Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming - PubMed (original) (raw)

Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming

Frederique Varoqueaux et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Synaptic vesicles must be primed to fusion competence before they can fuse with the plasma membrane in response to increased intracellular Ca2+ levels. The presynaptic active zone protein Munc13-1 is essential for priming of glutamatergic synaptic vesicles in hippocampal neurons. However, a small subpopulation of synapses in any given glutamatergic nerve cell as well as all gamma-aminobutyratergic (GABAergic) synapses are largely independent of Munc13-1. We show here that Munc13-2, the only Munc13 isoform coexpressed with Munc13-1 in hippocampus, is responsible for vesicle priming in Munc13-1 independent hippocampal synapses. Neurons lacking both Munc13-1 and Munc13-2 show neither evoked nor spontaneous release events, yet form normal numbers of synapses with typical ultrastructural features. Thus, the two Munc13 isoforms are completely redundant in GABAergic cells whereas glutamatergic neurons form two types of synapses, one of which is solely Munc13-1 dependent and lacks Munc13-2 whereas the other type employs Munc13-2 as priming factor. We conclude that Munc13-mediated vesicle priming is not a transmitter specific phenomenon but rather a general and essential feature of multiple fast neurotransmitter systems, and that synaptogenesis during development is not dependent on synaptic secretory activity.

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Figures

Figure 1

Figure 1

Mutation of the murine _Munc13_-2 gene. (A) Maps of the wild-type _Munc13_-2 gene, the respective targeting vector, and the resulting mutant gene. Positions of exons (black boxes) and restriction enzyme sites are indicated. Hatched bar, Position of probe used for Southern. Neo, Neomycin resistance gene; TK, thymidine kinase gene. (B) Southern blot analysis of the Munc13-2 KO mutations in mice by using mouse tail DNA (_Eco_RI digest) from the indicated Munc13-2 genotypes. (C) Munc13-2 protein expression in mice of the indicated genotypes as determined by Western blotting using an antibody that recognizes all Munc13-2 splice variants. (D) Expression of Munc13-1 and Munc13-2 protein in mice of the indicated genotypes as determined by Western blotting using an antibody to Munc13-1 (Upper) and an antibody that recognizes all Munc13-2 splice variants (Lower).

Figure 2

Figure 2

Complex functional redundancy of Munc13 isoforms. (A) Examples of EPSCs recorded from cells of the indicated genotypes. (B) Mean EPSC amplitudes measured in cells of the indicated genotypes. (C) Mean readily releasable vesicle pool sizes in cells of the indicated genotypes as estimated by the charge integral measured after release induced by application of 500 mOsm hypertonic sucrose solution for 4 s. (D) mEPSC activity recorded in cells of the indicated genotypes. Holding potential −70 mV. (E) Examples of IPSCs recorded from cells of the indicated genotypes. Munc13-1/2 DKO inhibitory neurons were identified after each experiment by detection of mIPSC events induced by application of α-latrotoxin. (F) Mean IPSC amplitudes measured in cells of the indicated genotypes. (G) mIPSC activity recorded in cells of the indicated genotypes. Pipette solution contained 140 mM KCl.

Figure 3

Figure 3

Normal expression of synaptic proteins in Munc13-1/2 DKO brains. Brain homogenates of the indicated genotypes were analyzed by Western blotting using specific antibodies to the indicated proteins.

Figure 4

Figure 4

Normal morphology of Munc13-1/2 DKO brains and cultured cells. (A) Nissl-stained sagittal section of brains from E18 mice of the indicated genotype. (Bar = 500 μm.) (B) Nissl-stained coronal sections of spinal cord from E18 mice of the indicated genotype. (Bar = 100 μm.) (C) Cultured hippocampal neurons from E18 mice of the indicated genotype (12 DIV) stained for the synapse specific marker synaptophysin. (Bar = 10 μm.) (D) Electron micrographs of synapses of the indicated genotypes. (Bar = 100 nm.)

Figure 5

Figure 5

Normal postsynaptic responsiveness in release-incompetent Munc 13-1/2 double-deficient neurons. (A) Examples of mEPSC activity in cells of the indicated genotypes after application of 1 nM α-latrotoxin for 1 min. Note that signals are blocked by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[_f_]quinoxaline (NBQX). Stars indicate mEPSC events. (Inset) Individual mEPSC (expanded scale). (B) Average mEPSC amplitude and frequency in cells of the indicated genotypes after treatment with 1 nM α-latrotoxin. (C) Examples of mIPSC activity in cells of the indicated genotypes after application of 1 nM α-latrotoxin for 1 min. Note that signals are blocked by bicuculline. Stars indicate mIPSC events. Inset shows individual mIPSC (expanded scale). (D) Average mIPSC amplitude and frequency in cells of the indicated genotypes after treatment with 1 nM α-latrotoxin. (E) Mean peak current responses evoked by exogenous application of GABA and kainate recorded from cells of the indicated genotypes.

Figure 6

Figure 6

No endocytosis in release-incompetent Munc13-1/2 DKO neurons. Localization of endocytotically active synapses by activity-dependent FM1-43 staining in cells of the indicated genotypes. Images show cells after staining and wash. (Bar = 10 μm.)

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