Rhythmic Changes in Synapse Numbers in Drosophila melanogaster Motor Terminals
Figure 3
Visualization of synapses in the MN5 terminal based on synaptic markers and LCM.
(A) Example of the staining of the MN5 neuromuscular junction with anti-Horseradish Peroxidase (HRP, blue) and anti-nc82 (BRP, magenta) in some of the thinnest branches, located deeply inside the muscle. The bar scale represents 10 µm. (B) Example of the same staining in a branch of this neuromuscular junction showing boutons with synapses (white arrowheads), and boutons without them (outlined arrowheads) at larger magnification. The bar scale represents 2 µm. (C) Triple staining done at CT19 and CT7 confirming the presence of synaptic vesicles (Syn, magenta) and synapses (BRP, green) in the presynaptic side of synapses in each bouton (see outlined boutons b1, b2 and b3). Orange dotted lines in b1, b2 and b3 show the places where the analysis of fluorescence intensity was done to confirm the presence of Synorf1 in the proximity of the synapses. Details of the signals obtained are shown for HRP (D), BRP (E) and Syn (F). In (E and F) orange outlines mark selected synapses (S1, S2 and S3) located in boutons marked as b1, b2 and b3. (G) The graph shows the profiles of fluorescence intensity measured for the immunofluorescence corresponding to HRP, BRP and Syn, detailed in (C), (E) and (F). Syn and BRP signals match their location in b1, b2 and b3 and confirm that synaptic vesicles are in the proximity of S1, S2 and S3 synapses. (H) Triple staining confirming the accumulation of glutamate receptors (GluRIIA, magenta) apposed to the accumulation of BRP signals (see outlined boutons b1, b2 and b3). Orange dotted lines in b1, b2 and b3 show the places where the analysis of fluorescence intensity was done to confirm the presence of GluRIIA in the proximity of the synapses. Details of the signals obtained are shown for HRP (I), BRP (J) and GluRIIA (K). In (J and K) orange outlines mark selected synapses (S1, S2 and S3) analyzed in b1, b2 and b3. (L) The graph shows the profiles of fluorescence intensity measured for HRP, BRP and GluRIIA, detailed in (H), (J) and (K). GluRIIA and BRP signals match their location in b1, b2 and b3 and confirm that the accumulations of glutamate receptors correspond with accumulations of BRP in the synapses marked S1, S2 and S3.