Layer 2/3 pyramidal neurons from the prefrontal cortical areas project to NAc ChIs. (A1 and A2) Low-magnification images from the prefrontal cortex MO-VO showing layer 2/3 neurons (A1) in contrast to layer five neurons that project to CPu ChIs (A2). (B) Experimental design of injections into the Cux2-CRE mouse line to label the medial orbital cortex MO layer 2/3 neurons that project to NAc ChIs. Mouse was first injected with the helper virus (mCherry) into the MO, followed by injection of RV-GFP into the NAc. (C) Immunocytochemistry of the MO cortex of the Camk2a-CRE mouse labeled the anti-mCherry and anti-GFP antibodies, showing that some cells infected with the helper virus (mCherry⁺) and rabies virus (GFP⁺) are double-labeled (mCherry⁺ GFP⁺, merged). (D) Representative cells in the layer 2/3 of the CaMK2a-CRE mouse prefrontal cortex labeled with the helper virus into the MO-VO followed by the RV-GFP injection into the NAc that were used for electrophysiology recordings. (E) Representative cells with traces of the firing response to a 250-pA step injection of current in layer 2/3 MO-VO pyramidal neurons infected with RV-GFP (GFP, green) and helper virus (mCherry, red). (F) The representative firing traces firing frequency of layer 2/3 MO-VO pyramidal neurons infected with RV-GFP (n = 4 neurons/3 mice) or helper virus (four neurons/three mice) in response to 50-pA current steps injections. (G and H) Histograms showing the membrane potential (F) and the voltage threshold (H) for action potential firing in layer 2/3 MO-VO pyramidal neurons infected with RV-GFP or helper virus.