Fig 2: AKT isoform expression in human brain tissue. Immunofluorescent staining using isoform-specific AKT antibodies and cell-type-specific markers in human brain tissue isolated from the temporal cortex shows an expression pattern like our mouse brain staining. AKT1 (red, left column) shows colocalization with the neuronal marker NeuN (green), AKT2 (red, middle column) shows colocalization with the astrocytic marker GFAP (white), and AKT3 (red, right column) shows diffuse staining in neuronal cell bodies and neuropil throughout the tissue. Bottom row: Human cortical tissue costained for NeuN (green) and GFAP (white) with AKT isoform-specific primary antibodies omitted but the secondary antibody included (red) to demonstrate the specificity of AKT isoform staining. Hoechst (blue), nuclear stain. Specimens were obtained from four subjects (N = 2/sex) considered as controls by the brain bank with a mean age of 58.75 years (Table 1), and images are representative of AKT isoform staining patterns for each subject.

Fig 3: Comparison of EYFP expression across different survival times after injection of anterograde AAVDJ/retrograde rAAV2retro versus anterograde AAV5/retrograde rAAV2retro.a The median intensity of EYFP-native fluorescence is summarized with a box and whisker plot. Wilcoxon rank sum test did not show significant difference among all samples. The number of double infected cells counted as EYFP immunopositive cells among the NeuN-immunopositive cells is indicated along the vertical axis. b The median intensity of EYFP immunofluorescence is summarized with a box and whisker plot. Wilcoxon rank sum test showed significant difference between AAV5 at 12 weeks and AAVDJ at 3 or 6 weeks. c Correlation between intensities of native fluorescence (horizontal axis) and immunofluorescence (vertical axis) for EYFP in individual neurons shown in (a) and (b).

Fig 4: Newborn neurons in the adult NAc.a Top: schematic of the experimental protocol. Bottom: representative picture of a coronal brain section from an 8-week-old mouse stained with antibodies against DCX (red), showing neuroblasts in the V-SVZ. Higher magnifications of the boxed areas (dashed yellow lines) are shown on the right. a1 High-magnification image from the area indicated with the yellow dashed rectangle in a, showing DCX and BrdU immunostainings. a2 High-magnification image from the area indicated with the yellow dashed rectangle in a1. Newborn neuroblasts (DCX+/BrdU+) in the ventral V-SVZ and NAc. a3–a5. Left: high-magnification maximum-projection images of newborn neuroblasts (DCX+/BrdU+) in the NAc. Right: orthogonal views. b TEM image showing a neuroblast in mitosis. b1 High-magnification image from the area indicated with the yellow dashed box in b. c Schematic of the experimental protocol. d Left: schematic illustrates the imaged brain area containing the NAc with both core and shell. Right: quantification of newborn neurons in the core and shell of the NAc in adult mice injected with BrdU as depicted in c (n = 8 mice). d Example of newborn cells labeled by BrdU+ and the mature neuronal marker NeuN+. e Example of newborn neurons triple-labeled with BrdU, NeuN, and the MSN marker DARPP-32. f, g Confocal pictures showing BrdU labeling in D1 receptor (EGFP in f) or D2 receptor (EGFP in g) expressing neurons. Abbreviations: ac, anterior commissure; dSt, dorsal striatum; lv, lateral ventricle; NAc, nucleus accumbens; Sp, septum. Scale bars in µm: 1000 (a), 100 (a1), 50 (a2), 5 (a3–a5, e–g), 2 (b), 500 nm (b1). See Supplementary Fig. 7.

Fig 5: Retrograde labelling with CTb in the L5 segment of a Tac1Cre mouse that had received injections of CTb into T13–L1 and AAV.flex.eGFP into L5. The section has been scanned to reveal CTb (red), NeuN (blue), and eGFP (green). There are numerous CTb-labelled neurons, and the CTb staining appears in the perikaryal cytoplasm of these cells. They are particularly numerous in laminae I–II, occupying the full mediolateral extent of the dorsal horn, and are scattered throughout the deeper laminae. This section is close to the AAV.flex.eGFP injection site, and there are many eGFP+ cells, particularly in laminae I and IIo. Note that in this section, the eGFP+ cells are not present in the extreme lateral part of the superficial dorsal horn, presumably resulting from lack of spread of the virus from the injection site. The dashed line represents the border between laminae II and III, and the box in the upper image shows the region illustrated in Fig. 3. The image is a projection of 11 optical sections at 1 µm z separation, with a pixel size of 0.297 µm. Scale bar = 100 µm