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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.

The inositol 1,4,5-trisphosphate receptor (IP3R) subtype IP3R1 is highly enriched in the brain, including hippocampal neurons. It plays an important function in regulating intracellular calcium concentrations. Residing on the smooth endoplasmic reticulum (sER), the IP3R1 mobilizes calcium into the cytosol upon binding the intracellular signaling molecule IP3, whose concentration is increased by stimulating certain metabotropic glutamate receptors. Increased calcium may mediate synaptic changes occurring during long-term plasticity, which includes molecular mechanisms underlying memory encoding. The exact synaptic localization of IP3R1 in the central nervous system (CNS) remains unclear. We hypothesized that IP3R1, in addition to its known expression in soma and dendritic shafts of hippocampal CA1 pyramidal neurons, also may be present in postsynaptic spines. Moreover, we hypothesized that IP3R1 may be present in presynaptic terminals as well, given the importance of calcium in regulating presynaptic neurotransmitter exocytosis. To test these two hypotheses, we used IP3R1 immunocytochemistry at the light and electron microscopical levels in the CA1 area of the hippocampus. Furthermore, we hypothesized that induction of long-term potentiation (LTP) would be accompanied by an increase in synaptic IP3R1 concentrations, thereby facilitating synaptic mechanisms of long term plasticity. To investigate this, we used quantitative immunogold electron microscopy to determine possible changes in IP3R1 concentration in sub-synaptic compartments before and five minutes after high frequency tetanizations. Firstly, our data confirm localization of IP3R1 in both presynaptic terminals and postsynaptic spines. Secondly, the concentration of IP3R1 after tetanization was significantly increased in the presynaptic compartment, suggesting a presynaptic role of IP3R1 in early phases of synaptic plasticity. It is therefore possible that IP3R1 is involved in modulating neurotransmitter release by regulating calcium homeostasis presynaptically.

In the original publication of the article the author name M. Schupps was incorrect.

The SNARE protein SNAP-25 is well documented as regulator of presynaptic vesicle exocytosis. Increasing evidence suggests roles for SNARE proteins in postsynaptic trafficking of glutamate receptors as a basic mechanism in synaptic plasticity. Despite these indications, detailed quantitative subsynaptic localization studies of SNAP-25 have never been performed. Here, we provide novel electron microscopic data of SNAP-25 localization in postsynaptic spines. In addition to its expected presynaptic localization, we show that the protein is also present in the postsynaptic density (PSD), the postsynaptic lateral membrane and on small vesicles in the postsynaptic cytoplasm. We further investigated possible changes in synaptic SNAP-25 protein expression after hippocampal long-term potentiation (LTP). Quantitative analysis of immunogold-labeled electron microscopy sections did not show statistically significant changes of SNAP-25 gold particle densities 1 h after LTP induction, indicating that local trafficking of SNAP-25 does not play a role in the early phases of LTP. However, the strong expression of SNAP-25 in postsynaptic plasma membranes suggests a function of the protein in postsynaptic vesicle exocytosis and a possible role in hippocampal synaptic plasticity.

The inositol 1,4,5-trisphosphate receptor (IP

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 (AZ). Little is known, however, about the ultrastructural localization of syntaxin-1 at the synapse. We have analyzed 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 AZ, supporting its role in transmitter release. Presynaptic plasma membrane lateral to the AZ, as well as presynaptic cytoplasmic (PreCy) vesicles were also labeled. However, syntaxin-1 was also significantly expressed in postsynaptic spines, where it was localized at the postsynaptic density (PSD), at postsynaptic lateral membranes and in postsynaptic cytoplasm. Postsynaptically, syntaxin-1 colocalized in the nanometer range with the N-methyl-D-aspartate (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 PSD.