Rescue of impaired fear extinction and normalization of cortico-amygdala circuit dysfunction in a genetic mouse model by dietary zinc restriction

Abstract

Fear extinction is impaired in neuropsychiatric disorders, including posttraumatic stress disorder. Identifying drugs that facilitate fear extinction in animal models provides leads for novel pharmacological treatments for these disorders. Zinc (Zn) is expressed in neurons in a cortico-amygdala circuit mediating fear extinction, and modulates neurotransmitter systems regulating extinction. We previously found that the 129S1/SvImJ mouse strain (S1) exhibited a profound impairment in fear extinction, coupled with abnormalities in the activation of the extinction circuit. Here, we tested the role of Zn in fear extinction in S1 and C57BL/6N reference strain (B6) by feeding the mice a Zn-restricted diet (ZnR) and testing for fear extinction, as well as neuronal activation of the extinction circuit via quantification of the immediate-early genes c-Fos and Zif268. Results showed that (preconditioning or postconditioning) ZnR completely rescued deficient extinction learning and long-term extinction retrieval in S1 and expedited extinction learning in B6, without affecting fear acquisition or fear expression. The extinction-facilitating effects of ZnR were associated with the normalization of Zif268 and/or c-Fos expression in cortico-amygdala regions of S1. Specifically, ZnR increased activity in infralimbic cortex, lateral and basolateral amygdala nuclei, and lateral central amygdala nucleus, and decreased activity in prelimbic and insular cortices and medial central amygdala nucleus. ZnR also increased activation in the main intercalated nucleus and decreased activation of the medial paracapsular intercalated mass in S1. Our findings reveal a novel role for Zn in fear extinction and further support the utility of the S1 model for identifying extinction facilitating drugs.

Figure 1.

Effect of ZnR on fear extinction using a paradigm consisting of 15 CS presentations during extinction retrieval. ZnR beginning 21 d before conditioning (Cond) rescued impaired extinction learning and retrieval in S1 mice and facilitated extinction learning in B6 mice, relative to control diet (Ctl), but did not lead to savings in extinction (re)learning during extinction retrieval (15 CS presentations) (n = 8 per group). Data are presented as means ± SEM. *p < 0.05, S1 control diet versus B6 control diet; ^#p < 0.05, S1 control diet versus S1 ZnR; ^†p < 0.05, B6 ZnR versus S1 control diet; ^§p < 0.05, B6 control diet versus B6 ZnR.

Figure 2.

Effect of ZnR on fear extinction using a paradigm consisting of one CS presentation during extinction retrieval. ZnR beginning 21 d before conditioning (Cond) rescued impaired extinction learning and retrieval in S1 mice and facilitated extinction learning in B6 mice, relative to control diet (Ctl) (n = 6 per group). Data are presented as means ± SEM. *p < 0.05, S1 control diet versus B6 control diet; ^#p < 0.05, S1 control diet versus S1 ZnR; ^†p < 0.05, B6 ZnR versus S1 control diet; ^§p < 0.05, B6 control diet versus B6 ZnR.

Figure 3.

Effect of ZnR on Zif268 expression in PFC following extinction retrieval. A, Representative coronal section from a control-fed S1 mouse stained for Zif268 immunoreactivity showing delineation of the prefrontal cortex regions quantified [delineation of regions was aided by use of a mouse atlas (Paxinos and Franklin, 2001)]. B–D, Control-diet (Ctl) S1 mice showed increased Zif268 expression in PrL (B), decreased expression in IL (C), and increased expression in AID (D), compared with ZnR S1 or B6 fed either control or ZnR diets. Scale bars, 500 μm (A), 200 μm (B, C), 100 μm (D). n = 6 per group. Data are presented as means ± SEM. AIV, Ventral insular cortex; Cg1, Cingulate cortex area 1; M1, primary motor cortex; M2, secondary motor cortex. **p < 0.01, B6 control diet versus S1 control diet; ^††p < 0.01, B6 ZnR versus S1 control diet; ^##p < 0.01, S1 ZnR versus S1 control diet.

Figure 4.

Effect of ZnR on Zif268 expression in amygdala following extinction retrieval. A, Representative coronal section from a control-fed S1 mouse stained for Zif268 immunoreactivity showing delineation of amygdala regions quantified [delineation of regions was aided by use of a mouse atlas (Paxinos and Franklin, 2001)]. B–F, Control-diet (Ctl) S1 mice showed decreased Zif268 expression in BA (B), Lad (C), Lav (D), and CeL (E), and increased expression in CeM (F), compared with ZnR S1 or B6 fed either control or ZnR diets. Scale bar, 500 μm (A), 50 μm (B, D), 200 μm (F). n = 6 per group. ACo, Anterior cortical amygdala; CeC, capsular subdivision of the central amygdala; MePD, posterodorsal division of the amygdala; MePV, posteroventral subdivision of the medial amygdala; PLCo, posterolateral cortical amygdala; opt, optic tract. Data are presented as means ± SEM. **p < 0.01, B6 control diet versus S1 control diet; ^††p < 0.01, B6 ZnR versus S1 control diet; ^##p < 0.01, S1 ZnR versus S1 control diet.

Figure 5.

Effect of ZnR on Zif268 expression in the ITC masses following extinction retrieval. A, Nissl stain of a control-fed S1 brain depicting ITC regions at bregma −1.58 mm. Scale bar, 100 μm. B, C, Control-diet (Ctl) S1 mice showed increased Zif268 expression in Imp (B) and decreased expression in the In (C) compared with ZnR S1 or B6 fed either control or ZnR diets. Scale bar, 25 μm. n = 6 per group. Data are presented as means ± SEM. **p < 0.01, B6 control diet versus S1 control diet; ^††p < 0.01 B6 ZnR versus S1 control diet; ^##p < 0.01, S1 ZnR versus S1 control diet.

Figure 6.

A, Effect of ZnR on fear conditioning (Cond) and fear expression using mild fear conditioning. ZnR beginning 21 d prior to fear conditioning did not enhance fear conditioning or fear expression in either S1 or B6 (n = 6 per group). Data are presented as means ± SEM. B, Effect of postconditioning ZnR on fear extinction. ZnR beginning after conditioning rescued impaired extinction learning and retrieval in S1, relative to control diet (Ctl) (n = 6–9 per group). Data are presented as means ± SEM. *p < 0.05, S1 control diet versus B6 control diet; ^#p < 0.05, S1 control diet versus S1 ZnR; ^†p < 0.05, B6 ZnR versus S1 control diet.