Background: Leuprolide acetate, a gonadotropin-releasing hormone agonist that may be used as a puberty blocker, is used to treat gender dysphoria in transgender and nonbinary adolescents. As adolescence is a time of increased mental health risk, it is important to understand the impact of pausing puberty on brain and behavioral development. We recently found that pausing puberty with leuprolide reduced anxiety-like behavior in both male and female rats. We also find that leuprolide treatment eliminates typical sex differences in rough-and-tumble play behavior that is observed during adolescent development. The current study now examines if leuprolide treatment alters mRNA levels of genes related to structural plasticity within the rodent adolescent amygdala, a brain region known to influence anxiety and rough-and-tumble behavior.
Methods: Juvenile Sprague Dawley rats (24 male and 24 female) were injected with either 25 ug/kg of leuprolide acetate or saline from postnatal day (P) 27-39. Behavioral assessments, including rough-and-tumble play and anxiety-like behaviors using the elevated plus maze, were conducted between P27-37 and P38-39, respectively. Brain and trunk blood samples were collected on P40 to measure sex steroid levels. Quantitative PCR was used to measure amygdala mRNA levels for the following genes: Glial fibrillary acidic protein (Gfap), an astrocyte structure marker; activity-regulated cytoskeleton-associated protein (Arc), an immediate early gene involved in cellular plasticity within the amygdala; and allograft inflammatory factor 1 (Aif), a marker indicative of present microglia. Results: Leuprolide eliminated sex differences in testosterone, androstenedione, and corticosterone levels (p<0.001). Control females exhibited higher Arc and Gfap levels than control males (p = 0.006 and p = 0.023, respectively), and leuprolide treatment eliminated these sex differences. Arc expression mRNA expression negatively correlated with serum testosterone and positively correlated with corticosterone and androstenedione levels (p = 0.027, p = 0.014, p = 0.006, respectively). Arc also negatively correlated with anxiety-like behavior (p = 0.005). Aif negatively correlated with pinning behavior (p = 0.010) and testosterone levels (p = 0.005).
Discussion: Our findings suggest that puberty blockers, such as leuprolide, reduce sex differences in markers of structural plasticity and astrocytes within the adolescent amygdala. These results indicate that sex differences in the amygdala, a region related to emotion and decision-making processes, continue to be refined during early adolescence. Furthermore, pausing puberty by leuprolide treatment can prevent further sex differences within the amygdala from developing. We find that leuprolide treatment lowers the levels of serum testosterone, which is associated with increased Gfap levels within the amygdala and reduced rough-and-tumble behavior in male but not female rats. Thus, astrocytes located in the amygdala may serve as biological targets for understanding the impact of hormonal blockade on sex differences in adolescent social development.
Conclusion: Our data highlight the significant role of puberty blockers in reducing sex differences in amygdala development and behavior during adolescence. These findings are crucial for comprehending the broader implications of puberty suppression in transgender and nonbinary youth, emphasizing the need for further research on the long-term effects of such treatments on mental health and neurodevelopment.
