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. 2020 Aug 17;30(16):3200–3211.e8. doi: 10.1016/j.cub.2020.05.077

Figure 3.

Figure 3

Functional Analyses of DAN Input Neurons

(A) Forty-nine GAL4 driver lines with identified DAN input neurons were used to drive UAS-CsChrimson and screened for memory implantation by pairing neuronal activation with odor exposure. Flies were starved 18–26 h prior to training and tested for immediate memory performance. Lines emphasized in this study (mean ± SEM; individual data points are displayed as dots, either P.I. > 0.1 or P.I. < −0.1, or connecting from a neuropil of prior interest) are labeled (Figure S3A, fully labeled version).

(B) Connectivity matrix between DANs ordered according to morphological cluster identity and neurons labeled in 10 GAL4 lines, corresponding to 11 input clusters (MBON-γ5β′2a and MBON-γ4γ5, SEZON01-03, LHON01-02, LHON-AD1b2, and OTHERS15-16). Valence of memory formed is reflected by input connectivity.

(C) Direct MBON-DAN connectivity matrix. We identified several MBONs to provide input to specific DANs (note: we traced all inputs to 7 PAM-γ5, and 2 β′2a DANs with extensive review; Figure S2; Methods S1). Numbers indicate total synapse counts between MBONs and DANs.

(D) Adding other traced DAN input neurons creates potential for indirect connectivity between some MBONs and specific subsets of DANs. Indirect connectivity matrix showing the number of DAN input neurons that are downstream of MBONs with at least 3 synapses between each. Columns are normalized by their sum.

(E) Olfactory learning with sucrose reinforcement. Schematic: experimental timeline and temperature shift protocol. Blocking neuron output during training abolished 30 min appetitive memory specifically in SEZON03-GAL4; UAS-Shits1 and R58E02-GAL4; UAS-Shits1 flies (mean ± SEM, p < 0.0241 and 0.0089, respectively; one-way ANOVA, with Dunnett’s post hoc test, n = 10).

(F) Olfactory learning with bitter (DEET) reinforcement. Schematic: experimental timeline and temperature shift protocol. Blocking neuron output during training impaired immediate aversive memory in MB320C-, SEZON01-, and SEZON02-GAL4; UAS-Shits1, but not in SEZON03; UAS-Shits1 flies (mean ± SEM, p < 0.0009, 0.0344, and 0.0170, respectively; one-way ANOVA with Dunnett’s post hoc test, n = 12).

(G) Connectivity matrix to specific branches of the PPL1-γ1pedc dendrite reveals classes of input neurons have branch specificity.

See also Figure S3, Data S1, and Video S3.