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

Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy

Background: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. New method: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. Comparison with existing method and results: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. Conclusion: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM

Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy

Background Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. New method To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. Comparison with existing method and results An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. Conclusion Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM

SNARE protein syntaxin-1 colocalizes closely with NMDA receptor subunit NR2B in postsynaptic spines in the hippocampus

Syntaxins are a family of membrane-integrated proteins that are instrumental in exocytosis of vesicles. Syntaxin-1 is an essential component of the presynaptic exocytotic fusion machinery in the brain and interacts with several other proteins. Syntaxin-1 forms a four-helical bundle complex with proteins SNAP-25 and VAMP2 that drives fusion of vesicles with the plasma membrane in the active zone. Little is known, however, about the ultrastructural localization of syntaxin-1 at the synapse. We have analysed the intrasynaptic expression of syntaxin-1 in glutamatergic hippocampal synapses in detail by using quantitative postembedding immunogold labeling. Syntaxin-1 was present in highest concentrations at the presynaptic active zone, supporting its role in transmitter release. Presynaptic plasma membrane lateral to the active zone, as well as presynaptic cytoplasmic vesicles were also labeled. However, syntaxin-1 was also significantly expressed in postsynaptic spines, where it was localized at the postsynaptic density, at postsynaptic lateral membranes and in postsynaptic cytoplasm. Postsynaptically, syntaxin-1 colocalized in the nanometer range with the NMDA receptor subunit NR2B, but only weakly with the AMPA receptor subunits GluA2/3. This observation points to the possibility that syntaxin-1 may be involved with NR2B vesicular trafficking from cytoplasmic stores to the postsynaptic plasma membrane, thus facilitating synaptic plasticity. Confocal immunofluorescence double labeling with PSD-95 and ultrastructural fractionation of synaptosomes also confirm localization of syntaxin-1 at the postsynaptic density