Dynamic expression of FKBP5 in the medial prefrontal cortex regulates resiliency to conditioned fear

Abstract

The factors influencing resiliency to the development of post-traumatic stress disorder (PTSD) remain to be elucidated. Clinical studies associate PTSD with polymorphisms of the FK506 binding protein 5 (FKBP5). However, it is unclear whether changes in FKBP5 expression alone could produce resiliency or susceptibility to PTSD-like symptoms. In this study, we used rats as an animal model to examine whether FKBP5 in the infralimbic (IL) or prelimbic (PL) medial prefrontal cortex regulates fear conditioning or extinction. First, we examined FKBP5 expression in IL and PL during fear conditioning or extinction. In contrast to the stable expression of FKBP5 seen in PL, FKBP5 expression in IL increased after fear conditioning and remained elevated even after extinction suggesting that IL FKBP5 levels may modulate fear conditioning or extinction. Consistent with this possibility, reducing basal FKBP5 expression via local infusion of FKBP5-shRNA into IL reduced fear conditioning. Furthermore, reducing IL FKBP5, after consolidation of the fear memory, enhanced extinction memory indicating that IL FKBP5 opposed formation of the extinction memory. Our findings demonstrate that lowering FKBP5 expression in IL is sufficient to both reduce fear acquisition and enhance extinction, and suggest that lower expression of FKBP5 in the ventral medial prefrontal cortex could contribute to resiliency to PTSD.

Figure 1.

Different FKBP5 levels in IL and PL of naïve rats. (A) Behavioral protocol and sacrifice time points for mRNA and protein extraction. (B) Expression of fear during behavioral training in all groups. Tissue punches were extracted at the time of sacrifice indicated by the arrows and RT-PCR and Western blots were used to detect FKBP5 expression. (C) FKBP5 mRNA expression in IL and PL in naïve rats normalized to B-actin. (D) FKBP5 protein relative to GAPDH is also higher in PL than in IL. Unpaired t-test (*) P < 0.01, n = 7–10 rats in each group.

Figure 2.

Fear conditioning and extinction alter FKBP5 mRNA and protein expression in IL. (A) FKBP5 mRNA expression from IL tissue punches extracted after behavioral training and normalized to B-actin. (B) FKBP5 mRNA expression in PL was also analyzed and compared with IL tissue punches. (C–F) FKBP5 protein expression in IL and PL tissue punches normalized to GAPDH and representative Western blots. (***) P < 0.001, (**) P < 0.01.

Figure 3.

Aversive associative learning increases IL expression of FKBP5 protein. (A) Behavior of groups that received paired (COND24) or unpaired (Unpaired24) tones and shocks. Animals were sacrificed the next day as indicated by arrows. (B) Western blots of FKBP5 protein in IL tissue punches relative to GAPDH. (C) FKBP5 protein expression in IL and PL tissue punches normalized to unpaired group from each structure. (**) P < 0.01.

Figure 4.

Knockdown of FKBP5 protein with FKBP5-shRNA plasmid infusions. (A) Localized expression of GFP in IL 5 d after bilateral infusion of Fkbp5-shRNA plasmid. (B) Expression of GFP from Fkbp5-shRNA plasmid in IL neurons. (C) FKBP5 protein from IL tissue punches extracted 2 or 5 d after infusion of Fkbp5–shRNA plasmids normalized to GAPDH. (D) Western blot showing reduced expression of FKBP5 in IL tissue punches from rats 5 d after infusion of scramble or Fkbp5-shRNA plasmids. (*) P < 0.05, (**) P < 0.01, n = 4–5 rats in each group. (CC) corpus callosum.

Figure 5.

Knockdown of FKBP5 in IL prior to conditioning reduces fear learning. (A) Timeline for behavioral training after IL infusion of Fkbp5–shRNA or control (scramble) plasmids 5 d prior to COND. (B) Cannula placement for scramble (black dots) and Fkbp5–shRNA (red dots) rats. (C) Freezing of rats infused with Fkbp5–shRNA or scramble plasmid into IL. n = 11–12 for each group, (*) P < 0.05; # P = 0.09.

Figure 6.

Reducing IL FKBP5 after fear conditioning facilitates extinction recall. (A) Timeline for behavioral training after IL infusion of Fkbp5-shRNA or control (scramble) plasmids 5 d prior to EXT. (B) IL cannula placement for scramble (black dots) and Fkbp5-shRNA (red dots) rats and GFP expression. (C) Freezing of rats infused with Fkbp5–shRNA or scramble plasmid into IL prior extinction. (D) Average freezing of IL-infused rats to two recall tones on day 9. n = 9–13 for each group, (*) P < 0.05.

Figure 7.

Reducing FKBP5 in PL does not affect fear conditioning or extinction. (A) Cannula placement for scramble (black dots) and Fkbp5–shRNA (red dots) rats. (B) Localized expression of GFP in PL 5 d after bilateral infusion of Fkbp5–shRNA plasmid. (C) Freezing of rats infused with Fkbp5–shRNA or scramble plasmid into PL. (D) Average freezing of PL-infused rats to two recall tones on day 3. n = 10–12 for each group, unpaired t-test P > 0.05.