Prefrontal inputs to the amygdala instruct fear extinction memory formation

Abstract

Persistent anxiety after a psychological trauma is a hallmark of many anxiety disorders. However, the neural circuits mediating the extinction of traumatic fear memories remain incompletely understood. We show that selective, in vivo stimulation of the ventromedial prefrontal cortex (vmPFC)-amygdala pathway facilitated extinction memory formation, but not retrieval. Conversely, silencing the vmPFC-amygdala pathway impaired extinction formation and reduced extinction-induced amygdala activity. Our data demonstrate a critical instructional role for the vmPFC-amygdala circuit in the formation of extinction memories. These findings advance our understanding of the neural basis of persistent fear, with implications for posttraumatic stress disorder and other anxiety disorders.

Fig. 1. Bidirectional control of the vmPFC-amygdala circuit.

(A and C) Examples (top) and virus localization population heat maps representing all mice used in the current study (bottom; red and blue respectively represent maximum and minimum areas of cumulative virus localization across mice) of ChR2-AAV–expressing (A) and ArchT-AAV–expressing (C) glutamatergic neurons in vmPFC. Scale bar, 500 μm. (B and D) Raster plots and firing rate from single units (top), and z-scored population activity (bottom) showing blue-light (473 nm, 10 mW, 20 Hz, 5-ms pulses) increased (B) and green-light (532 nm, 10 mW, continuous) decreased (D) in vivo vmPFC unit activity in ChR2-AAV– and ArchT-AAV–expressing neurons. (E) Example of slice containing ChR2-AAV–expressing vmPFC axons in the BL and cartoon depicting the procedure for recording blue light–evoked activity at BL pyramidal neurons. (F) Blue light shone on ChR2-AAV–expressing vmPFC axons in the BL increased EPSC amplitude at BL pyramidal neurons in a light intensity–dependent manner (scale bar: y axis, 500 pA; x axis, 50 ms). (G) Application of the AMPA receptor blocker CNQX abolished light-evoked EPSCs at BL pyramidal neurons (scale bar: y axis, 300 pA; x axis, 50 ms). (H and J) Examples of infected axons immunolabeled with anti–green fluorescent protein (GFP)/Alexa 488 antibodies (green) in the BL and BM of ChR2-AAV–expressing (H) and ArchT-AAV–expressing (J) vmPFC projection neurons. ICNs are immunolabeled with anti–Forkhead box protein P2 (FoxP2)–tetramethyl rhodamine isothiocyanate (TRITC) antibodies (red) (scale bars, 100 μm). (I and K) Cartoon depicting optic-fiber placement (top) and localization (bottom) targeting ChR2-AAV–expressing (I) and ArchT-AAV–expressing (K) vmPFC axons, representing all mice used in the current study. Data are means ± SEM.

Fig. 2. vmPFC-amygdala circuit stimulation facilitates extinction formation.

(A and B) Blue light shone on ChR2-AAV–expressing vmPFC axons in the amygdala during full (50-trial) extinction training (A) did not change freezing during training or subsequent (light-free) retrieval (B). (C and D) Light shone during partial (10-trial) extinction training (C) decreased freezing during (light-free) retrieval (D). (E and F) Light shone during retrieval [after partial (10-trial, light-free) extinction training] (E) did not alter freezing (F). For virus expression and optical fiber locations, see Fig. 1, A, H, and I. Extinction trial-block = 5 CS (conditioned stimulus) presentations.

Fig. 3. vmPFC-amygdala circuit silencing impairs extinction formation and BL recruitment.

(A and B) Green light shone on ArchT-AAV–expressing vmPFC axons in the amygdala during extinction training (A) increased freezing during training or subsequent (light-free) retrieval (B). (C and D) Light shone during retrieval (after light-free extinction training) (C) did not alter freezing (D). (E to G) Green light shone during extinction training (E) decreased the number of Zif268⁺ cells in the LA (F) and BL (G) (example images shown on the right; scale bar, 50 μm). For virus expression and optical fiber locations, see Fig. 1, C, J, and K. Extinction trial-block = 5 CS presentations.