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 47Anxiety disorders are characterized by a common symptom that promotes 48 overgeneralization of fear in non-threatening environments. Dysregulation of the 49 amygdala, anterior cingulate cortex (ACC), or hippocampus has been hypothesized to 50 contribute to increased fear associated with anxiety disorders. Our findings show that 51 the ACC and HPC projections to the basolateral amygdala regulate generalized fear in 52 non-threatening, environments. However, descending ACC projections control fear 53 generalization independent of time, whereas HPC projections play a strictly time-54 dependent role in regulating generalized fear. Thus, dysfunctional ACC/HPC signaling 55 to the BLA may be a predominant underlying mechanism of non-specific fear associated 56 with anxiety disorders. Our data have important implications for predictions made by 57 theories about aging memories and interactions between the hippocampus and cortical 58 regions. 59 Surgical Procedures 126Mice were anesthetized with a subcutaneous injection of a Ketamine (75 mg/kg) 127 + Xylazine (10 mg/kg) + Acepromazine (2 mg/kg) cocktail. Following administration of 128 anesthesia, mice were mounted on a stereotaxic apparatus (David Kopf Instruments, 129 Tujunga, CA). The scalp of each mouse was retracted; the skull was adjusted so that 130 bregma and lambda were on the same horizontal plane (within .05mm of each other). 131Two 0.33 gauge infusion needles were guided to the appropriate coordinates relative to 132 bregma and small bilateral burr holes were drilled. Coordinates for the following brain 133 regions were ACC: .08 mm AP, ±.07 mm ML, -3.6 mm DV from bregma at a 14° angle; 134 vHPC: -3.2 mm AP, ±3.3 mm ML, -4.25 mm DV from bregma. AAV8-CaMKIIa-135 hM4D(Gi)-mCherry virus (hM4D) (Addgene) or a control virus under the same promoter, 136 AAV8-CamKIIa-EGFP (EGFP) (Addgene) was bilaterally infused at 0.1µL/minute to a