Figure 6: Axo-axonal lateral neuromodulation underlies mAChR-B effects.
A. Experimental configuration. GCaMP6f was expressed in all αβγ KCs. The SPARC method was used to drive CsChrimson in a small subset of αβγ KCs. Responses to MCH and OCT of γ KCs that do not express CsChrimson were examined with and without optogenetic activation of the KC subpopulation.
B. Example of a fly brain with the strategy presented in A. Maximum intensity projection of 28 confocal sections (1 μm) through the central brain of a fly carrying MB247-LexA-LexAop-GCaMP6f, and CsChrimson::tdTomato in stochastically distributed subsets of neurons within the MB247-GAL4 driver line transgenes.
C, F, I, L. ΔF/F of GCaMP6f signal in the γ KC lobe for control (C, F) and KD (I, L) flies, during presentation of odor pulses (OCT, C, I; MCH, F, L; horizontal black lines) without (black) or with (orange) optogenetic activation of the KC subpopulation expressing CsChrimson. MB247-LexA was used to drive LexAop-GCaMP. MB247-GAL4 was used to drive UAS-mAChR-B RNAi 1 and TI{20XUAS-SPARC2-I-Syn21-CsChrimson::tdTomato-3.1}CR-P40. Data are mean (solid line) ± SEM (shaded area). GCaMP signals were taken from regions not expressing CsChrimson (see Figure S4C).
D, E, G, H, J, K, M, N. Peak “on” response (D, G, J, M), and the integral of the odor response (E, H, K, N) of the traces presented in C, F, I, L before (left) or after (right) optogenetic activation. A significant decrease in odor responses is observed only in control flies, indicating lateral neuromodulation. This lateral neuromodulation is absent in KD flies not expressing mAChR-B. n for control and KD flies, respectively: OCT, 7, 6; MCH, 7, 7. * p < 0.05, ** p < 0.01, *** p < 0.001, (Wilcoxon matched-pairs signed rank test). For detailed statistical analysis see Table S1.