Stress and sex-dependent effects on conditioned inhibition of fear.

Anxiety and stress-related disorders are highly prevalent and are characterized by excessive fear to threatening and nonthreatening stimuli. Moreover, there is a large sex bias in vulnerability to anxiety and stress-related disorders-women make up a disproportionately larger number of affected individuals compared with men. Growing evidence suggests that an impaired ability to suppress fear in the presence of safety signals may in part contribute to the development and maintenance of many anxiety and stress-related disorders. However, the sex-dependent impact of stress on conditioned inhibition of fear remains unclear. The present study investigated sex differences in the acquisition and recall of conditioned inhibition in male and female mice with a focus on understanding how stress impacts fear suppression. In these experiments, the training context served as the "fear" cue and an explicit tone served as the "safety" cue. Here, we found a possible sex difference in the training requirements for safety learning, although this effect was not consistent across experiments. Reductions in freezing to the safety cue in female mice were also not due to alternative fear behavior expression such as darting. Next, using footshock as a stressor, we found that males were impaired in conditioned inhibition of freezing when the stress was experienced before, but not after, conditioned inhibition training. Females were unaffected by footshock stress when it was administered at either time. Extended conditioned inhibition training in males eliminated the deficit produced by footshock stress. Finally, exposing male and female mice to swim stress impaired safety learning in male mice only. Thus, we found sex × stress interactions in the learning of conditioned inhibition and sex-dependent effects of stress modality. The present study adds to the growing literature on sex differences in safety learning, which will be critical for developing sex-specific therapies for a variety of fear-related disorders that involve excessive fear and/or impaired fear inhibition.

Presynaptic GABAB receptor inhibition sex dependently enhances fear extinction and attenuates fear renewal.

Anxiety and trauma-related disorders are highly prevalent worldwide, and are associated with altered associative fear learning. Despite the effectiveness of exposure therapy, which aims to reduce associative fear responses, relapse rates remain high. This is due, in part, to the context specificity of exposure therapy, which is a form of extinction. Many studies show that fear relapses when mice are tested outside the extinction context, and this is known as fear renewal. Using Pavlovian fear conditioning and extinction, we can study the mechanisms underlying extinction and renewal. The aim of the current experiment was to identify the role of presynaptic GABAB receptors in these two processes. Previous work from our lab showed that genetic deletion or pharmacological inhibition of GABAB(1a) receptors that provide presynaptic inhibition on glutamatergic terminals reduces context specificity and leads to generalization. We therefore hypothesized that inactivation of these presynaptic GABAB receptors could be used to reduce the context specificity associated with fear extinction training and suppress renewal when mice are tested outside of the extinction context. Using CGP 36216, an antagonist specific for presynaptic GABAB receptors, we blocked presynaptic GABAB receptors using intracerebroventricular injections during various time points of extinction learning in male and female mice. Results showed that blocking these receptors pre- and post-extinction training led to enhanced extinction learning in male mice only. We also found that post-extinction infusions of CGP reduced renewal rates in male mice when they were tested outside of the extinction context. In an attempt to localize the function of presynaptic GABAB receptors within regions of the extinction circuit, we infused CGP locally within the basolateral amygdala or dorsal hippocampus. We failed to reduce renewal when CGP was infused directly within these regions, suggesting that presynaptic inhibition within these regions per se may not be necessary for driving context specificity during extinction learning. Together, these results show an important sex-dependent role of presynaptic GABAB receptors in extinction and renewal processes and identify a novel receptor target that may be used to design pharmacotherapies to enhance the effectiveness of exposure therapy.

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

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.

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

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.

Basolateral amygdala Thy1-expressing neurons facilitate the inhibition of contextual fear during consolidation, reconsolidation, and extinction.

Disrupted fear inhibition is a characteristic of many anxiety disorders. Investigations into the neural mechanisms responsible for inhibiting fear will improve understanding of the essential circuits involved, and facilitate development of treatments that promote their activity. Within the basolateral amygdala (BLA), Thy1-expressing neuron activity has been characterized by us and others as promoting fear inhibition to discrete fear cues by influencing consolidation of cued fear learning or cued fear extinction. Here, we evaluated how activating BLA Thy1-expressing neurons using DREADDs affected the consolidation, expression, reconsolidation, and extinction of contextual fear. Using an inhibitory avoidance paradigm, our present findings indicate a similar involvement of BLA Thy1-expressing neuron activity in the consolidation and extinction, but not expression, of fear. Importantly, our data also provide the first evidence for involvement of these neurons in inhibiting fear reconsolidation. Therefore, these data enhance our understanding of the roles that Thy1-expressing neurons within the BLA play in inhibiting fear when examining avoidance, in addition to the already established role in Pavlovian fear paradigms. Future investigations should further explore the circuits responsible for these contextual effects modulated by BLA Thy1 neuron activation, and could promulgate development of therapies targeting these neurons and their downstream effectors.

Hippocampal GABAB(1a) Receptors Constrain Generalized Contextual Fear.

Many anxiety disorders are characterized by generalization of fear responses to neutral or ambiguous stimuli. Therefore, a comprehensive understanding of the mechanisms contributing to generalized fear is essential for formulating successful treatments for anxiety disorders. Previous research shows that GABA-mediated presynaptic inhibition has a critical role in cued fear generalization, as animals with genetically deleted presynaptic GABAB(1a) receptors cannot discriminate between CS+ and CS- tones. Work from our laboratory has further identified that GABAB(1a) receptors are necessary for maintaining contextual memory precision, thereby constraining generalized contextual fear. We previously found that GABAB(1a) KO mice show generalized fear to a neutral context 24 h after training, but not 2 h after training. A similar pattern was observed with object location and recognition, suggesting that this receptor subtype affects consolidation and/or retrieval of precise contextual and spatial memories. Here we sought to specifically examine the involvement of GABAB(1a) receptors in consolidation or retrieval of a precise fear memory. To do so, we infused a selective GABAB(1a) receptor antagonist, CGP 36216, intracerebroventricularly (ICV), or locally into the dorsal hippocampus, ventral hippocampus, or anterior cingulate cortex (ACC), during consolidation and retrieval of context fear training. Blockade of GABAB(1a) receptors through ICV, dorsal hippocampal, or ventral hippocampal infusions 'after' training (consolidation) resulted in fear generalization to the neutral context when mice were tested 24, but not 6 h after training. Post-training infusions of CGP into the ACC, however, did not promote generalized fear. In addition, ICV, dorsal hippocampal, ventral hippocampal, or ACC infusions immediately 'before' testing (retrieval) did not result in context fear generalization. These data suggest that GABA-mediated presynaptic inhibition is not critical for retrieval of precise contextual memory, but rather has an important role in the long-term consolidation of precise contextual memories and constrains generalized fear responses.