Fig 2: CRF-R1 signaling pathway in NAc of adult rats. Measurement of NAc extracellular neurotransmitter levels using in vivo microdialysis, the vertical gray bars indicate the time of NAc local infusion of 70 mM K+-aCSF, and the horizontal black lines indicate the time of intra-NAc infusion of the antagonist, vehicle, or co-infusion of antagonist with Forskolin. (A) NAc DA levels in the presence of aCSF (blue dotted line; n = 9), 10 µM of CP154,526 (orange dotted line; n = 9) and 10 µM of CP154,526 + 50 µM of Forskolin (green dotted line; n = 6). DA levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (2, 21) = 3.278, p = 0.0577), a main effect of time (F (9, 189) = 32.31, p < 0.0001), and treatment × time interaction (F (18, 189) = 3.246, p < 0.0001; CP154,526 vs. aCSF, **** p < 0.0001, * p = 0.0210, CP154,526 + Forskolin vs. aCSF, n.s. p > 0.9999, Bonferroni post hoc test). (B) NAc glutamate levels in the presence of aCSF (blue dotted line; n = 9), 10 µM of CP154,526 (orange dotted line; n = 9) and 10 µM of CP154,526 + 50 µM of Forskolin (green dotted line; n = 6). Glutamate levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (2, 21) = 2.060, p = 0.1524), a main effect of time (F (9, 189) = 9.253, p < 0.0001), and treatment × time interaction (F (18, 189) = 1.803, p = 0.0274; CP154,526 vs. aCSF, **** p < 0.0001, CP154,526 + Forskolin vs. aCSF, ** p= 0.0025, Bonferroni post hoc test). (C) NAc GABA levels in the presence of aCSF (blue dotted line; n = 9), 10 µM of CP154,526 (orange dotted line; n = 9) and 10 µM of CP154,526 + 50 µM of Forskolin (green dotted line; n = 6). GABA levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (2, 21) = 0.03616, p = 0.9645), a main effect of time (F (9, 189) = 19.32, p < 0.0001), and no treatment × time interaction (F (18, 189) = 0.1907, p > 0.9999; CP154,526 vs. aCSF, n.s. p > 0.9999, CP154,526 + Forskolin vs. aCSF, n.s. p > 0.9999, Bonferroni post hoc test).

Fig 3: CRF-R1 signaling pathway in NAc of juvenile rats. Measurement of NAc extracellular neurotransmitter levels using in vivo microdialysis, the vertical gray bar indicates the time of NAc local infusion of 70 mM K+-aCSF, and the horizontal black lines indicate the time of intra-NAc infusion of the antagonist, vehicle, or co-infusion of antagonist with Forskolin. (A) NAc DA levels in the presence of aCSF (blue; n = 5), 10 µM of CP154,526 (orange; n = 6), and 10 µM of CP154,526 + 50 µM of Forskolin (green; n = 5). DA levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (2, 13) = 1.279, p = 0.3110), a main effect of time (F (9, 117) = 25.19, p < 0.0001), and treatment × time interaction (F (18, 117) = 1.914, p = 0.0209; CP154,526 vs. aCSF, **** p< 0.0001, CP154,526 + Forskolin vs. aCSF, **** p< 0.0001, Bonferroni post hoc test). (B) NAc glutamate levels in the presence of aCSF (blue; n = 5), 10 µM of CP154,526 (orange; n = 6) and 10 µM of CP154,526 + 50 µM of Forskolin (green; n = 6). Glutamate levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (2, 14) = 2.756, p = 0.0979), a main effect of time (F (9, 126) = 12.04, p < 0.0001), and treatment × time interaction (F (18, 126) = 2.207, p = 0.0058; CP154,526 vs. aCSF, ** p= 0.0098, **** p< 0.0001, CP154,526 + Forskolin vs. aCSF, ** p= 0.0079, Bonferroni post hoc test). (C) NAc GABA levels in the presence of aCSF (blue; n = 5), 10 µM of CP154,526 (orange; n = 6) and 10 µM of CP154,526 + 50 µM of Forskolin (green; n = 6). GABA levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (2, 14) = 0.1510, p = 0.8612), a main effect of time (F (9, 126) = 34.07, p < 0.0001), and no treatment × time interaction (F (18, 126) = 0.3438, p = 0.9942; CP154,526 vs. aCSF, n.s. p > 0.9999, CP154,526 + Forskolin vs. aCSF, n.s. p > 0.9999, Bonferroni post hoc test).

Fig 4: CRF-R1 localization in juvenile versus adult rats. Comparison of CRF-R1 localization between juvenile (left) and adult (right) rats using immunofluorescence. The right panel is a 1.3 × zoom of the selected region on the left, showing every separate channel and its respective merge image. The scale bar represents 15 µm in every image. (A) The figure shows patterns of the label for CRF-R1 and TH in the NAc of juvenile and adult rats. Arrowheads show TH-positive axons, and asterisks show puncta pattern distribution of CRF-R1. (B) The figure shows patterns of the label for CRF-R1 and GAD67 in the NAc of juvenile and adult rats. Arrowheads show GAD67-positive somas and axons, and asterisks show puncta pattern distribution of CRF-R1. (C) The figure shows patterns of the label for CRF-R1 and TH in the VTA of juvenile and adult rats. Arrowheads show TH-positive somas and axons, and asterisks show puncta pattern distribution of CRF-R1.

Fig 5: CRF-R1 modulates neurotransmitter levels in NAc of adult rats. Measurement of NAc extracellular neurotransmitter levels using in vivo microdialysis, the vertical gray bars indicate the time of NAc local infusion of 70 mM K+-aCSF, and the horizontal black lines indicate the time of intra-NAc infusion of the antagonist or vehicle. (A) NAc DA levels in the presence of aCSF (blue; n = 9) and 10 µM of CP154,526 (orange; n = 9). DA levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed a main effect of treatment (F (1, 8) = 6.258, p = 0.0369), a main effect of time (F (9, 72) = 28.51, p < 0.0001), and treatment × time interaction (F (9, 72) = 6.401, p < 0.0001; **** p < 0.0001, * p =0.0210, CP154,526 vs. aCSF, Bonferroni post hoc test). (B) NAc glutamate levels in the presence of aCSF (n = 9) and 10 µM of CP154,526 (n = 9). Glutamate levels were normalized to the average of the first 3 values for each condition and expressed percentage. Two-way ANOVA revealed a main effect of treatment (F (1, 8) = 16.01, p = 0.0039), a main effect of time (F (9, 72) = 6.566, p < 0.0001), and treatment × time interaction (F (9, 72) = 6.337, p < 0.0001; * p = 0.0140, **** p < 0.0001, CP154,526 vs. aCSF, Bonferroni post hoc test). (C) NAc GABA levels in the presence of aCSF (n = 9) and 10 µM of CP154,526 (n = 9). GABA levels were normalized to the average of the first 3 values for each condition and expressed as percentage. Two-way ANOVA revealed no effect of treatment (F (1, 8) = 0.01630, p = 0.9016), a main effect of time (F (9, 72) = 13.39, p < 0.0001), and no treatment × time interaction (F (9, 72) = 0.07740, p = 0.9999; n.s. p > 0.9999, CP154,526 vs. aCSF, Bonferroni post hoc test).