Fig. 3: AMPH upregulates Netrin-1 in the NAc of mid-adolescent females, counteracting its downregulation of Dcc levels in the VTA.

a Netrin-1 is highly expressed in the PFC, with lower expression in the NAc. DCC is expressed in a complementary pattern, with DCC-expressing dopamine axons segregated to the NAc. b When Dcc is reduced in dopamine neurons of adolescent male mice, their axons fail to recognize the NAc as their final target and instead grow ectopically to the PFC⁵³. c Reducing Netrin-1 expression in the NAc during adolescence also results in ectopic growth of Dcc-expressing dopamine axons to the PFC in male mice⁶². d Experimental timeline for early adolescent treatment. e Experimental timeline for mid-adolescent treatment. f AMPH in early adolescence downregulates NAc Netrin-1 levels in males, but not in females (Table 3A, B). g AMPH in mid-adolescencence no longer downregulates Netrin-1 in the NAc, however mid-adolescent females show significant Netrin-1 upregulation in response to AMPH (Fig. 1i) (Table 3C, D). All graphs of Western blots are normalized to the saline condition for each age and sex. h Dcc mRNA expression in the VTA and Netrin-1 protein levels in the NAc of female mice treated with AMPH in mid-adolescence show a strong and significant negative correlation (Table 3E). i Top, Netrin-1 protein in the NAc of female mice was downregulated using an shRNA approach before treatment with AMPH or saline in mid-adolescence. Left, Netrin-1 shRNA virus was well expressed in the NAc of female mice. Right, Adult female mice treated with AMPH in mid-adolescence were not different from their saline-treated counterparts when receiving a scrambled control virus, in agreement with the results in Fig. 2h. However, adult females had an increased expanse of dopamine input to the PFC as adults when Netrin-1 in the NAc was downregulated with shRNA before treatment with AMPH in mid-adolescence (Table 3F). All bar graphs are presented as mean values ± SEM. Source data are provided as a Source Data file. *p < 0.05, **p < 0.01.