Lack of medial prefrontal cortex activation underlies the immediate extinction deficit

Abstract

We conducted a series of experiments to investigate the neural basis of the immediate extinction deficit, the lack of extinction when the interval between fear memory acquisition and extinction is short. In experiment 1, rats were given extinction training composed of 15 conditioned stimuli (CSs) either 15 min (immediate extinction: I-EXT) or 24 h (delayed extinction: D-EXT) after five tone-shock pairings. In the retention test performed 48 h after conditioning, I-EXT group exhibited significantly higher freezing than D-EXT group. In experiment 2, functional activation in the medial prefrontal cortex (mPFC) was detected using c-fos immunoreactivity. The number of Fos-positive neurons in the mPFC was significantly lower in I-EXT group than in D-EXT group. In experiment 3, rats received immediate extinction with microstimulation of the infralimbic region (IL) of the mPFC, either contingently paired or unpaired with the CS. In a subsequent retention test, the paired stimulation group exhibited decreased freezing relative to the unpaired stimulation group. Together, our results suggest that the immediate extinction deficit may be linked to the lack of neuronal activity in the IL.

Figure 1.

Immediate extinction deficit. A, Percentage of freezing during conditioning. Rats were given five CS–US conditioning trials. All groups exhibited a similar increase in freezing across trials. B, Percentage of freezing during extinction training. The immediate extinction group (I-EXT) and the delayed extinction group (D-EXT) received 15 CS-only trials, 15 min and 24 h after fear conditioning, respectively. Both groups showed similarly attenuated freezing at the end of the extinction training. The immediate no-extinction group (I-NO-EXT) and the delayed no-extinction group (D-NO-EXT) were placed in the extinction context for the same amount of time but without CS presentation at the corresponding time points. All tests were performed in a novel context. C, Averaged percentage of freezing across three test trials. The D-EXT group showed significantly lower freezing relative to the other groups. *p < 0.05. All data are presented as means ± SEM.

Figure 2.

Fos expression in the mPFC following immediate extinction. A, Representative photomicrographs showing Fos expression in the IL, PL, and M2. Fos expression was identified using CY3 fluorescence (red), and nuclei were identified by DAPI staining (blue). Scale bar, 10 μm. B, Schematic diagram illustrating the IL, PL, and M2. C–E, Percentages of Fos-IR neurons in the IL, PL, and M2. Fos-IR neurons in both the IL and PL were found in significantly greater numbers in the D-EXT group than in other groups. Numbers of Fos-IR neurons in the I-EXT group were not different from the controls. D-EXT, Delayed extinction group; I-EXT, immediate extinction group; I-NO-EXT, immediate no extinction group; D-NO-EXT, delayed no extinction group; I-UNPAIRED, immediate unpaired conditioning group; D-UNPAIRED, delayed unpaired conditioning group. *p < 0.05. All data are presented as means ± SEM.

Figure 3.

Effects of IL stimulation during immediate extinction training on long-term extinction memory. A, A representative image showing a coronal section from a rat with a stimulating electrode in the IL. B, Location of stimulation sites in the IL for the immediate unpaired-stimulation group, immediate paired-stimulation group, immediate stimulation-only group, delayed unpaired-stimulation group, and immediate M2-stimulation group [modified from Paxinos and Watson (1998)]. C, Percentage of freezing during conditioning. Rats were given five CS–US conditioning trials. All groups showed robust freezing in the last conditioning trial. D, Percentage of freezing during extinction training. The immediate paired-stimulation group (I-PAIRED) and M2-stimulation group (M2-STIM) received IL or M2 stimulation at 100 ms after CS onset during immediate extinction training, respectively. The immediate unpaired-stimulation group (I-UNPAIRED) and delayed unpaired-stimulation groups (D-UNPAIRED) received explicitly unpaired IL stimulation and tones at corresponding time points. The immediate no-stimulation and delayed no-stimulation groups received extinction training at 15 min (I-NO-STIM) or 24 h (D-NO-STIM) after conditioning, but never received IL stimulation. In addition, the stimulation-only group (I-STIM) received IL stimulation in the absence of CS presentation. All groups showed a similar pattern of reduced freezing, except for the I-STIM group. E, Averaged percentage of freezing across three test trials. The I-PAIRED group froze significantly less than other groups, but did not differ from the D-NO-STIM or D-UNPAIRED groups. *p < 0.05. All data are presented as means ± SEM.