Anterior Cingulate Cortex and Ventral Hippocampal Inputs to the Basolateral Amygdala Selectively Control Generalized Fear

Abstract

A common symptom of anxiety disorders is the overgeneralization of fear across a broad range of contextual cues. We previously found that the ACC and ventral hippocampus (vHPC) regulate generalized fear. Here, we investigate the functional projections from the ACC and vHPC to the amygdala and their role in governing generalized fear in a preclinical rodent model. A chemogenetic approach (designer receptor exclusively activated by designer drugs) was used to inhibit glutamatergic projections from the ACC or vHPC that terminate within the BLA at recent (1 d) or remote (28 d) time points after contextually fear conditioning male mice. Inactivating ACC or vHPC projections to the BLA significantly reduced generalized fear to a novel, nonthreatening context but had no effect on fear to the training context. Further, our data indicate that the ACC-BLA circuit supports generalization in a time-independent manner. We also identified, for the first time, a strictly time-dependent role of the vHPC-BLA circuit in supporting remote generalized contextual fear. Dysfunctional signaling to the amygdala from the ACC or the HPC could underlie overgeneralized fear responses that are associated with anxiety disorders. Our findings demonstrate that the ACC and vHPC regulate fear expressed in novel, nonthreatening environments via projections to the BLA but do so as a result of training intensity or time, respectively.SIGNIFICANCE STATEMENT Anxiety disorders are characterized by a common symptom that promotes overgeneralization of fear in nonthreatening environments. Dysregulation of the amygdala, ACC, or hippocampus (HPC) has been hypothesized to contribute to increased fear associated with anxiety disorders. Our findings show that the ACC and HPC projections to the BLA regulate generalized fear in nonthreatening, environments. However, descending ACC projections control fear generalization independent of time, whereas HPC projections play a strictly time-dependent role in regulating generalized fear. Thus, dysfunctional ACC/HPC signaling to the BLA may be a predominant underlying mechanism of nonspecific fear associated with anxiety disorders. Our data have important implications for predictions made by theories about aging memories and interactions between the HPC and cortical regions.

Keywords: ACC; DREADD; amygdala; anxiety disorders; context generalization; hippocampus.

Figure 1.

Inactivation of the ACC eliminates time-dependent generalized context fear. A, All mice underwent context fear conditioning, which consisted of five unsignaled footshocks (1 s, 1.0 mA), each separated by 90 s, in the training context, which included the conditioning chamber with a polka-dot insert attached to the rear Plexiglas wall, white noise (70 db), dim illumination, and the stainless-steel grid floors were cleaned with 70% ethanol. One day or 28 d after training, mice were either placed back in the training context or a distinct novel context, which included the conditioning chamber with a small exhaust fan, and flat brown Plexiglas floors, which were cleaned with 2% Quatricide. There was no visible illumination (illuminated only with an infrared light), and no polka-dot wall insert. B, There was no effect of CNO alone on context-dependent fear behavior. As a CNO control experiment, naive mice were context fear-conditioned and given an intraperitoneal injection of CNO or saline 30 min before testing either 1 or 28 d after training. Percent freezing levels of animals that received saline (filled symbols) or CNO (open symbols) during recent (circles) and remote (squares) tests in the training or neutral context were analyzed (± SEM). Two-way ANOVA identified a significant main effect of context at the recent time point (F_(1,12) = 96.40, p < 0.001), but not at the remote time point; mice froze significantly more in the training context than the novel context at 1 but not 28 d after training. ***p < 0.001, significantly different from animals tested in training context. C, On the first day of the experimental procedures, pAAV-CaMKIIa-hM4D(Gi)-mCherry virus (hM4D) or pAAV-CaMKIIa-EGFP (EGFP) was bilaterally infused into the ACC. All behavioral tests were completed 7 weeks after viral infusions. For the recent test, mice were tested 1 d after training, (D) whereas mice tested at the remote time were tested 28 d after training. All mice were given an intraperitoneal injection of CNO 30 min before testing. E, Analysis of transgene expression in all hM4D infusions into the ACC for mice tested with systemic injection of CNO. No expression was observed outside of the ACC for systemic inactivation. Dark red represents minimum spread observed and included in analysis. Red represents typical spread observed. Light red represents maximum spread observed and included in behavioral analysis. F, Representative image of pAAV-CaMKIIa-hM4D(Gi)-mCherry expression in the ACC. Expression of mCherry was observed throughout the ACC and was typical of a membrane bound fluorophore. White arrows indicate fiber tracts exiting the ACC toward the corpus callosum. G, hM4D mice administered CNO froze significantly less than EGFP control mice in the novel context only during the remote test, suggesting that inactivation of the ACC eliminates generalized fear at a remote time point. Percent freezing levels of EGFP (○) and hM4D (●) mice during recent (left) and remote (right) tests in the training or neutral context were analyzed (± SEM). Two-way ANOVA identified a significant main effect of context at the recent time point (F_(1,16) = 64.2, p < 0.001) and at the remote time point (F_(1,17) = 52.3, p < 0.001); mice froze more in the training context than the novel context. However, there was a significant context × treatment interaction only at the remote time point (F_(1,17) = 4.64, p < 0.05). **p < 0.01. ***p < 0.001. H, Representative image of pAAV-CaMKIIa-hM4D(Gi)-mCherry expression in the BLA in a mouse that had virus infused into the ACC. Robust expression of mCherry was observed in the external capsule fibers entering the BLA.

Figure 2.

Inactivation of ACC CaMKIIα projections in the BLA eliminates time-dependent generalized fear. A, To identify whether the ACC regulates fear generalization via CaMKIIα projections to the BLA, pAAV-CaMKIIa-hM4D(Gi)-mCherry virus (hM4D) or pAAV-CaMKIIa-EGFP (EGFP) was bilaterally infused into the ACC followed by cannulations targeting their axon terminals in the BLA. B, All behavioral tests were completed 7 weeks after viral infusions. Cannulations for the BLA were completed 1 week before behavioral training procedures. Mice were tested 1 d or (C) 28 d after training. All mice were given a local infusion of CNO into the BLA 5 min before testing to inactivate ACC CaMKIIα projections. D, Analysis of transgene expression in all hM4D mice tested with inactivation of BLA terminals. One mouse was excluded from analysis due to significant hM4D expression in the motor cortex. Dark red represents minimum spread observed and included in analysis. Red represents typical spread observed. Light red represents maximum spread observed and included in behavioral analysis. E, Cannulation targets within the BLA. Black dots indicate animals included in behavioral analyses. Red Xs indicate missed targets and used in a site-specific control analysis. F, hM4D mice with inactivated CaMKIIα projections from the ACC to the BLA froze significantly less than EGFP mice in the novel context, but not in the training context only at the remote test. Percent freezing levels of EGFP (○) and hM4D (●) mice during recent (left) and remote (right) tests in the training or neutral context 5 min after a microinfusion of CNO were analyzed (± SEM). A two-way ANOVA identified a significant effect of context at the recent test (F_(1,27) = 47.1, p < 0.001) and remote test (F_(1,35) = 15.6, p < 0.001). As observed previously, there was a significant interaction only at the remote test (F_(1,35) = 6.71, p < 0.05). Thus, inactivation of ACC CaMKIIα projections to the BLA eliminated time-dependent generalized fear. G, hM4D mice with extra-BLA infusions did not show a reduction in freezing in the novel context. Percent freezing levels of hM4D mice tested in the neutral context with missed BLA targeting compared with hM4D mice with specific targeting in the BLA was analyzed (± SEM). A nonparametric Mann–Whitney t test showed a significant effect of CNO infusion target (p < 0.05). **p < 0.01.

Figure 3.

Inactivation of ACC to BLA CaMKIIα projections eliminates time-independent generalized fear. A, Hybrid B6S1 mice were tested for contextual fear after training with either 3, 1 mA shocks or 5, 1 mA shocks. Percent freezing levels of 3 shock (○) and 5 shock (●) trained mice in the training context were analyzed (± SEM). A two-way ANOVA identified significant shock × context interaction (F_(1,19) = 5.42, p < 0.05), showing that 5-shock training, but not 3-shock training, significantly increased freezing in the novel context at the 24 h test. B, All behavioral tests were completed 7 weeks after viral infusions. Cannulations for the BLA were completed 1 week before behavioral training procedures. In this experiment, rapid generalization was induced using a hybrid mouse line. Mice were tested once in each context at 1 d or (C) 28 d after training with a 72 h intertest interval. All mice were given a local infusion of CNO into the BLA 5 min before testing to inactivate ACC CaMKIIα projections. D, As done previously, mice were infused with the hM4D or EGFP virus into the ACC with cannulations targeting the BLA. Viral spread analysis of all hM4D mice tested using a within-subject design with inactivation of BLA terminals identified no expression outside of the ACC. Dark red represents minimum spread observed and included in analysis. Red represents typical spread observed. Light red represents maximum spread observed and included in behavioral analysis. E, Cannulation targets were analyzed to correct placement into the BLA. No mice had targets localized outside of the BLA in this experiment. F, At recent and remote tests, inactivating CaMKIIα projections from the ACC to the BLA significantly reduced freezing to the novel context. Percent freezing levels of EGFP (○) and hM4D (●) mice during within-subject recent (left) or remote (right) tests in the training and neutral context 5 min after a microinfusion of CNO were analyzed (± SEM). A two-way ANOVA identified significant main effects of context at the recent (F_(1,10) = 64.8, p < 0.001) and remote tests (F_(1,13) = 17.9, p < 0.001). However, for the first time, there was a significant interaction at the recent (F_(1,10) = 5.35, p < 0.05) and remote times (F_(1,13) = 4.93, p < 0.05), suggesting that ACC CaMKIIα projections to the BLA control a time-independent form of generalization. *p < 0.01, ***p < 0.001.

Figure 4.

Inactivation of the vHPC eliminates time-dependent context fear generalization. A, On the first day of the experimental procedures, pAAV-CaMKIIa-hM4D(Gi)-mCherry virus (hM4D) or pAAV-CaMKIIa-EGFP (EGFP) was bilaterally infused into the vHPC. All behavioral tests were completed 7 weeks after viral infusions. For the recent test, mice were tested 1 d after training, (B) whereas mice tested at the remote time were tested 28 d after training. All mice were given an intraperitoneal injection of CNO 30 min before testing. C, hM4D mice administered CNO froze significantly less than EGFP control mice in the novel context only. Percent freezing levels of EGFP (○) and hM4D (●) mice during recent (left panel) and remote (right panel) tests in the training or neutral context were analyzed (± SEM). Two-way ANOVA identified a significant main effect of context at the recent time point, F_(1,21) = 70, p < 0.001, and at the remote time point F_(1,16) = 40.9, p < 0.001; mice froze more in the training context than the novel context. However, there was a significant context × treatment interaction only at the remote time point F_(1,16) = 15.9, p < 0.01. ***p < 0.001, suggesting that the vHPC also regulates time-dependent generalized fear. D, Analysis of transgene expression in hM4D infusions into the vHPC for mice tested with systemic injection of CNO. No expression was observed outside of the vHPC. Dark red: minimum spread observed and included in analysis; red: represents typical spread observed; light red: maximum spread observed and included in behavioral analysis. E, Representative photomicrograph of pAAV-CaMKIIa-hM4D(Gi)-mCherry expression in the vHPC. Robust transgene expression was observed throughout the vHPC and typical of a membrane-bound fluorophore. Inset, 20× magnification. White arrows indicate examples of somatic transgene expression.

Figure 5.

CaMKIIα projections from the vHPC to the BLA regulate time-dependent generalization. A, To identify whether the vHPC regulates fear generalization via its CaMKIIα projections to the BLA, pAAV-CaMKIIa-hM4D(Gi)-mCherry virus (hM4D) or pAAV-CaMKIIa-EGFP (EGFP) was bilaterally infused into the vHPC followed by cannulations targeting the BLA. B, All behavioral tests were completed 7 weeks after viral infusions. Cannulations for the BLA were completed 1 week before behavioral training procedures. Mice were tested 1 d or (C) 28 d after training. All mice were given a local infusion of CNO into the BLA 5 min before testing. D, Viral spread analysis of all hM4D mice tested with inactivation of BLA terminals. Dark red represents typical minimum spread observed and included in analysis. Red represents spread observed. Light red represents maximum spread observed and included in behavioral analysis. E, Cannulation targets within the BLA. Black dots indicate animals included in behavioral analyses. Red Xs indicate missed targets and used in a site-specific control analysis. F, hM4D mice with inactivated CaMKIIα projections from the vHPC to the BLA froze significantly less than EGFP mice in the novel context, but not in the training context. Percent freezing levels of EGFP (○) and hM4D (●) mice during recent (left) and remote (right) tests in the training or neutral context 5 min after a microinfusion of CNO were analyzed (± SEM). A two-way ANOVA identified a significant effect of context at the recent test (F_(1,20) = 68.6, p < 0.001) and remote test (F_(1,24) = 13.3 p < 0.01). As observed previously, there was a significant interaction only at the remote test (F_(1,24) = 4.34, p < 0.05). G, hM4D mice with off-target infusions did not show a reduction in freezing in the novel context. Percent freezing levels of hM4D mice tested in the neutral context with missed BLA targeting compared with hM4D mice with specific targeting in the BLA was analyzed (± SEM). A nonparametric Mann–Whitney t test showed a significant effect of CNO infusion target (p < 0.05). *p < 0.05.

Figure 6.

The vHPC coordinates time-dependent generalization. A, As done previously, mice were infused with hM4D or EGFP virus into the vHPC with cannulations targeting the BLA. Viral spread analysis of all hM4D mice tested using a within-subject design with inactivation of BLA terminals identified no expression outside of the vHPC. Dark red represents minimum spread observed and included in analysis. Red represents typical spread observed. Light red represents maximum spread observed and included in behavioral analysis. B, Cannulation targets were again analyzed to correct placement into the BLA. There were no missed targets outside of the BLA in this experiment. C, All behavioral tests were completed 7 weeks after viral infusions. Cannulations for the BLA were completed 1 week before behavioral training procedures. In this experiment, rapid generalization was induced using a hybrid mouse line. Mice were tested once in each context at 1 d or (D) 28 d after training with a 72 h intertest interval. All mice were given a microinfusion of CNO into the BLA 5 min before testing. E, Inactivating CaMKIIα projections from the vHPC to the BLA significantly reduced freezing to the novel context only at the remote test. These data suggest that glutamatergic projections from the vHPC to the BLA selectively control time-dependent generalized fear. Percent freezing levels of EGFP (○) and hM4D (●) mice during within-subject recent (left) or remote (right) tests in the training and neutral context 5 min after a local infusion of CNO were analyzed (± SEM). A two-way ANOVA identified significant main effects of context at the recent (F_(1,13) = 19, p < 0.001) and remote tests (F_(1,9) = 80.9, p < 0.001). After induced generalization, there was a significant interaction only at the remote test (F_(1,9) = 14.6, p < 0.01). ***p < 0.001.