Anterior cingulate cortex and dorsal hippocampal glutamate receptors mediate generalized fear in female rats

Abstract

Exhibiting fear to non-threatening cues or contexts-generalized fear-is a shared characteristic of several anxiety disorders, which afflict women more than men. Female rats generalize contextual fear at a faster rate than males and this is due, in part, to actions of estradiol in the dorsal CA1 hippocampus (dCA1). To understand the mechanisms underlying estradiol's effects on generalization, we infused estradiol into the anterior cingulate cortex (ACC) or ventral CA1 hippocampus (vCA1) of ovariectomized (OVX) female rats. Estradiol acts within the ACC, but not the vCA1, to promote generalized fear. We next examined if AMPA or NMDA receptor antagonists (NBQX, APV) infused into the dCA1 or the ACC of female rats could block generalized fear induced by systemic injections of estradiol. Immediate pre-testing infusions of NBQX or APV into either region eliminated estradiol-induced generalization. Specific blockade of GluN2B receptors with infusions of Ro 25-6981 into the dCA1 or ACC also eliminated generalized fear. Our results suggest that in addition to the dCA1, the ACC is an important locus for the effects of estradiol on fear generalization. Moreover, within these regions, AMPA and NMDA-GluN2B receptors are necessary for estradiol-induced generalization of fear responses, suggesting a critical involvement of glutamatergic transmission. Furthermore, we identified a novel role for GluN2B in mediating the effects of estradiol on generalized fear in female rats. These data potentially implicate GluN2B receptors in more general forms of memory retrieval inaccuracies, and form the foundation for exploration of glutamate receptor pharmacology for treatments of anxiety disorders involving generalization.

Keywords: Anterior cingulate cortex; Estradiol; Females; Generalization; Glutamate; Hippocampus.

Fig. 1.

Estradiol acts within the ACC to promote generalized fear. A. Local infusion site verification for the ACC and vCA1. Black dots represent infusion sites for one or more animals. Drawings are adapted from Swanson, (2018). B. Schematic of the experimental paradigm. All animals were trained in an inhibitory avoidance chamber and 24 h later received local infusion of either estradiol or vehicle. Animals were tested for generalization 24 h after infusions in either the training or the neutral context. C. Estradiol infusions into the ACC induces context generalization. Animals infused with estradiol displayed generalization as they had similar latencies to cross in the training and neutral context whereas vehicle animals had significantly shorter latencies to cross in the neutral than the training, suggesting context discrimination. D. Estradiol infusions into the vCA1 does not induce generalization. Vehicle- and estradiol-treated animals had significantly shorter latencies to cross in the neutral context than the training context, suggesting no generalization. E. Open field locomotor behavior. Estradiol and vehicle treated animals had similar distances traveled and spent equivalent amounts of time in the center and surround of the arena, suggesting estradiol treatment does not influence locomotion. Data are expressed as mean ± SEM *p < 0.05 **p < 0.01.

Fig. 2.

AMPA and NMDA receptors mediate the effects of estradiol on generalized fear. A. Local infusion site verification for the dCA1 and ACC. Black dots represent infusion sites for one or more animals. Drawings are adapted from Swanson, (2018). B. Schematic of the experimental procedure. All animals were trained in an inhibitory avoidance chamber. Twenty-four hours after training animals received peripheral injections of either estradiol or vehicle. Twenty-four hours after injections animals received local infusions of either APV, NBQX, or vehicle and were then tested for generalization in either the training or the neutral context. C. Blocking AMPA or NMDA receptors within the dCA1 eliminates estradiol-induced generalization. Estradiol alone induced context generalization, but when either the NMDA antagonist (APV) or the AMPA antagonist (NBQX) were infused into the dCA1, estradiol-induced generalization was significantly attenuated. D. Estradiol treatment induced context generalization. Blocking either NMDA receptors with APV or AMPA receptors with NBQX in the ACC eliminated estradiol-induced generalization. Data are expressed as mean ± SEM *p < 0.05 **p < 0.01.

Fig. 3.

GluN2B receptors mediate the effects of estradiol on generalized fear. A. Local infusion site verification for the dCA1 and ACC. Black dots represent infusion sites for one or more animals. Drawings are adapted from Swanson, (2018). B. Schematic of the experimental procedure. All animals were trained in an inhibitory avoidance chamber. Twenty-four hours after training animals received peripheral injections of either estradiol or vehicle. Twenty-four hours after injections animals received local infusions of either Ro 25–6981, or vehicle and were then tested for generalization in either the training or the neutral context. C. Estradiol treatment alone induced contextual fear generalization when compared to vehicle-treated animals. Estradiol-induced generalization was blocked when GluN2B receptors were blocked in the dCA1. D. Estradiol treatment induced contextual fear generalization when compared to vehicle-treated animals. Estradiol-induced generalization was reduced when GluN2B receptors were blocked in the ACC. Data are expressed as mean ± SEM *p < 0.05 **p < 0.01.