Fig. 2
In vitro and in vivo anterograde monosynaptic labeling by H129-dgK-G4 and its AAV helper. A. In vitro transneuronal labeling of H129-dgK-G4. 1 × 106 fetal mouse cortical neurons were sequentially plated in both chambers of the microfluidic plates at day 1 and day 5, and cultured for additional 14 days to allow synapse formation in the afferent chamber. The indicated viruses were inoculated to the efferent side with the dosage of 1 × 106 pfu for H129-derived tracers and 8 × 109 vg for AAV2/9-mCh-gK. The labeled neurons in the afferent chamber were examined at 2 days post infection (dpi). The dotted lines indicate the borders between chambers and the microchannels, and shown are the representative images from 6 microfluidic plates per group performed in 3 independent experiments. B–E. In vivo anterograde monosynaptic labeling of H129-dgK-G4 with the helper AAV2/9-mCh-gK. In the simplified visual circuit pathway (B, upper panel), neurons in the lateral geniculate nucleus (LGN) receive the projection from the retina, and innervate the neurons in layer 4 of the primary visual cortex (V1(L4)). AAV2/9-mCh-gK (1.0 × 1012 vg/ml, 100 nl) and H129-dgK-G4 (5.0 × 108 pfu/ml, 100 nl) were sequentially injected into LGN at day 1 and day 22, and the brains were collected at day 27 after perfusion (B, lower panel). Shown are the representative images of the injection site (C), V1 (D), and retinas (E) from the same mouse. The white arrow indicates the cells colabeled by mCherry and GFP, representing the potential tracing starter neurons