Nature research paper: Synaptic gradients transform object location to action
P2XWhole-cell patch-clamp recordings from the GF were carried out in 2–4-day-old female flies as described above. For P2Xreceptor activation of LC4 or LPLC2 VPNs, a glass capillary pulled to a 1-μm diameter was positioned on the VPN dendrites, which expressed both GFP and the P2Xreceptor, approximately 50 μm below the surface of the brain. ATP was microinjected under the control of a Picospritzer .
. Female flies carrying the recombined constructs were crossed into male flies with VPN-specific LexA driver lines and hsFLP recombinase. At 48 h after puparium formation, pupae were heat-shocked for 15 min in 37 °C water bath.Unless otherwise specified, dissected flies were aged 3–4 days post eclosion. Brains were dissected in ice-cold Schneider’sMedium , and fixed in acid-free glyoxal containing 5% sucrose overnight at 4 °C. Brains were rinsed repeatedly with PBST containing 0.
. The technique for subsequent mounting in DPX was adapted from the Janelia protocol for mounting the central nervous system of adultin DPX. After being washed to remove residual secondary antibodies, brains were additionally fixed with PBS containing 4% paraformaldehyde for 3 h at room temperature, rinsed with PBS and mounted on 22 × 22-mm square No. 1.5H cover glass previously coated with poly--lysine with added 0.
To assess the spatial proximity between presynaptic sites of individual LC4 or LPLC2 neurons and DN dendrites , Brp puncta in single VPN cells were reconstructed using the Imaris Spots tool, followed by identification of their centroids, as well as centroids of reconstructed dendritic filaments. Distance between Brp puncta and DN dendrite centroids was measured along the sagittal diameter of the glomerulus or along three cardinal axes of the glomerulus .
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