0.5 µg of shRNA plasmid were injected twice 24h apart at the speed of 0.5 µl/min into rat brain to target infralimbic cortex.

Adolescent rats are prone to impaired fear extinction, suggesting that mechanistic differences in extinction could exist in adolescent and adult rats. Since the infralimbic cortex (IL) is critical for fear extinction, we used PCR array technology to identify gene expression changes in IL induced by fear extinction in adolescent rats. Interestingly, the ephrin type B receptor 2 (EphB2), a tyrosine kinase receptor associated with synaptic development, was downregulated in IL after fear extinction. Consistent with the PCR array results, EphB2 levels of mRNA and protein were reduced in IL after fear extinction compared with fear conditioning, suggesting that EphB2 signaling in IL regulates fear extinction memory in adolescents. Finally, reducing EphB2 synthesis in IL with shRNA accelerated fear extinction learning in adolescent rats, but not in adult rats. These findings identify EphB2 in IL as a key regulator of fear extinction during adolescence, perhaps due to the increase in synaptic remodeling occurring during this developmental phase.