Figure 4 |. Functional plasticity in the mushroom body calyx associated with long-term memory.

(A) Two-photon in vivo imaging setup. Schematic of a fly placed on a custom-made holder under a two-photon microscope equipped with a 40× 1.1 NA water immersion objective. The odor is delivered for 5s with a moisturized, constant air stream through a 1.2mm cannula. (Central panel) Z series of the entire MBC volume of flies expressing post-synapse-tagged Homer-GCaMP3 imaged during odor application at 1Hz (10 optical sections per volume, 4μm step size). (Right panel) A single slice of the image stack shown in the middle panel. Scale bar = 10 μm. (B) Representative optical section from a volumetric time series showing false-coloured response of KC dendrites to 5 s exposure to EtOH (top) or cVA + EtOH (bottom). (C) Magnification of the white square area in (B). 5×5 μm2 ROIs were classified as cVA-responsive (red) if they were only active during cVA + EtOH application, but did not respond to EtOH alone. ROIs that responded to both conditions were classified as Carrier. Scale bar = 5 μm. (D) The fraction of cVA responsive ROIs increased after LTM acquisition compared to the mock control (box plot represent first and third quartiles, thick lines mark the medians, and whiskers represent data range. *p < 0.05, n = 7). (E) Dynamics of ΔF/F% changes over time in KCs of MB247-homer::GCaMP3 flies after mock training (top) or LTM acquisition (bottom). Each row of the heat map represents average responses per animal of all cVA responsive ROIs (red) or of all carrier EtOH responsive ROIs (grey) within one MBC over time. Each column represents one 1s. Flies are first exposed to the EtOH (5s) and then to cVA + EtOH (5s) as indicated by the dashed lines. (F) Plot of average calcium dynamics over time of cVA responsive ROIs during 5 s stimulation with EtOH or with cVA in EtOH (dashed lines) in mock-trained (top) or cVA CS+ (bottom) flies (n = 7). Data represented as mean ± std.