α-Syn accumulates in synaptic boutons at the Drosophila NMJ. (A) Third instar larval stage (L3) Drosophila (left) was used to investigate presynaptic terminals (boutons) terminating at the NMJ (middle). Immunohistochemistry assay reveals the muscle fibres (grey) stained with phalloidin that binds to actin; the axons descending from the motor neuron and terminating onto the muscles are labelled with anti-HRP (green); and the presynaptic terminal of the motor neuron labelled with anti-BRP (red). (B) Representative image of NMJ of nSyb>EGFP and nSyb>WT-α-syn-EGFP larvae immunostained with anti-GFP (green), anti-CSP (cyan) and anti-HRP (magenta). Arrowheads indicate accumulation of WT-α-syn-EGFP in synaptic boutons, which are immunolabelled with anti-CSP while control EGFP is homogeneously expressed in synaptic boutons and axonal regions devoid of CSP immunoreactivity; dashed boxes show a higher magnification of the areas indicated by the arrowheads. (C) Quantitative analysis of the ratio of GFP fluorescence intensity between boutons and axons; ****P = 0.0001; mean ± SEM shown for each genotype (n = 5–6 flies/genotype). Statistical analyses were performed using unpaired two-tailed t-test. Scale bars: 10 µm.