Quantitative analysis of synaptic boutons in Drosophila primary neuronal cultures

Little information is currently available for structural and quantitative aspects of Drosophila central synapses due to difficulties in accessing those synapses in the tiny fly brain. Here, we developed a new approach to quantitatively analyze central synapses using Drosophila primary neuronal cultures. Two different markers were used to identify synaptic boutons: GFP marking with a synaptotagmin (Syt)::eGFP transgene and anti-Syt antibody. These markers clearly recognized puncta-like synaptic boutons and both signals were well overlapped. In addition, these puncta signals were completely absent in neuronal cultures derived from a Syt null mutant SytAD4, firmly demonstrating that anti-Syt(+) puncta are presynaptic terminals. Since anti-Syt signals were stronger and extensive, it was chosen to quantify synaptic boutons in the neuronal culture. Using an image analysis software Image J, synaptic boutons were quantified on the basis of the size and intensity of anti-Syt(+) signals. The number of synaptic boutons in wild type neurons increased by 27% between 3 and 9 days in culture. This increase was much greater (142%) in neuronal cultures derived from a FasIIe86 mutant known to show alterations in synapse growth and stabilization. A parallel increase in neurite length was also observed in both wild type and FasIIe86 neurons. Interestingly, the number of GABAergic synaptic boutons did not increase during this time, indicating distinctive mechanisms underlying development and maintenance of specific types of boutons. Our results successfully showed that Drosophila synaptic boutons can be quantified and thus we can examine genes and signaling pathways regulating structural properties of central synapses in Drosophila.