Parvalbumin-Positive Interneurons in the Medial Prefrontal Cortex Regulate Stress-Induced Fear Extinction Impairments in Male and Female Rats

Abstract

Stress has profound effects on fear extinction, a form of learning that is essential to behavioral therapies for trauma-related and stressor-related disorders. Recent work reveals that acute footshock stress reduces medial prefrontal cortex (mPFC) activity that is critical for extinction learning. Reductions in mPFC activity may be mediated by parvalbumin (PV)-containing interneurons via feedforward inhibition imposed by amygdala afferents. To test this hypothesis, footshock stress-induced Fos expression was characterized in PV⁺ and PV^- neurons in the prelimbic (PL) and infralimbic (IL) cortices. Footshock stress increased the proportion of PV⁺ cells expressing Fos in both male and female rats; this effect was more pronounced in IL compared with PL. To determine whether PV⁺ interneurons in the mPFC mediate stress-induced extinction impairments, we chemogenetically silenced these neurons before an immediate extinction procedure in PV-Cre rats. Clozapine-N-oxide (CNO) did not affect conditioned freezing during the extinction procedure. However, CNO exacerbated extinction retrieval in both male and female rats with relatively high PL expression of designer receptors exclusively activated by designer drugs (DREADD). In contrast, in rats with relatively high IL DREADD expression, CNO produced a modest facilitation of extinction in the earliest retrieval trials, but in male rats only. Conversely, excitation of IL PV interneurons was sufficient to impair delayed extinction in both male and female rats. Finally, chemogenetic inhibition of IL-projecting amygdala neurons reduced the immediate extinction deficit in male, but not female rats. These results reveal that PV interneurons regulate extinction learning under stress in a sex-dependent manner, and this effect is mediated by amygdaloprefrontal projections.SIGNIFICANCE STATEMENT Stress significantly impairs the memory of fear extinction, a type of learning that is central to behavioral therapies for trauma-based and anxiety-based disorders (e.g., post-traumatic stress disorder). Here we show that acute footshock stress recruits parvalbumin (PV) interneurons in the medial prefrontal cortex (mPFC) of male and female rats. Silencing mPFC PV interneurons or mPFC-projecting amygdala neurons during immediate extinction influenced extinction retrieval in a sex-dependent manner. This work highlights the role for PV-containing mPFC interneurons in stress-induced impairments in extinction learning.

Keywords: fear extinction; parvalbumin; prefrontal cortex; rat; sex; stress.

Figure 1.

Footshock stress during fear conditioning induces Fos expression in mPFC PV interneurons in male and female rats. A, Experimental design for assessing footshock stress-induced activation of mPFC PV interneurons (experiment 1). Animals underwent 5 trials of tone-shock conditioning (Shock; males, n = 4; females, n = 4), tone-only exposure (No-shock; males, n = 4; females, n = 4), or remained in the home cage (Home; males, n = 3; females, n = 4). Ninety minutes after the session, animals were perfused and brains were removed for immunohistochemistry (IHC; PV and Fos). B, Percentage of freezing (mean ± SEM) during the first (Tr 1) and last (Tr 5) conditioning trials. Fear conditioning increased conditioned freezing compared with No-shock controls. C, Representative image (10×) of a coronal section of the mPFC showing PV and Fos staining in a shocked male. D, E, Outlines depict the IL (D) and PL (E) subdivisions of the mPFC. F, Density of PV⁺ cells (mean ± SEM) plotted by region, sex, and experimental group. G, Density of Fos⁺ PV^– cells (mean ± SEM) plotted by region, sex, and experimental group. H, Percentage of colabeled PV⁺ cells (mean ± SEM) plotted by region, sex, and experimental group. ***p < 0.001, ****p < 0.0001.

Figure 2.

Schematic representation of viral expression for all of the animals in each experiment (viral expression common to all rats is indicated by the most darkly shaded areas). A, Maximal viral spread of DIO-hM4Di-mCherry in the mPFC for each subject (experiment 2). B, Maximal viral spread of S5E2-Gq-dTomato in the IL for each subject (experiment 3). C, Representative immunofluorescent image of CAV2-Cre-GFP in the IL (top panel) and maximal viral spread of DIO-hM4Di-mCherry in the BLA for each subject (bottom panels; experiment 4).

Figure 3.

Chemogenetic inhibition of mPFC PV interneurons before immediate extinction in male and female rats. A, Viral strategy for expressing hM4Di selectively in mPFC PV interneurons of PV-Cre transgenic rats. B, PV-immunostained tissue of the mPFC showing colabeling of PV and mCherry expression (the rightmost mCherry-expressing cell also colocalized with a faint green signal not visible in the image). Scale bar, 50 µm. C, Experimental design for assessing the contribution of mPFC PV interneurons to the IED (experiment 2a). Animals were injected with VEH or CNO before conditioning (a stressor) and immediate extinction, then were tested for extinction retrieval 48 h later. D, Percentage of freezing (mean ± SEM) across conditioning, immediate extinction, and retrieval, plotted by sex and drug group [VEH males (♂), n = 22; CNO ♂, n = 23; VEH females (♀), n = 19; ♀ CNO, n = 19]. Treatment with CNO resulted in nonsignificant increases in freezing during retrieval. Elements of this figure were created with bioRENDER.

Figure 4.

The effect of chemogenetic mPFC PV inhibition on stress-induced extinction impairment depends on sex and localization of hM4Di expression. A, Representative coronal sections through the mPFC showing examples of subjects with relatively high or low IL hM4Di-mCherry expression. Scale bar, 500 µm. B, Percentage of freezing (normalized to baseline; mean ± SEM) during early retrieval blocks, split by high or low IL hM4Di expression. C, Percentage of freezing (normalized to baseline; mean ± SEM) during early retrieval in male and female rats with relatively high IL expression. CNO-treated males, but not females, with high IL hM4Di expression showed an initial modest reduction in freezing relative to vehicle controls. D, Percentage of freezing (normalized to baseline; mean ± SEM) during early retrieval in male and female rats with relatively low IL expression. CNO-treated males and females with low IL hM4Di expression showed higher levels of freezing relative to vehicle controls.

Figure 5.

Chemogenetic inhibition of mPFC PV interneurons reduces shock-induced Fos expression in those neurons. A, Experimental design for DREADD validation using immunohistochemical (IHC) Fos staining (experiment 2b). B, Percentage of freezing (mean ± SEM) across the conditioning session plotted by sex and drug group [VEH males (♂), n = 6; CNO ♂, n = 7; VEH females (♀), n = 6; CNO ♀, n = 6]. Treatment with CNO before conditioning had no effect on freezing across the conditioning session. C, Number of mCherry⁺ cells in the IL and PL cortex (symbols represent data from individual subjects). There was significantly greater mCherry expression in the PL compared with the IL, with no differences in expression across groups. D, Percentage of colabeled mCherry⁺ (mean ± SEM) in the IL and PL, plotted by sex and drug group. Treatment with CNO before conditioning significantly reduced the proportion of mCherry⁺ cells expressing Fos. *p < 0.05, ***p < 0.001.

Figure 6.

Chemogenetic excitation of IL PV interneurons before delayed extinction (under low stress). A, Viral strategy for expressing hM3Dq selectively in IL PV interneurons of wild-type rats (experiment 3). B, mPFC tissue showing dTomato expression in the IL. Scale bar, 500 µm. C, PV-immunostained tissue of the IL showing colabeling of PV and dTomato expression in separate pilot animals. Scale bar, 100 µm. D, Percentage of freezing (mean ± SEM) across conditioning, extinction, and retrieval, plotted by sex and drug group [VEH males (♂), n = 6; CNO ♂, n = 5; VEH females (♀), n = 9; CNO ♀, n = 4]. Treatment with CNO before delayed extinction resulted in impairment of extinction retrieval. Elements of this figure were created with bioRENDER.

Figure 7.

Chemogenetic inhibition of BLA neurons projecting to the IL cortex reduces stress-induced extinction impairment in male, but not female, rats. A, Viral strategy for expressing hM4Di selectively in IL-projecting BLA neurons (experiment 4). B, Representative histology showing hM4Di-mCherry expression in the BLA and CEA. Scale bar, 200 µm. C, Percentage of freezing (mean ± SEM) across conditioning and immediate extinction in males, plotted by drug group (VEH males, n = 10; CNO males, n = 13). D, Percentage of freezing with (normalized to baseline; mean ± SEM) during retrieval in males, plotted by drug group. E, Percentage of freezing (mean ± SEM) across conditioning and immediate extinction in females, plotted by drug group (VEH females, n = 15; CNO females, n = 13). F, Percentage of freezing (normalized to baseline; mean ± SEM) during retrieval in females, plotted by drug group. Elements of this figure were created with bioRENDER. ♂, Male; ♀, female.