Fig. 2.

Positive experience activates the VTA-BLA-NAc circuit.

(A) Left, The experimental timeline; Right, sexual behavior, including mounting latency (221.7 ± 53.35, n = 7), mounting frequency (11.57 ± 1.962, n = 7) and mounting duration (33.24 ± 6.888, n = 7). Intraperitoneal injection of 4-OHT induced hM3D-mCherry labeling in activated cells during neutral experience (B) or positive experience (B′) in the VTA, SN and IPN of Fos-CreERT2 mice. Intraperitoneal injection of clozapine-N-oxide (CNO) activated c-Fos expression in hM3D-mCherry-labelled neurons (neutral experience group, C and D; positive experience group, C′ and D′). C-Fos expression in BLA (E, E′), NAc (F, F′), mPFC (G, G′) and DG (H, H′) after reactivating hM3D-mCherry-labelled VTA neurons. Statistical analysis of the histological data revealed that positive experience induced more hM3D-mCherry-labelled VTA neurons compared with neutral experience (I, two-tailed unpaired t-test with Welch's correction, t_(4.607) = 5.852, P = 0.0027), and that the c-Fos positive VTA cells of positive experience group after CNO injection were more than those of neutral experience group (I′, Mann-Whitney U test, P = 0.0079) although most VTA cells were hM3D-mCherry-labelled in both groups (I″, neutral 81.53 ± 5.510 %, positive 86.41 ± 1.888 %). Few cells were labelled with hM3D-mCherry (B, B′) and only a small number of cells were c-Fos-positive after CNO injection (C, C′) in SN and IPN in both groups. Statistical analysis revealed no significant difference in the number of c-Fos-positive cells in SN (J) and IPN (K) between neutral and positive experience groups. The number of c-Fos-positive cells after clozapine-N-oxide injection was significantly different in BLA (L, two-tailed unpaired t-test with Welch's correction, t_(4.159) = 5.265, P = 0.0056) and NAc (M, two-tailed unpaired t-test with Welch's correction, t_(4.461) = 2.870, P = 0.04) between neutral and positive experience groups, but not in mPFC (N, Mann-Whitney U test, P = 0.5476) and DG (O, two-tailed unpaired t-test, t₍₈₎ = 0.8750, P = 0.4071). Neutral experience group: n = 5; positive experience group: n = 5. (P) Diagram illustrating virus injection in target areas. (Q) Representative coronal slice showing the expression of AAV-hSyn-WGA-Cre-T2A-ZsGreen (green) 3 weeks after virus injection into the VTA. (R) Representative coronal slice showing the expression of AAV-CAG-DIO-mCherry (red) 3 weeks after virus injection into the BLA. (S) Representative coronal slice showing the strong mCherry–positive (red) fibers in NAc 3 weeks after virus injection into the BLA. (T) Diagram illustrating virus injection in target areas and subsequent positive experience. (U) Representative image showing the injection sites in the NAc. Representative images showing the expression of GFP (V) and c-Fos (V′) in BLA. (V″) Representative image showing c-Fos expression in BLA merged with GFP. (W) Statistical analysis showing the percentage of c-Fos positive BLA neurons that were also GFP labelled (69.81 ± 1.731 %, n = 4). Representative images showing the expression of GFP (X) and c-Fos staining (X′) in NAc. (X″) Representative merged images. (N) Statistical analysis showing the percentage of c-Fos positive VTA neurons that were also GFP labelled (14.25 ± 3.080 %, n = 4). *P < 0.05, **P < 0.01. Data are presented as means±SEM. Annotation (AP): distance from the bregma (mm). Scale bars correspond to 100 μm. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)