Figure 7. Measuring postsynaptic acetylcholine receptor clusters with Dα7-GFP.
(A) Diagram of the GFP-tagged Dα7 acetylcholine receptor subunit used for AChR visualization. (B) High magnification confocal z-stack images of PNs in the DA1 and VA1d glomeruli expressing Dα7-GFP and mtdT, stained with antibodies against GFP (green), mtdT (red), and endogenous Brp (blue). Patches of mtdT labeling represent ascending PN axons within the plane of the glomerulus. Insets show a high magnification single optical section demonstrating the punctate nature of Dα7-GFP. (C) Representative high magnification single optical sections of Mz19-positive PNs in the DA1 glomerulus expressing Dα7-GFP and stained with antibodies against GFP (green) and endogenous Brp (red). The majority of GFP-positive puncta are apposed to or colocalized with endogenous Brp (yellow), consistent with their association with bona fide active zones. Insets show high magnification of a single optical section where asterisks denote apposed Dα7 puncta. Brp puncta without apposition likely belong to synapses not labeled by the PN-GAL4 driver. Scale bar = 5 μm (2 μm for inset). (D) Representative high magnification single optical sections of Mz19-positive PNs and Or67d-positive ORNs in the DA1 glomerulus expressing Dα7-GFP in the PNs and Brp-Short-mStraw in the ORNs using two binary expression systems. Most ORN active zones (labeled by Brp-Short, red) are apposed to or colocalized (yellow) with PN Dα7 puncta (green), further supporting that these puncta label bona fide synaptic contacts. Insets show high magnification of a single optical section where asterisks denote apposed Brp-Short::Dα7 pairs. Brp puncta without apposition likely belong to ORN synapses with neurons other than PNs and Dα7 puncta without apposition likely correspond to PN postsynaptic sites apposed to synaptic contacts from neurons other than ORNs. (E) Quantification of Dα7 AChR puncta (green, left axis) and neurite volume (black, right axis) in the DA1 and VA1d glomeruli of both male and female adult flies. Statistical comparisons between males and females of a single genotype were done by student's t test. ***p < 0.001. (F) Quantification of AChR density in male (blue) and female (pink) adults based on the data from (C). Significance was assessed with a one-way ANOVA and corrected for multiple comparisons by a posthoc Tukey's multiple comparisons test. *p < 0.05. n.s. = not significant. In all cases, data represent mean ± SEM and n ≥ 11 animals, 21 antennal lobes. Scale bar = 5 µm.