Figure 5.

Experimental design and confirmation of specific tracing of LMVN GABAergic axons

(A) Schematic diagram of Cre-inducible AAV vector expressing palGFP under control of EF1α promoter. Other abbreviations are defined in Figure 1A.

(B) Schematic of unilateral injection of AAV. The outline is the same as Figure 1B. Green vertical line and circle indicate the injection capillary and target, respectively.

(C) Representative projected z stack confocal images of IHC for palGFP (Left, green) and FISH for Gad1 mRNA (Middle, red) in the LMVN of the GAD1-Cre mouse injected with Cre-inducible AAV vector expressing palGFP. Merged image of (Left) and (Middle) displays colocalization (Right).

(D) Representative images of the coronal brain section of a GAD1-Cre mouse injected unilaterally with Cre-inducible AAV vectors encoding palGFP into the LMVN. (Left) Wide-field images of the coronal sections. The value in the upper left corner indicates the AP coordinates from bregma (mm). AAV vectors were injected into the right side (a white frame). The contralateral projections were observed on the left side. (Right) Magnified images corresponding to a white frame in the left wide-field image.

(E) Confocal fluorescence images of the coronal sections in which a high density of GFP-positive axons was detected. Each value in the upper side indicates the AP coordinates from bregma (mm). Fluorescence images are captured in z stack and projected with maximum intensity. The right side of the image corresponds to the ipsilateral side of the injection. Anatomical abbreviations are listed in Table 1.