Combining laser scanning confocal microscopy and electron microscopy to determine sites of synaptic contact between two identified neurons

Here we report a double labelling method for correlative confocal and electron microscopy (EM) which allows selective characterisation of structural relationships between two single identified neurons in the same preparation. Using the lobster stomatogastric nervous system, we labelled pairs of identified, synaptically-connected neurons by intracellular injection of Lucifer Yellow (LY) in one neuron and a mixture of Rhodamine (Rdh) and Horseradish Peroxidase (HRP) in its partner. First, whole-mounts of LY- and Rdh-stained neurons were visualized using laser scanning confocal microscopy (LSCM) in order to isolate neuropilar regions of possible synaptic contact. Second, after conventional treatment for electron microscopy (LY was revealed with immunogold and HRP with DAB), areas of close appositions were viewed in EM. This technique allowed us to determine all the regions of close contact between two cells, and then to use electron microscopy to determine the presence or absence of synaptic contact within each of these restricted areas. These techniques enabled us to show that there were few areas of apposition and that only an extremely small proportion of these areas was in fact regions of synaptic contact between the two labelled neurons.