Infralimbic BDNF signaling is necessary for the beneficial effects of extinction on set shifting in stressed rats

Abstract

Current pharmacotherapies for posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) are ineffective for many patients, and often do not restore cognitive dysfunction associated with these disorders. Behavioral therapies, such as exposure therapy, can be effective for treatment-resistant patients. The mechanisms underlying exposure therapy are not well-understood. Fear extinction as an intervention after chronic stress can model the beneficial effects of exposure therapy in rats. Extinction requires neuronal activity and protein synthesis in the infralimbic (IL) cortex for its beneficial effects. We hypothesized that extinction requires Brain-Derived Neurotrophic Factor (BDNF) activity in the IL cortex to reverse stress-induced cognitive flexibility impairments. Extinction learning reversed set-shifting deficits induced by Chronic Unpredictable Stress (CUS), tested 24 h after extinction. Blocking BDNF signaling in the IL cortex during extinction by local administration of a neutralizing antibody prevented the beneficial effects of extinction on set shifting after stress. Extinction induced activation of the BDNF TrkB receptor, and signaling pathways associated with BDNF (Akt and Erk). Administration of exogenous BDNF into IL cortex in the absence of extinction was sufficient to reverse the effects of stress on set shifting. The effects of extinction were prevented by blocking either Erk or Akt signaling in the IL cortex, whereas the effects of exogenous BDNF were dependent on Erk, but not Akt, signaling. Our observations suggest that BDNF-Erk signaling induced by extinction underlies plastic changes that can reverse or counteract the effects of chronic stress in the IL cortex.

Fig. 1. Extinction induces the phosphorylation of the BDNF receptor TrkB at Y515, but not Y816 in the IL.

A Phosphorylation of TrkB at Y515 is increased in the IL 30 min after the end of extinction compared to tone controls (*p = 0.0018, n = 14–18/group). Insets show data for males and females separately; a student’s t-test showed an effect of extinction on Y515 in males (t₁₇= 3.568, p = 0.0024) but not in females alone. B Phosphorylation of TrkB at Y816 was unchanged 30 min after the end of extinction compared to tone controls (p = 0.6292, n = 12/group), insets show males and females separately. Bars represent SEM. C Fear conditioning (left) and extinction (right) curves showing % time freezing during the presentation of the tone. Bars represent SEM.

Fig. 2. Infralimbic BDNF is necessary for the effects of extinction on set shifting in stressed animals.

A Fear extinction curves in stressed and non-stressed rats, showing percent time freezing during tone presentation. Bars represent SEM. B Extinction induced an increase in phosphorylation of TrkB at Y515 compared to tone controls (*p = 0.0315; 8–9 rats/group). Insets show males and females separately. C CUS impaired set shifting on the AST (CUS/Tones/IgG compared to No Stress/Extinction/IgG, *p = 0.0003), and extinction rescued set shifting in stressed rats (CUS/Extinction/IgG compared to CUS/Tones/IgG, +p = 0.0091). Blocking BDNF in the IL at the time of extinction attenuated the beneficial effect of extinction in stressed rats (CUS/Extinction/anti-BDNF compared to CUS/Extinction/IgG, #p = 0.0218). The BDNF antibody alone had no effect in non-stressed control rats (No stress/Extinction/anti-BDNF compared to No stress/Extinction/IgG, p = 0.9739). Insets show males and females separately. In males, multiple comparisons showed a difference between No stress/extinction/control IgG vs CUS/extinction/anti-BDNF. In females, multiple comparisons showed differences between No stress/extinction/control IgG vs CUS/tones/IgG. Bars represent SEM.

Fig. 3. Extinction induces the phosphorylation of Erk but not Akt in the IL.

A Extinction increased phosphorylation of Erk in the IL 30 min after extinction compared to tone controls (*p = 0.0021, n = 12–14/group). Insets show males and females separately. ANOVA on male data showed a main effect of extinction on pErk (F_1,30= 8.469, p = 0.0067), a main effect of stress (F_1,30= 5.136, p = 0.038), and a difference between Tones CUS vs Extinction Control (p = 0.0074). Extinction induced an increase in pErk in females and a difference between Tones-CUS vs Extinction-CUS p = 0.0023). B Extinction did not significantly increase the phosphorylation of Akt in the IL 30 min after the end of extinction compared to tone controls (p = 0.1639, n = 11–14/group). Insets show males and females separately.

Fig. 4. Erk and Akt signaling are required in the IL cortex for the beneficial effects of extinction on set shifting in stressed animals.

Microinjections of either LY200492 or PD98059 into the IL cortex immediately after extinction blocked the effects of extinction on set-shifting 24 h later in stressed rats. CUS induced a deficit in set shifting (CUS Tones Veh vs Ctrl Ext Veh, +p = 0.0026). Both PD98059 and LY294002 attenuated the improvement in set-shifting induced by extinction in stressed animals (CUS Ext Veh vs CUS Ext PD98059: #p = 0.0031; CUS Ext Veh vs CUS Ext LY294002: *p = 0.0389). Insets show males and females separately.

Fig. 5. Exogenous BDNF administered in the IL cortex reverses the effects of stress on set shifting, and these effects are dependent on Erk signaling.

CUS-BDNF animals performed better than CUS-vehicle-treated animals (+p = 0.0349, n = 6 rats/group). Stressed rats that received BDNF and the PI3k inhibitor LY294002 performed better than CUS-Veh animals (*p = 0.0408), and were not significantly different than CUS-BDNF alone (p = 0.9616). By contrast, stressed rats treated with BDNF and the Erk inhibitor PD90859 performed worse than CUS-BDNF rats (#p = 0.0385). Insets show males and females separately. In males, Holm–Sidak tests showed a difference between CUS-Veh and CUS/BDNF/LY294002 (p = 0.0312). Bars represent SEM. Bottom left is a representative example of injection site localization by dye injection into the IL.