Article Figures & Data
Figures
- Fig. 1.
Targeting strategy and identification of the Munc13-3 deletion mutation in mice.A, Structure of the targeted region of the murine Munc13-1 gene, targeting vector, and mutated gene resulting from homologous recombination. Exons are indicated by black boxes (numbers give corresponding base pairs in the rat Munc13-3 cDNA), and the location of the probe used for Southern analysis of genomic DNA digested with BamHI is indicated by a hatched bar. The location of the neomycin resistance gene (Neo) and two copies of the HMV thymidine kinase gene (TK) in the targeting vector are indicated by gray boxes.BglII/BamHI indicates fused site. The positions of the BamHI sites upstream of theProbe and at the very 3′ end of the gene representation are not part of genomic clones used for the construction of the targeting vector and were not mapped. B, Southern analysis of genomic DNA from different Munc13-3 genotypes. DNA was digested with BamHI, separated electrophoretically, blotted onto nylon filters, and probed with the probe shown above. Bands representing the wild-type (WT) and knock-out (KO) allele are indicated. C, Western blot analysis of cerebellum homogenates from different Munc13-3 genotypes. Brain homogenates were separated by SDS-PAGE, blotted onto nitrocellulose filters, and probed with an antibody directed against the N terminus of Munc13-3 (Augustin et al., 1999b). Note the complete absence of Munc13-3 in knock-out brain (arrowhead).
- Fig. 2.
Normal cytoarchitecture in Munc13-3-deficient mice. A, In situhybridization for Munc13-3 in mouse brain. Like in rat, Munc13-3 mRNA expression is almost exclusively restricted to the cerebellum in mouse brain. B, C, Cresyl violet stainings of paraffin sections through wild-type and Munc13-3-deficient cerebellum. Note the normal cytoarchitecture and cell density in Munc13-3-deficient cerebellum. D, Frozen section through wild-type cerebellum stained with an antibody specific to Munc13-3 (Augustin et al., 1999b). Munc13-3 is localized to the molecular layer of the cerebellum, which mainly contains synaptic neuropil with parallel fiber–Purkinje cell synapses. E, Frozen section through Munc13-3 mutant cerebellum, demonstrating the complete absence of Munc13-3 protein and thus the specificity of the immunostaining procedure. F, Staining for Munc13-1 protein in rat cerebellum using a specific monoclonal antibody according to published procedures (Betz et al., 1998). wm, White matter;gcl, granule cell layer; ml, molecular layer. Scale bar: A, 2 mm; B–E, 100 μm; F, 75 μm.
- Fig. 3.
Increased paired-pulse facilitation in parallel fiber–Purkinje cell synapses in Munc13-3-deficient mice.A, Sample synaptic responses taken from wild-type (+/+) and Munc13-3-deficient (−/−) Purkinje cells after paired stimulation (75 msec interval) of parallel fibers. Note the increase in paired-pulse facilitation in mutant cells. B, Magnitude of the paired-pulse facilitation as a function of interstimulus intervals. Ratios of the second to first EPSC amplitude (mean ± SEM) from wild-type (open circles; 8 animals, 17 cells) and mutant (filled circles; 9 animals, 19 cells) mice are plotted as a function of interstimulus interval. Note significant increases in the paired-pulse facilitation ratio in Munc13-3-deficient Purkinje cells (p < 0.05) for intervals up to 200 msec. C, Amplitudes of successive EPSCs plotted as a function of stimulus number during repetitive stimulation at 14 Hz from wild-type (open circles; 9 animals, 9 cells) and mutant (filled circles; 10 animals, 10 cells) animals. No depression is observed in mutant or wild-type cells. Error bars indicate SE.
- Fig. 4.
Unaltered spontaneous inhibitory neurotransmission in Purkinje cells from Munc13-3-deficient cerebellum. General features of miniature inhibitory synaptic currents of Munc13-3-deficient Purkinje cells. A, Examples for inhibitory synaptic responses in mutant (right) and control (left) cells. Experimental recordings consisted of six sweeps per cell, each with 10 sec length. B, Isolated single events from A. The thick gray lines show the averaged miniature IPSCs. C–E, Frequency, amplitude, and rise time of miniature IPSCs remain unimpaired in Munc13-3-deficient mice. Four-hundred eighty single events from six cells (wild-type) and 540 single events from seven cells (mutant), respectively, were used to compile these histograms. Error bars indicate SEM.
- Fig. 5.
Normal ultrastructure of synapses in Munc13-3-deficient cerebellum. Two brains per genotype were analyzed. For the determination of synapse densities, 42 photographs (124 synapses; 79 and 45 synapses per animal, respectively) from +/+, and 47 photographs (142 synapses; 80 and 62 synapses per animal, respectively) from −/− cerebella were examined. For the ultrastructural comparison, 24 +/+ (13 and 11 synapses per animal, respectively) and 25 −/− (14 and 11 synapses per animal, respectively) synapses were analyzed. Error bars indicate SEM.
- Fig. 6.
Deficits in motor learning in Munc13-3-deficient mice. A, Falling latency of naı̈ve wild-type and mutant mice measured with the rotating rod apparatus at 40 rpm. Note that falling latency was not significantly affected by the lack of Munc13-3. Mean values are given (n = 11). Error bars indicate SEM.B, Behavioral analysis of wild-type (+/+) and mutant (−/−) mice in the rotating rod motor learning task.Points represent the mean of the rotation speed learned to criterion (n = 11). Error bars indicate SEM. Note that wild-type mice achieved a significantly (p < 0.05) higher rotation speed than mutants compared from the 11th session onward.
Tables
Protein level (% of wild-type control) Munc13-1* 104 ± 2 Munc13-2 94 ± 4 Munc13-3 0 α-SNAP 81 ± 5 Amphiphysin 95 ± 10 Arf6 105 ± 4 Complexin I* 110 ± 4 Complexin II* 107 ± 1 Doc2α 87 ± 2 Dynamin 105 ± 3 GDI 95 ± 8 Mint I 114 ± 4 Msec7 101 ± 4 Munc18-1 87 ± 5 Neuroligin 1/3 105 ± 7 NMDA RI 101 ± 4 NSF 99 ± 10 PSD95 83 ± 4 Rab3A* 108 ± 7 Rabphilin 86 ± 8 SAP102 111 ± 5 SNAP25 88 ± 6 SV2B 117 ± 4 SV2C 87 ± 5 Synapsin I 89 ± 5 Synapsin II 109 ± 3 Synaptobrevin II 91 ± 8 Synaptogyrin* 104 ± 5 Synaptophysin 100 ± 8 Synaptoporin* 87 ± 10 Synaptotagmin I 92 ± 5 Syntaxin IA/B 104 ± 6 Data are given as mean ± SEM (n = 3;
↵* n = 5). Munc13-3 levels in hetergozygous mutant cerebella were not significantly different from wild-type controls (113 ± 6; n = 3), indicating a lack of a gene dosage effect.
