippled and solid lines indicate the exons that encode the respective ubMunc13-2 sequence stretch. The black and red coloring indicates exons in which forward and reverse primers for the PCR analysis of Munc13-2 KO retina cDNA were located (Table 1). The blue coloring indicates exons that are deleted by the Munc13-2 KO strategy. C1, C1 domain; C2, C2 domain; MHD, Munc13 homology domain (Koch et al., 2000); MUN, Mun domain (Li et al., 2011). B, ubMunc13-2 transcripts found in Munc13-2 KO retina. Predicted molecular weights of the corresponding protein products are indicated on the left. Note that the sequences 5′ to exon 9 were not PCR amplified and have been added to the image, assuming that the corresponding transcripts were generated from the standard and only known transcriptional start site. MCS, Multicloning site of the Munc13-2 KO targeting vector; ORF, open reading frame.

Figure 4.

Generation of Munc13-2-EYFP and Munc13-3-EGFP knock-in mice. A, E, Strategy for the generation of the Munc13-2-EYFP (A) and Munc13-3-EGFP (E) knock-in mutations in mouse embryonic stem cells. In each case, wild-type gene, targeting vector, mutated gene after homologous recombination (m_n), and mutated gene after Cre recombination (m) are schematized. The gray boxes, black triangles, and a black horizontal bar indicate exons, loxP sites, and the probe used for Southern analysis of mutated genes. Sections of genomic sequence that are not shown here are indicated by an open arrowhead. NEO, Neomycin resistance gene; pBlue, pBluescript KS; TK, herpes simplex virus thymidine kinase. B, F, Southern blot analysis of Munc13-2 (B) or Munc 13-3 (F) wild-type and mutated genes using DraI- or BglII-digested mouse tail DNA and the probes indicated in A and E. C, D, G, Western blot analysis of brain homogenates from wild-type mice (+/+) and mice carrying the mutated genes after Cre recombination (m/m). Brains were homogenized by Ultra-Turrax, and soluble (s) and insoluble (p) protein fractions were separated by ultracentrifugation (436,000 × g). Proteins (10 μg per lane) were analyzed by SDS-PAGE and immunoblotting with antibodies to ubMunc13-2 (C), bMunc13-2 (D), or Munc13-3 (G). Note that Munc13-2 and Munc13-2-EYFP (C) and Munc13-3 and Munc13-3-EGFP (G) protein levels were almost identical in either mouse strain. H, I, Maximum projection Z-stacks of images acquired with an Apotome microscope. H, The distribution patterns of Munc13-EYFP fusion proteins correlates well with these obtained upon immunolabeling (Fig. 2). I, Colabeling for EYFP and ubMunc13-2 in Munc13-2-EYFP mice overlapped extensively at the OPL (top) but only partially at the IPL (bottom). Scale bars: H, 10 μm; I, 5 μm.

Figure 5.

ubMunc13-2 localization at photoreceptor ribbon synapses. The subcellular distribution of ubMunc13-2 immunoreactivity was analyzed by STED/confocal microscopy (single-scan, in A) and postembedding immunoelectron microscopy (B, C). A, High-resolution fluorescence microscopy shows that ubMunc13-2 (in red) is distributed along the base of a photoreceptor ribbon, labeled by CtBP2 (in green), likely at the CAZ. B, At the ultrastructural level, gold particles (red circles indicate ribbon active zone-associated gold particles; blue circles indicate cytoplasmic, non-ribbon-associated gold particles) confirm the subcellular location of ubMunc13-2 at the CAZ and at the presynaptic membrane, within 250 nm of the CAZ at both rod (inset; R1) and cone (insets; C1, C2) photoreceptor ribbons. C, Relevant features visible on this electron micrograph are illustrated in D. E, Distribution (percentage) of gold particles in the various subregions of the rod photoreceptor presynaptic compartment (number of gold particles, 139 observed at a total of 150 rod synapses). Scale bars: STED, 1 μm; EM, 100 nm.

Figure 6.

Deletion of Munc13-2 is detrimental to proper synaptic transmission but not to retinal organization. ERG recordings were performed in scotopic and photopic conditions. A, Intensity–response curves did not differ statistically for the a-waves (squares) of wild-type and Munc13-2 KO mice. In contrast, the b-wave (circles) amplitudes were slightly, but significantly reduced in the Munc13-2 KO mice compared with those of wild-type controls (mean ± SEM; n = 5; *0.05 ≥ p > 0.01; **0.01 ≥ p > 0.001; one-way ANOVA), under scotopic (dark-adapted) conditions. B, Under photopic conditions, differences in b-wave amplitudes in response to high flash intensities became increasingly smaller upon increasing background illumination (open symbols, wild type; filled symbols, Munc13-2 KO). C–E, High-resolution, single-plane confocal images. C, Prototypical photoreceptor triads comprising photoreceptor terminals (labeled with VGluT1), bipolar dendrites (labeled with PKCα), and horizontal cell processes (labeled with calbindin) were observed with the expected lamination at the OPL of wild-type and Munc13-2 KO retinas. D, In PKCα-labeled bipolar dendrites, metabotropic glutamate receptors (labeled with mGluR6) are concentrated in apposition to ribbon-bearing presynaptic active zones (labeled with bassoon) in wild-type and Munc13-2 KO retinas. E, L-Type voltage-gated calcium channels Cav1.4 are distributed at the base of CtBP2-immunoreactive photoreceptor ribbons in wild-type and Munc13-2 KO mice. Scale bar, 20 μm.

Figure 7.

Tomographic reconstruction of wild-type and Munc13-2-deficient photoreceptor synapses. A, Color-coded models representing rod photoreceptor ribbon synapses from wild-type and Munc13-2 KO retinas tomographically reconstructed from 150-nm-thick sections. B, Single tomographic slice (isotropic voxel size, 1.3 nm) of a rod photoreceptor ribbon synapse from wild-type (left) or Munc13-2 deletion mutant (right) retinas, at the level of the active zone. C, Detail from B showing docked synaptic vesicles at the plasma membrane of wild-type or Munc13-2 deletion-mutant synapses. D, Quantitative analysis of synaptic vesicle docking. The numbers of vesicles docked at the plasma membrane (PM) forming the presynaptic invagination (rendered in red; A), tethered to the ribbon (rendered in orange; A) or to its base (rendered in white; A) were comparable in wild-type (n = 15 synapses/tomograms) or Munc13-2 KO (n = 14 synapses/tomograms) synapses. For clarity, small sketches representing the different populations of vesicles scrutinized are added below the respective graphs. Error bars indicate SEM. Scale bars: B, 200 nm; C, 50 nm.