Fig. 4. Labeling POm- or vM1-derived inputs in S1BF neurons using cFork system. (A) Schematic diagram illustrating the experimental strategy used to demonstrate simultaneous dual color labeling of postsynaptic neurons based on synaptic connections with presynaptic inputs. AAV1s with different recombinases (AAV1-Cre or AAV1-FlpO) were injected into two presynaptic inputs (presynaptic #1 and #2) independently. Postsynaptic neurons receiving multiple presynaptic inputs, including presynaptic #1 and #2, were designed to express our dual gene expression cassette (cDIO-EGFP-fDIO-mScarlet) included in the AAV-cGreen-fRed construct. (B) Experimental scheme for simultaneous labeling of POm- or vM1-S1BF connectivity. AAV1-hSyn-Cre and AAV1-hSyn-FlpO were injected into two different presynaptic inputs of S1BF, POm and vM1, respectively. AAV5-cGreen-fRed was injected into the postsynaptic S1BF. (C) Representative confocal image showing S1BF cortical neurons receiving synaptic inputs from POm (green) or vM1 (red). Fluorescent signals were amplified by fluorescent immunostaining with anti-GFP or anti-RFP antibodies. The white dotted line indicates the boundary of L4; scale bar: 200 μm. Representative S1BF cells expressing both EGFP and mScarlet are denoted as C1 and C2 (right panels); scale bar: 50 μm.
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