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. 2023 Sep 18;12:e85756. doi: 10.7554/eLife.85756

Figure 4. UpWind Neurons (UpWiNs) integrate excitatory and inhibitory synaptic inputs from mushroom body output neurons (MBONs).

(A) Functional connectivity between MBON-α3 and UpWiNs. Chrimson88-tdTomato was expressed in MBON-α3 by MB082C split-GAL4, and the photostimulation responses were measured by whole-cell current-clamp recording in randomly selected UpWiNs labeled by R64A11-LexA. 3 out of 11 neurons (7 flies) showed excitatory response. Mean voltage traces from individual connected (orange) and unconnected UpWiNs (gray) are overlaid. The connection was strong enough to elicit spikes (black; single-trial response in one of the connected UpWiNs). Magenta vertical line indicates photostimulation (10 ms). (B) Functional connectivity between MBON-α1 and UpWiNs. Chrimson88-tdTomato expression in MBON-α1 was driven by MB310C split-GAL4. 4 out of 17 neurons (12 flies) showed inhibitory response. Mean voltage traces from individual connected (green) and unconnected UpWiNs (gray) are overlaid. (C) Integration of synaptic inputs from MBON-α3 and MBON-α1. Population responses of UpWiNs were measured by two-photon calcium imaging at the junction between dendrites and axonal tracts (mean ∆F/F ± SEM) while photostimulating MBON-α3 (orange; n=5), MBON-α1 (green; n=11) or both (black; n=7). Expression of GCaMP6s was driven by R64A11-LexA, and Chrimson88-tdTomato by G0239-GAL4 (MBON-α3) and/or MB310C (MBON-α1). Photostimulation: 1 s (magenta). While activation of MBON-α1 did not evoke detectable inhibition in the calcium signal, it effectively canceled the excitation by MBON-α3.

Figure 4—source data 1. The values used for Figure 4A.
Figure 4—source data 2. The values used for Figure 4B.
Figure 4—source data 3. The values used for Figure 4C.

Figure 4.

Figure 4—figure supplement 1. Excitatory interconnections between UpWind Neurons (UpWiNs).

Figure 4—figure supplement 1.

(A) Expression of Chrimson-tdTomato (red) and GCaMP6s (green) in UpWiNs. To express them in mutually exclusive subsets of UpWiNs, GCaMP expression was driven by a broad UpWiN driver 64A11-LexA, while Chrimson-tdTomato and LexAp65-DBD2-RNAi were driven in a subset of UpWiNs by SS67249. White box in the left image indicates an example axonal region of interest (ROI), which is zoomed in on the right image. Scale bar, 5 µm. (B) Thresholded images of GCaMP and Chrimson-tdTomato fluorescence. A small number of voxels that showed co-expression of GCaMP and Chrimson, presumably due to incomplete RNAi, were excluded from analysis. (C) Two-photon imaging of GCaMP6s signals (mean ∆F/F ± SEM; n=8). 1 s photostimulation (magenta) evoked calcium responses in both axonal and dendritic ROIs.
Figure 4—figure supplement 1—source data 1. The values used for Figure 4–figure supplement 1.