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. 2018 Mar 28;38(13):3318–3332. doi: 10.1523/JNEUROSCI.2173-17.2018

Figure 1.

Figure 1.

IC excitatory and inhibitory neurons as starter cells for rabies-based monosynaptic tracing. A, Characterization of Vglut2-Cre mice with Vglut2 ISH (top row) and VGAT-Cre mice with VGAT ISH (bottom row). Almost all EGFP-positive neurons expressed Vglut2 mRNA in Vglut2-Cre mice (top, arrowheads) and VGAT mRNA in VGAT-Cre mice (bottom, arrowheads). B, Construction of the AAV helper virus and rabies virus (left) and time course of the experiment (right). C, Schematic diagram of extrinsic connections to the IC. Cre-expressing–infected cells express EGFP-TVA (green dots) and RG. Neurons that contact these neurons are infected retrogradely and express DsRed alone (red dots, input neurons). Neurons expressing both EGFP and DsRed are the starter neurons (yellow dots). D Left, An image of the IC showing the input (red) and starter neurons (yellow) superimposed on the drawing of the IC subdivisions. The ICC (central IC) is the core, and the ICD (dorsal IC) and ICX (external IC) are grouped as the shell. The DsRed fluorescence intensity was enhanced to emphasize the labeled fibers primarily located in the ICD and ICX. Right, Starter neurons were identified by colocalization of EGFP (green) and DsRed (red) protein. E, Injection of AAV-DIO-RG and RV without prior AAV-DIO-EGFP-TVA injection in the Vglut2-Cre mice resulted in no DsRed-labeled neurons. F, Injection of AAV-DIO-EGFP-TVA and RV without prior AAV-DIO-RG injection in the Vglut2-Cre mice resulted in no DsRed-labeled input neurons. G, Injecting AAV-DIO-EGFP-TVA, AAV-DIO-RG, and RV to the wild-type mice and performed ISH for VGAT mRNA to identify the GABAergic neurons in the injection site. H, Non-Cre, nonspecific-labeled RV neurons that expressed DsRed in the injection site. I, GABAergic neurons that identified by ISH. J, Six RV neurons (arrowheads) among the 27 RV neurons were GABAergic. K, The 19.9% proportion of Cre-independent RV neurons was GABAergic (14 brain slices collected from 4 mice). Scale bars: A, H, I, J, 20 μm; D, Left, 250 μm; D, Right, 50 μm; E, F, 200 μm.