Fig 1.
Specificity of in-house VAMP2 antibody.
(A) Brain homogenates from wild type (WT1–2) and knockout (KO1–3) mice. The VAMP2 band at 18kDa is absent from brains of the three knockout mice. Protein loaded 30 μg. (B) Western blots from wild type rat brain. BH: brain homogenate; S: crude synaptosomes; SV: synaptic vesicles. Only one band was seen in each preparation. Protein loaded 10 μg. (C) Quantitative analysis of immunogold labeling with anti-VAMP2 of presynaptic cytoplasm at excitatory synapses in hippocampus, preincubated with VAMP2 protein. Ultrastructural quantification of VAMP2 immunogold labeling of presynaptic cytoplasm at excitatory synapses in hippocampus of VAMP2 KO mice. Statistical significance (p < 0.001). (D) Electron micrographs showing labeling for VAMP2. (D1) Standard labeling, i.e. normal immunogold without pre-incubation with antigen. (D2) Pre-incubation with VAMP2 antigen. (D3) Standard labeling of wildtype. (D4) Labeling of VAMP2-KO tissue. Scale bar: 125 nm.
Fig 2.
Regional VAMP2 expression in the rat brain.
(A) Staining of homogenates from different brain regions with anti-VAMP2, i.e. cortex (Cx), thalamus (Th), hippocampus [64], brain stem (Bs), cerebellum (Cb), and spinal cord (SpC). Protein loaded 7,5 μg. (B) Staining of different brain regions with anti-beta-tubulin. Protein loaded: 5 μg. (C) Quantitation of intensities of bands seen above, indicating highest concentration in the hippocampus, lowest in the spinal cord. Fluorescence intensity of anti-VAMP2 staining was normalized to anti-beta-tubulin staining. (D) Vibratome sections immunostained for VAMP2. Anti-VAMP2 antibody produced immunoperoxidase-staining of neurons in hippocampus, cerebellum, cerebral cortex and thalamus (D1–D3). In the hippocampus, the somata and proximal dendrites of pyramidal neurons were strongly stained in sub-regions of CA1 (D4) and CA3 (D5), as were the granule cells in dentate gyrus (D6). Strong immunostaining was found in purkinje cell somata and the proximal dendrites in the cerebellar cortex (D9), and in the pyramidal cells of the cerebral cortex (D7). Immunolabeling was also observed in thalamic neurons (D8). Immunoperoxidase staining. Scale bar: (D1): 1000 μm. (D2): 2000 μm, (D3): 500 μm, (D4–D8): 20 μm, (D9): 50 μm.
Fig 3.
Electron micrographs showing VAMP2 immunogold labeling of vesicles in asymmetric synapses from the CA1 region of the rat hippocampus.
(A) Gold particles are present over pre- and postsynaptic vesicles (arrowheads), as well as some at synaptic plasma membranes. Scale bar: 200 nm. (B) Selected postsynaptic vesicles (arrowheads) in higher magnification. Presynaptic vesicles are also seen. Synaptic cleft is marked with an asterisk. Scale bar: 75 nm.
Fig 4.
Confocal images of dissociated hippocampal cultures.
Double labeling with anti-TUJ1 (A) and anti-VAMP2 (B) demonstrates that VAMP2 is located along the dendrites and gives characteristic punctate labeling (C). Double labeling with antibodies against synaptophysin (P38) (D) and VAMP2 (E) indicates that VAMP2 is colocalized with the presynaptic marker (F). Double labeling for PSD-95 (G) and VAMP2 (H) shows partial colocalization postsynaptic (I). Small, high-resolution pictures of single synapses from (F) and (I) are shown in the right part of these images, respectively. Scale bar: 20 μm.
Fig 5.
Quantitation of synaptic VAMP2 immunogold labeling.
(A) Schematic drawing showing regions of interest in electron micrographs. PreCy: Presynaptic Cytoplasm. PreL: Presynaptic Lateral plasma membrane. AZ: Active Zone. PoCy: Postsynaptic Cytoplasm. PoL: Postsynaptic Lateral membrane. PSD: PostSynaptic Density. DCy: Dendrite Cytoplasm. DM: Dendritic plasma Membrane. AM: Astrocyte plasma Membrane. (B) Transverse histogram depicting the mean number of gold particles at every 30 nm distance from the center of the synaptic cleft; negative values are postsynaptic, positive values are presynaptic. The peak is about 200 nm from the center of the synaptic cleft, but significant levels are seen also in postsynaptic cytoplasm. (C) Mean immunogold labeling over cytoplasmic regions of interest. (D) Mean immunogold labeling over plasma membrane regions of interest.
Fig 6.
Vesicular colocalization of VAMP2 and AMPA receptor subunits.
(A) Electron micrographs showing double immunogold labeling of VAMP2 (10 nm gold particles) and GluA1 (20 nm gold) in spine and dendrite. (B) Quantitative analysis of colocalization between VAMP2 and GluA1 in spines and dendrites. (C) Electron micrographs showing double immunogold labeling of VAMP2 (10 nm gold particles) and GluA2 (20 nm gold particles). (D) Quantitative analysis of colocalization between VAMP2 and GluA2 in spines and dendrites. (E–K) Immunoprecipitation (IP) of synaptic vesicles with magnetic beads coated with antibody, along with brain homogenate (BH), synaptic vesicle (SV) preparations and flow-through (FT), all subjected to gel electrophoresis and western blotting [65]. (E) IP with anti-GluA1, WB with anti-GluA1. (F) IP with anti-GluA1, WB with anti-VAMP2. (G) IP with anti-GluA2, WB with anti-GluA2. (H) IP with anti-GluA2, WB with anti-VAMP2. (I) IP with anti-VAMP2, WP with anti-VAMP2. (J) IP with anti-VAMP2, WB with anti-GluA1. (K) IP with anti-VAMP2, WB with anti-GluA2. (L) WB of brain homogenate fractions stained with anti-synaptophysin (P38) antibody. (M) WB of brain homogenate fractions stained with phosphate-activated glutaminase [39] antibody. (N) Electron micrograph of vesicle preparation similar to the ones used in (E-M). Scale bars: 100 nm (A spine, B), 125 nm (A dendrite), 100 nm (N).
Fig 7.
Control immunolocalization of plasma membrane AMPA receptor subunits (external epitopes) in hippocampal neuronal cultures.
(A) Beta-tubulin (green, anti-TuJ1) labeling of dendrite, after anti-GluA1 external epitope [66] labeling and subsequent plasma membrane permeabilization. Note punctate GluA1 [66] labeling along the dendrite. (B) Synaptophysin (P38, green) and GluA1 [66]. (C) Synaptophysin (green) and GluA2 [66]. (D) Labeling with anti-GluA1. (E) Labeling with anti-GluA1, but the antibody was preincubated with the peptide antigen before staining. (F) Labeling with anti-GluA2. (G) Labeling with anti-GluA2, but the antibody was preincubated with the peptide antigen before staining. (H) Immunolabeling of non-permeabilized neuronal cultures with ani-GluA1 (green). (I) Immunolabeling with the postsynaptic marker anti-PSD95 [66] after permeabilization of neuronal cultures. (J) Merging of (H) and (I) showing postsynaptic labeling of GluA1. Scale bar (in A, valid for all images): 10 μm.
Fig 8.
Cleavage of VAMP2 with tetanus toxin disrupts synaptic insertion of GluA1, but not GluA2, in the plasma membrane of dissociated hippocampal cultures.
(A and B) VAMP2 immunofluorescence after permeabilization, in (B) also after tetanus toxin treatment. Note the effect of tetanus toxin, with disruption of VAMP2. (C and D) Immunofluorescence against the external epitope of GluA1, in (D) also after tetanus toxin treatment. (E and F) Immunofluorescence against the external epitope of GluA2, in (F) also after tetanus treatment. (G) Graphical depiction of intensity of punctate, external GluA1 labeling, showing a significant reduction of synaptic GluA1 after tetanus toxin treatment. (H) Graphical depiction of intensity of punctate, external GluA2 labeling, showing a slight, but significant increase of synaptic GluA2 after tetanus toxin treatment. Scale bar: 10 μm. Asterisks denote statistical significant difference (p < 0.001).
Fig 9.
The effect on plasma membrane expression of AMPA receptor subunits in hippocampal culture after presynaptic silencing with Bafilomycin (BAF).
(A) Control synapses immunolabeled with anti-GluA1 in non-permeabilized culture. (B) As in (A), but treated with Bafilomycin (BAF) before fixation. (C) Control synapses immunolabeled with anti-GluA2 in non-permeabilized culture. (D) As in (C), but treated with Bafilomycin (BAF) before fixation. (E) Quantification of difference in maximal punctate GluA1 labeling intensities between control and Bafilomycin-treated cultures. There is no significant difference. (F) Quantification of difference in maximal punctate GluA2 labeling intensities between control and Bafilomycin-treated cultures. There is a slight, but significant decrease in labeling intensities after Bafilomycin treatment. Asterisk denotes statistical significance (p < 0.001). Scale bar: 10 μm.