Different methods of fear reduction are supported by distinct cortical substrates

Abstract

Understanding how learned fear can be reduced is at the heart of treatments for anxiety disorders. Tremendous progress has been made in this regard through extinction training in which the aversive outcome is omitted. However, current progress almost entirely rests on this single paradigm, resulting in a very specialized knowledgebase at the behavioural and neural level of analysis. Here, we used a dual-paradigm approach to show that different methods that lead to reduction in learned fear in rats are dissociated in the cortex. We report that the infralimbic cortex has a very specific role in fear reduction that depends on the omission of aversive events but not on overexpectation. The orbitofrontal cortex, a structure generally overlooked in fear, is critical for downregulating fear when novel predictions about upcoming aversive events are generated, such as when fear is inflated or overexpected, but less so when an expected aversive event is omitted.

Keywords: extinction; fear; infralimbic; learning; neuroscience; orbitofrontal; rat.

Figure 1.. The IL is not necessary for overexpectation.

Location of cannula placements for (A) drug- and (B) vehicle-infused rats in the IL cortex in the overexpectation experiment as verified based on the atlas of Paxinos and Watson, 1997. The symbols represent the most ventral point of the cannula track for each rat and distances are indicated in mm from bregma. (C) Behavioural design for Overexpectation. Behavioural data are represented as mean + SEM percent levels of freezing during the cue period for D) Conditioning, (E) Overexpectation and, (F) Test for Overexpectation of the target stimulus. Overexpectation-M/B (OE-M/B, filled black), n = 13; overexpectation-vehicle (OE-VEH, open black), n = 11; control-M/B (CON-M/B, filled burgundy), n = 11; control-vehicle (CON-VEH open burgundy), n = 14.

Figure 2.. The IL is necessary for extinction.

Using the same animals, the location of cannula placements reallocated for (A) Drug- and (B) Vehicle-infused rats in the IL extinction experiment as verified based on the atlas of Paxinos and Watson, 1997. The symbols represent the most ventral point of the cannula track for each rat and distances are indicated in millimetres from bregma. (C) Behavioural design for Extinction. Behavioural data are represented as mean + SEM percent levels of freezing during the cue period for (D) Conditioning, (E) Extinction, and (F) Test for Extinction of the target stimulus. Extinction-M/B (filled black), n = 11; extinction-vehicle (open black), n = 11; control-M/B (filled burgundy), n = 8; control-vehicle (open burgundy), n = 7.

Figure 3.. The lOFC is necessary for overexpectation.

Location of cannula placements for (A) drug- and (B) vehicle-tinfused rats in the lOFC overexpectation experiment as verified based on the atlas of Paxinos and Watson, 1997. The symbols represent the most ventral point of the cannula track for each rat and distances are indicated in millimetres from bregma. (C) Behavioural design for Overexpectation. Behavioural data are represented as mean + SEM percent levels of freezing during the cue period for (D) Conditioning, (E) Overexpectation and, (F) Test for Overexpectation of the target stimulus. Overexpectation-M/B (filled black), n = 12; overexpectation-vehicle (open black), n = 11; control-M/B (filled burgundy), n = 12; control-vehicle (open burgundy), n = 9.

Figure 4.. The lOFC is not necessary for extinction by omission.

Using the same animals, the location of cannula placements reallocated for (A) drug- and (B) vehicle-infused rats in the lOFC extinction experiment as verified based on the atlas of Paxinos and Watson, 1997. The symbols represent the most ventral point of the cannula track for each rat and distances are indicated in millimetres from bregma. (C) Behavioural design for Extinction. Behavioural data are represented as mean + SEM percent levels of freezing during the cue period for (D) Conditioning, (E) Extinction, and (F) Test for Extinction of the target stimulus. Extinction-M/B (filled black), n = 10; extinction-vehicle (open black), n = 12; control-M/B (filled burgundy), n = 11; control-vehicle (open burgundy), n = 11.

Figure 5.. Inactivating the lOFC impairs but does not abolish extinction learning.

Location of cannula placements for (A) drug- and (B) vehicle-infused rats in the lOFC extinction experiment as verified based on the atlas of Paxinos and Watson, 1997. The symbols represent the most ventral point of the cannula track for each rat and distances are indicated in millimetres from bregma. (C) Behavioural design for Extinction. Behavioural data are represented as mean + SEM percent levels of freezing during the cue period for (D) Conditioning, (E) Extinction, and (F) Test for Extinction of the target stimulus. Extinction-M/B (filled black), n = 16; extinction-vehicle (open black), n = 16; control-M/B (filled burgundy), n = 11; control-vehicle (open burgundy), n = 12.

Figure 6.. The lOFC is not required for subsequent extinction learning.

Using the same animals, the location of cannula placements reallocated for (A) drug- and (B) vehicle-infused rats in the lOFC re-extinction experiment as verified based on the atlas of Paxinos and Watson, 1997. The symbols represent the most ventral point of the cannula track for each rat and distances are indicated in millimetres from bregma. (C) Behavioural design for Extinction. Behavioural data are represented as mean + SEM percent levels of freezing during the cue period for D) Conditioning, (E) Extinction, and F) Test for Re-Extinction of the target stimulus. Extinction-M/B (filled black), n = 16; extinction-vehicle (open black), n = 16; control-M/B (filled burgundy), n = 12; control-vehicle (open burgundy), n = 11.

Figure 7.. Behavioural sequence for Experiments 1–3.

(A) In Experiments 1 and 2, rats are trained to associate two individual cues (tone and light) with a shock during Phase 1 and their performance during this phase was used to determine group allocation such that fear acquisition was similar between the groups. Immediately prior to overexpectation training (Phase 2), the IL (Experiment 1) or lOFC (Experiment 2) was pharmacologically silenced with muscimol and baclofen (0.1 mM muscimol-1 mM baclofen, M/B). Rats in the overexpectation group (OE) received compound presentations of the two cues followed by the delivery of a shock. Rats in the control group (Control) were handled. All rats were then tested for conditioned responding (freezing) the following day to either the tone or the light (counterbalanced). Following the overexpectation experiment, the same rats received conditioning to a novel stimulus (white-noise or steady light, counterbalanced) paired with shock during Phase 3. Rats were reassigned to one of the four groups (counterbalanced for training and drug history) based on their responding during Conditioning. Prior to extinction in Phase 4, rats received an infusion of M/B or vehicle into the IL or lOFC. Rats in the extinction condition (EXT) received non-reinforced presentations of the target cue. Rats in the control condition (Control) were handled. All rats were then tested for conditioned responding to the target cue (counterbalanced) the following day. (B) In Experiment 3, rats were trained to associate a cue (steady light or white-noise, counterbalanced between rats) with shock during Phase 1. Prior to extinction training (Phase 2), rats received an infusion of M/B or vehicle into the lOFC. Rats in the extinction condition (EXT) received non-reinforced presentations of the fear-conditioned cue. Rats in the control condition (Control) were handled. All rats were then tested for conditioned responding to the target cue (counterbalanced) the following day. Following initial extinction, the same rats took part in a subsequent extinction experiment. Rats received conditioning to a novel stimulus (tone or flashing light, counterbalanced) paired with shock during Phase 3. Rats were reassigned to groups based on their responding during Conditioning. Immediately prior to extinction training in Phase 4, the lOFC was pharmacologically silenced in the same manner as described above. Again, rats in the extinction condition (EXT) received non-reinforced presentations of the target stimulus while rats in the control condition (Control) were handled. All rats were then tested for conditioned responding to the target cue the following day.