Fig. 4: DN1as inhibits a subgroup of DN3s including SMP232.
a Representative PER and GFP double staining of DN1a-spl > trans-Tango brain. Anti-PER (cyan) and anti-GFP (DN3: yellow) staining were visualized in the brain of flies. Yellow arrows indicate DN3s. Scale bar, 20 μm. b Direct synaptic contacts between DN1s and DN3s revealed by restricted trans-Tango. The experimental and control brains were stained with anti-GFP (yellow) and anti-RFP (cyan), respectively, to identify that the DN3s were postsynaptic to DN1as. Anti-GFP-labeled neurons are downstream of DN1as (upper). Anti-RFP-labeled neurons are non-downstream neurons (lower). The yellow arrow indicates DN3s; Scale bars, 50 μm. c Anatomic characterization of the SMP232-like DN3s postsynaptic to DN1as revealed by restricted trans-Tango in (b). The left panel is the EM reconstruction of DN3s and the right panel is a GFP staining of downstream of DN1a by restricted trans-Tango. Purple arrow indicates SMP232-like DN3s; Scale bar, 50 μm. d SMP232-like DN3s are labeled by intersection between GluClα-GAL4 and AstC-LexA. The red arrow indicates SMP232-like DN3s; Scale bar, 20 μm. e Double staining of GFP (cyan) and RFP (yellow) in AstC-LexA > LexAop-GFP, DN1a-spl > UAS-tdTomato brain. Scale bar, 20 μm. f Representative pseudocolored images of calcium responses of DN3s before and after ATP perfusion activation of DN1as (left); Scale bars, 20 μm. Representative GCaMP traces (ΔF/F) of DN3s in response to DN1as activation (right, n = 8). Con: grey; Exp: green. g Quantification of relative max calcium activities of DN3s in (f). Data are presented as mean in MaxΔF/F0 ± SEM in the histogram; two-tailed t-test; ****p < 0.0001, p = 4.95044E-07. h Model of two inhibitory DN1as-DN3s and DN1as-LNds circuits based on our functional imaging data.
