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Neurons communicate with each other via synapses where the electrochemical signal is converted into a biochemical one. Information is transferred by the release of neurotransmitters into the synaptic cleft and is received on the post-synaptic side by corresponding receptor proteins (Sudhof, 2004). During this process, the synaptic vesicles fuse with the pre-synaptic membrane, so that the inner vesicle membrane is transiently exposed to the extracellular space.
In cultured hippocampal neurons, luminal protein epitopes can thus be bound by antibodies added to the culture medium, which are then incorporated into the synaptic vesicles during the subsequent clathrin-mediated endocytosis (Kraszewski et al., 1995).
With this approach, active synapses with ongoing vesicle recycling can be selectively labelled (figure 1). If for example an antibody against Synaptotagmin1 is applied, all active synapses are labelled independently of their neurotransmitter phenotype (figure 2). In contrast, an antibody against the vesicular GABA transporter (VGAT) allows selective labelling of exclusively active inhibitory synapses (Martens et al. 2008).
When primary-labelled antibodies are used, direct visualization of recycling vesicles is possible without additional fixation, permeabilization or application of any secondary reagents.
Alternatively, primary antibodies can be pre-incubated with NanoTags secondary FluoTags to form primary-secondary antibody complexes. These complexes can then be used like primary labelled antibodies for the direct labeling of living neurons.
Figure 1: Labeling of recycling synaptic vesicles with fluorochromated antibodies directed against luminal epitopes of synaptic vesicle proteins.
Figure 2: Labeling of recycling vesicles in cultured hippocampus neurons (cat. no. 105 311AF). The living cells were incubated for 30 min with the labeled antibody in culture medium.
After endocytosis, the lumen of the synaptic vesicle is reacidified by a proton ATPase. The resulting proton gradient is required for reloading the synaptic vesicles with neurotransmitter molecules mediated by specific vesicular neurotransmitter transporters to prepare them for the next round of fusion (Gasnier, 2000).
pH-sensitive fluorescent dyes such as CypHer5E and AcidiFluor Orange are fluorescent only in an acidic environment. Corresponding antibody conjugates are therefore ideally suited to detect recycling synaptic vesicles in living cells since they only light up after complete antibody internalization and reacidification of the synaptic vesicle lumen (figure 2).
Synaptic Systems offers a unique portfolio of antibodies directed against luminal epitopes of synaptic vesicle proteins that are suitable for activity dependent labeling of recycling vesicles. A panel of different fluorophore conjugates is available to match your exact needs.
Explore the possibilities of these exclusive reagents.
