Fig. 4: Input-defined postsynaptic neurons’ anterograde monosynaptic tracing with H129Amp tracer system together with other tracers.
a Schematic illustration of the experiment setup and tracing strategy. The H129Amp tracer system (H129Amp-CTG 1.5 × 108 pfu/ml and helper 1.5 × 108 pfu/ml, in 300 nl) was injected into the L-AC (AP: −2.80 mm; ML: −4.13 mm; DV: −2.38 mm) of wild-type C57BL/6 mice, and AAV2/9-DIO-mCh-gK (1.0 × 1012 vg/ml, 100 nl) was simultaneously injected into the R-AC (AP: −2.80 mm; ML: +4.13 mm; DV: −2.38 mm). At Day 21, H129-dgK-G4 (5.0 × 108 pfu/ml, 100 nl) was injected into the R-AC of the same mice. Brains were collected at Day 26, and images were obtained after cryosection and DAPI counterstaining. H129Amp-CTG propagates in the L-AC neurons (1st order) with helper assistance, and transmits through the first synapse to the 2nd-order neuron in the R-AC. There, H129Amp-CTG expresses Cre to initiate AAV2/9-DIO-mCh-gK expressing mCherry and gK, labeling the neurons and supporting H129-dgK-G4 propagation, respectively. The newly produced H129-dgK-G4 then transmits through the second synapse to the 3rd-order neurons, labeling them with GFP. b–e Representative input-defined postsynaptic neurons’ anterograde monosynaptic tracing result at Day 26. Shown are the representative images of the 2nd-order brain region, R-AC (b), and the 3rd-order regions, including L-AC, R-MG, and R-LA (c–e). The 1st-order starter neurons were not visible anymore and the potential 2nd−order starter neurons are labeled by both GFP and mCherry (merged as yellow), indicated with white arrowheads (b). Images with higher magnifications of the boxed areas are presented in the lower panels.
