22 research outputs found
Changes in concentrations of NMDA receptor subunit GluN2B, Arc and syntaxin-1 in dorsal hippocampus Schaffer collateral synapses in a rat learned helplessness model of depression
Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-D-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activityregulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.publishedVersio
Activity-regulated cytoskeletal-associated protein (Arc) in presynaptic terminals and extracellular vesicles in hippocampal synapses
The activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is a neuron-specific immediate early gene (IEG) product. The protein regulates synaptic strength through modulation of spine density and morphology, AMPA receptor endocytosis, and as being part of a retrovirus-like inter-cellular communication mechanism. However, little is known about the detailed subsynaptic localization of the protein, and especially its possible presynaptic localization. In the present study, we provide novel electron microscopical data of Arc localization at hippocampal Schaffer collateral synapses in the CA1 region. The protein was found in both pre-and postsynaptic cytoplasm in a majority of synapses, associated with small vesicles. We also observed multivesicular body-like structures positive for Arc. Furthermore, the protein was located over the presynaptic active zone and the postsynaptic density. The relative concentration of Arc was 25% higher in the postsynaptic spine than in the presynaptic terminal. Notably, small extracellular vesicles labeled for Arc were detected in the synaptic cleft or close to the synapse, supporting a possible transsynaptic transmission of the protein in the brain
Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits
Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism
Regional VAMP2 expression in the rat brain.
<p>(A) Staining of homogenates from different brain regions with anti-VAMP2, i.e. cortex (Cx), thalamus (Th), hippocampus [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140868#pone.0140868.ref064" target="_blank">64</a>], brain stem (Bs), cerebellum (Cb), and spinal cord (SpC). Protein loaded 7,5 μg. (<i>B</i>) 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 (<i>D1–D3</i>). In the hippocampus, the somata and proximal dendrites of pyramidal neurons were strongly stained in sub-regions of CA1 (<i>D4</i>) and CA3 (<i>D5</i>), as were the granule cells in dentate gyrus (<i>D6</i>). Strong immunostaining was found in purkinje cell somata and the proximal dendrites in the cerebellar cortex (<i>D9</i>), and in the pyramidal cells of the cerebral cortex (<i>D7</i>). Immunolabeling was also observed in thalamic neurons (<i>D8</i>). Immunoperoxidase staining. Scale bar: (<i>D1</i>): 1000 μm. (<i>D2</i>): 2000 μm, (<i>D3</i>): 500 μm, (<i>D4–D8</i>): 20 μm, (<i>D9</i>): 50 μm.</p
Vesicular colocalization of VAMP2 and AMPA receptor subunits.
<p>(<i>A</i>) 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. (<i>C</i>) 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. (<i>E</i>–<i>K</i>) 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140868#pone.0140868.ref065" target="_blank">65</a>]. (<i>E</i>) IP with anti-GluA1, WB with anti-GluA1. (<i>F</i>) IP with anti-GluA1, WB with anti-VAMP2. (<i>G</i>) IP with anti-GluA2, WB with anti-GluA2. (H) IP with anti-GluA2, WB with anti-VAMP2. (<i>I</i>) IP with anti-VAMP2, WP with anti-VAMP2. (<i>J</i>) IP with anti-VAMP2, WB with anti-GluA1. (<i>K</i>) IP with anti-VAMP2, WB with anti-GluA2. (<i>L</i>) WB of brain homogenate fractions stained with anti-synaptophysin (P38) antibody. (<i>M</i>) WB of brain homogenate fractions stained with phosphate-activated glutaminase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140868#pone.0140868.ref039" target="_blank">39</a>] antibody. (<i>N</i>) Electron micrograph of vesicle preparation similar to the ones used in (<i>E</i>-<i>M</i>). Scale bars: 100 nm (<i>A spine</i>, <i>B</i>), 125 nm (<i>A dendrite</i>), 100 nm (<i>N</i>).</p
Specificity of in-house VAMP2 antibody.
<p>(<i>A</i>) 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. (<i>B</i>) 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. (<i>C</i>) 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 (<i>p</i> < 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.</p
The effect on plasma membrane expression of AMPA receptor subunits in hippocampal culture after presynaptic silencing with Bafilomycin (BAF).
<p>(<i>A</i>) Control synapses immunolabeled with anti-GluA1 in non-permeabilized culture. (<i>B</i>) As in (<i>A</i>), but treated with Bafilomycin (BAF) before fixation. (<i>C</i>) Control synapses immunolabeled with anti-GluA2 in non-permeabilized culture. (<i>D</i>) As in (<i>C</i>), but treated with Bafilomycin (BAF) before fixation. (<i>E</i>) Quantification of difference in maximal punctate GluA1 labeling intensities between control and Bafilomycin-treated cultures. There is no significant difference. (<i>F</i>) 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 (<i>p</i> < 0.001). Scale bar: 10 μm.</p
Confocal images of dissociated hippocampal cultures.
<p>Double labeling with anti-TUJ1 (<i>A</i>) and anti-VAMP2 (<i>B</i>) demonstrates that VAMP2 is located along the dendrites and gives characteristic punctate labeling (<i>C</i>). Double labeling with antibodies against synaptophysin (P38) (<i>D</i>) and VAMP2 (<i>E</i>) indicates that VAMP2 is colocalized with the presynaptic marker (<i>F</i>). Double labeling for PSD-95 (<i>G</i>) and VAMP2 (<i>H</i>) shows partial colocalization postsynaptic (<i>I</i>). Small, high-resolution pictures of single synapses from (<i>F</i>) and (<i>I</i>) are shown in the right part of these images, respectively. Scale bar: 20 μm.</p
Quantitation of synaptic VAMP2 immunogold labeling.
<p>(<i>A</i>) 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. (<i>B</i>) 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. (<i>C</i>) Mean immunogold labeling over cytoplasmic regions of interest. (<i>D</i>) Mean immunogold labeling over plasma membrane regions of interest.</p