Predator exposure is a life-threatening experience and elicits innate fear behaviors as well as learned fear responses to the context in which the predator was encountered (Blanchard et al., 1989; Blanchard et al., 2001; Ribeiro-Barbosa et al., 2005). Recent studies revealed a cortical network that is responsive to predatory threats and exerts a critical role in processing fear memory. Thus, the caudal prelimbic area (PL), rostral part of the anterior cingulate area (ACA), medial visual area (VISm), and the ventral part of retrosplenial area (RSPv) form a highly interconnected circuit that presents a differential increase in Fos expression in response to predator exposure. Cytotoxic lesions of the elements of this cortical circuit apparently had no impact on innate fear responses during predator exposure but had a profound impact on contextual fear memory largely disrupting learned contextual fear responses in a predator-related environment (de Andrade Rufino et al., 2019).

The ACA occupies a central position in this cortical network and establishes dense bidirectional connections with all other elements of the circuit. Importantly, lesions in the ACA had a larger impact on decreasing learned contextual fear responses versus lesions of the other elements of this cortical network (de Andrade Rufino et al., 2019). However, at this point, it is not clear how the ACA is involved in processing predator-related fear memory.

Previous studies in the literature using fear conditioning to physically aversive stimuli (i.e., footshock) reported important roles for the ACA in fear memory. The ACA seems to be necessary for the acquisition of contextual fear. Pretraining inactivation of the ACA blocked fear acquisition (Tang et al., 2005; Bissière et al., 2008), and pretraining activation of the ACA using a mGluR agonist (Bissière et al., 2008) enhanced fear learning thus suggesting an involvement of the ACA in the acquisition of fear responses.

Of relevance, the insertion of an empty temporal gap, or trace, between the CS and UCS makes learning the association critically dependent on the prefrontal cortex. Recording studies in trace fear conditioning revealed units called “bridging cells” in the prefrontal cortex that maintain firing during the trace intervals, perhaps reflecting the maintenance of attentional resources during the CS-USC interval (Gilmartin et al., 2014). These findings from trace fear conditioning suggest that the prefrontal cortex strengthens the predictive value of the available cue to influence memory storage. The ACA is also required for memory consolidation. Infusion of the protein-synthesis inhibitor anisomycin in the ACA impairs memory consolidation of recent and remote memory (Einarsson and Nader, 2012), and virally mediated disruption of learning-induced dendritic spine growth in the ACA impairs memory consolidation (Vetere et al., 2011). In fact, the ACA has been largely associated with the encoding and retrieval of contextual fear memory at remote time points (Frankland et al., 2004; Kitamura et al., 2017; Abate et al., 2018).

Here, we examined how that ACA mediates predator fear memory. To this end, we exposed mice to cats and investigated innate and contextual fear responses. We started by asking whether the ACA is involved in the acquisition and/or expression of predator fear memory and applied pharmacogenetic silencing in the ACA during exposure to the predator or to the context. Next, combining retrograde tracing and Fos protein immunostaining, we examined the pattern of activation of ACA source of inputs during acquisition and expression of predator fear responses. We could see how the ACA would be able to combine predator and contextual cues particularly during the acquisition phase. To complement these findings, during the cat exposure, we applied optogenetic inhibition to the anteromedial thalamic nucleus>ACA path, which putatively relays information related to the predator cues, and examined the effect on the acquisition of predator fear memory.

Next, using optogenetic silencing and functional tracing combining Fluoro Gold and Fos immunostaining, we examined how the ACA entrains selected targets to influence acquisition or expression of predator fear memory. The data help to clarify how the ACA influences both the acquisition and expression of predator fear memory. Overall, the ACA offers predictive relationships between the threatening stimuli and the context to influence memory storage in amygdalar and hippocampal circuits. It also has a role in memory retrieval and the expression of contextual fear. Our results open interesting perspectives for understanding how the ACA is involved in processing contextual fear memory to predator threats as well as other ethologic threatening conditions such as those seen in the confrontation with a conspecific aggressor during social disputes.

Combining the use of pharmaco- and optogenetic circuit manipulation tools and functional tracing, we untangled the ACA role in processing contextual fear memory to a predator threat.

To test the acquisition and expression of predator contextual fear responses, we used a two-phase paradigm composed of cat exposure (acquisition phase), and, on the following day, exposure to the context where the predator had been previously encountered (expression phase) (see Appendix – Behavioral Protocol). Notably, the defensive responses in mice differ somewhat from those seen in rats exposed to cats and then to the predator-associated context. During cat exposure, rats spend close to 90% of the time freezing and 2% of the time risk assessing the environment Ribeiro-Barbosa et al., 2005; mice froze for approximately 30% of the time and risk assessed the environment for close to 30% of the time (see Appendix – Behavioral Protocol). During exposure to the predatory context, rats only froze a little and then spent time risk assessing the predator-related context for 70% of the time (Ribeiro-Barbosa et al., 2005), which is different from mice that presented no freezing and a relatively weaker risk assessment response—close to 50% of the time (see Appendix 1—table 1).

To uncover whether the ACA response is specific to threat and threat paired context, we examined the ACA activity in four different conditions: exposure to the cat, the cat-related context, the novel non-threat stimulus (plush cat), and the context paired with non-threat stimulus (see Appendix – Comparison of ACA activity). This analysis revealed that ACA Fos expression in response to the cat and cat-related context was significantly higher relative to its expression level in response to exposure to a novel non-threat stimulus and to the context paired with a non-threat stimulus (see Appendix 1—figure 3).

Pharmacogenetic inhibition of the ACA during cat exposure did not change innate responses but significantly reduced contextual fear responses. This suggests that the ACA is involved in the acquisition of predator-related contextual fear responses. In line with these results, studies using fear conditioning to physically aversive stimuli (i.e., foot shock) have also supported the idea that the ACA appears to be necessary for the acquisition of contextual fear (Tang et al., 2005; Bissière et al., 2008).

Our functional tracing analysis combining retrograde tracer in the ACA and Fos immunostaining revealed that the ACA is particularly influenced by afferent sources of inputs conveying contextual information and predator cues during the acquisition phase. Among the cortical inputs, the ventral retrosplenial (RSPv) and medial visual (VISm) areas presented the largest percentage of FG retrogradely labeled cells expressing Fos (around 50% of the retrogradely labeled cells). The retrosplenial cortex is critical for the recognition of an environment directly paired with an aversive event (Robinson et al., 2018). Recent studies using two-photon imaging revealed that landmark cues served as dominant reference points and anchored in the retrosplenial cortex the spatial code, which is the result of local integration of visual, motor, and spatial information (Fischer et al., 2020). The VISm is necessary for the visuospatial discrimination used in the integration of allocentric visuospatial cues (Sánchez et al., 1997; Espinoza et al., 1999) and may as well be involved in integrating contextual cues. Therefore, the results suggest that cortical fields projecting to the ACA particularly active during the acquisition phase of predator fear memory are involved in computing contextual landmarks.

In the thalamus, our functional tracing revealed that the CL and AMv nuclei contained more than 50% of the FG retrogradely labeled cells expressing Fos during the acquisition phase of predator fear memory. Both the CL and AMv are likely to convey information regarding the predatory threat. The CL receives projections from the dorsal part of the periaqueductal gray (Kincheski et al., 2012), and the AMv receives substantial inputs from the dorsal premammillary nucleus (Canteras and Swanson, 1992). The dorsal premammillary nucleus is part of the predator-responsive circuit of the medial hypothalamus (Gross and Canteras, 2012) and is the most responsive site to a live predator or its odor (Cezario et al., 2008; Dielenberg et al., 2001).

In order to test whether the information relayed through the projection from the anteromedial thalamic nucleus (AM) to the ACA influences the acquisition of predator fear memory, we silenced the AM>ACA pathway during cat exposure. Photoinhibition of the AM>ACA projection during cat exposure did not change innate fear responses but significantly reduced contextual fear responses. This suggests that silencing the AM>ACA projection impairs the acquisition of contextual fear response to a predator threat. In line with this result, studies from our group have shown that pharmacological inactivation of the AM drastically reduced the acquisition but not the expression of contextual fear responses to predator threats (de Lima et al., 2017). Therefore, the AM>ACA pathway does not seem involved in the expression of contextual fear. It is noteworthy that anterior thalamic lesions slow down the acquisition of contextual fear conditioning but do not affect contextual fear memory tested in the short term (Dupire et al., 2013; Marchand et al., 2014).

Our findings collectively support the idea that the ACA integrates contextual and predator cues during the acquisition phase to provide predictive relationships between the context and the threatening stimuli and influence memory storage. The ventral hippocampus and BLA appear as critical sites for memory storage of contextual fear to predator threats. Ventral hippocampal cytotoxic lesions significantly reduced conditioned defensive behaviors during re-exposure to the predator-associated context (Pentkowski et al., 2006). Likewise, cytotoxic NMDA lesions in the BLA impaired contextual fear responses to predator threat (Martinez et al., 2011; Bindi et al., 2018). In line with these ideas, recent findings from our lab indicate that cycloheximide (a protein synthesis inhibitor) injection in the BLA or the ventral hippocampus impairs contextual fear responses to predatory threat (F. Reis and N.S. Canteras personal observations). Therefore, we investigated how the ACA would facilitate associative plasticity and memory storage in the BLA and hippocampus. To this end, we used optogenetic silencing and functional tracing combining Fluoro Gold and Fos immunostaining to examine how the ACA entrains selective targets to influence acquisition or expression of predator fear memory.

Among the ACA targets, we start by exploring projections to the BLA and the perirhinal region (PERI). Previous model of the prefrontal regulation of memory formation suggests that the prefrontal cortex is likely to influence memory storage in the amygdala and hippocampus using two branches (Gilmartin et al., 2014). One direct branch is to the BLA and conveys information about the predictive value of the relevant clues during learning. The other branch is to the perirhinal cortices, which occupies a strategic position in this network influencing both the hippocampus and the amygdala. Tract-tracing studies revealed that the PERI provides dense projections to the BLA and the hippocampal formation (Shi and Cassell, 1999). Photoinhibition of both ACA>BLA and ACA>PERI pathways during cat exposure significantly reduced contextual fear responses thus suggesting a role in the acquisition of predator fear memory. In contrast, photoinhibition of both pathways did not influence the expression of contextual fear memory. Corroborating these findings, our functional tracing revealed that both ACA>BLA and ACA>PERI paths are significantly more active during the acquisition compared to the expression of predator contextual fear memory. Triple retrograde tracing experiments revealed that the ACA cells projecting to the BLA and the PERI were mostly found in the supragranular layers (layers II and III), and close to 50% of these cells provided a branched projection to the BLA and PERI (see in the Appendix, Triple retrograde tracing and Appendix 1—figure 9).

The POST in turn is necessary for normal acquisition of contextual and auditory fear conditioning (Robinson and Bucci, 2012). Thus, we tested the ACA>POST pathway as a putative candidate to influence predator fear memory. However, the ACA>POST pathway’s photoinhibition did not impair either the acquisition or the expression of contextual fear memory. Notably, we showed that the position of the optic fibers was consistent across these experiments and suitable to silence the ACA>POST pathway. In favor of the lack of effect of the ACA>POST pathway, our functional tracing examined the percentage of Fos positive cells in the ACA projecting to the POST and revealed a relatively low percentage of double-labeled cells both in the acquisition and the expression of predatory contextual fear memory. Moreover, among the ACA paths that received optogenetic silencing in the present investigation, we noted that the ACA>POST projection presented the smallest density of terminals (see in the Appendix, Quantification of ACA terminal fields and Figure 8).

Pharmacogenetic inhibition of the ACA during the exposure to the predatory context significantly reduced contextual fear responses. This result further suggests that the ACA is also involved in the expression of predator-related contextual fear responses. Exposure to the predatory context yielded a different activation pattern among the sources of inputs to the ACA compared to those seen during cat exposure. Unfortunately, our functional tracing does not offer any plausible hint regarding the putative source of inputs to the ACA driving retrieval of memory traces to influence contextual fear expression. In this regard, we note that the ACA is involved in the consolidation of contextual fear conditioning to physically aversive stimuli (Einarsson and Nader, 2012). Future work could investigate whether the ACA is also involved in the consolidation of contextual fear memory to predator threat and how this consolidation would influence the retrieval predator fear memory in the ACA.

The dorsolateral periaqueductal gray (PAGdl) appears to be a likely ACA target to influence the expression of contextual fear responses to predator threats. Exposure to the predatory context yields a significant Fos expression in the dorsal PAG (Cezario et al., 2008)—a region where electrical, pharmacological, and optogenetic stimulation have been shown to produce freezing, flight, and risk assessment behavior in the absence of a predatory threat (Bittencourt et al., 2004; Assareh et al., 2016; Deng et al., 2016). The present findings showed that photoinhibition of the ACA>PAGdl pathway during cat exposure did not change innate or contextual fear responses. Conversely, photoinhibition of the ACA>PAGdl projection during predatory context altered contextual fear responses. In line with these findings, the functional tracing revealed that the ACA contained a significantly larger proportion of double-labeled Fos/FG cells for the PAGdl FG injected animals in response to the context versus the cat exposure. Collectively, our findings revealed that the ACA>PAGdl pathway is significantly more active during exposure to the predatory context and influences the expression of contextual fear to predator threat. Triple retrograde tracing experiments revealed that ACA cells projecting to the PAGdl are in the infragranular layers (layers V and VI) and do not overlap with the cells projecting to the BLA or PERI (see in the Appendix, Triple retrograde tracing and Appendix 1—figure 9).

Finally, it is noteworthy that all ACA paths involved in the acquisition or expression of contextual fear responses were differentially activated between acquisition and expression of fear memory and are unlikely to provide a condition to produce a state-dependent effect on contextual fear memory. However, regardless of endogenous activity patterns, there is a hypothetical possibility of a disruptive effect if memory is encoded in the absence of a given pathway and tested in its presence. In this case, it is important to bear in mind that as much as the optogenetic silencing of a given pathway is effective, it is unlikely to provide a complete inactivation of the entire pathway.

Overall, the ACA can provide predictive relationships between the context and the predator threat and influences fear memory acquisition through projections to the BLA and PERI and the expression of contextual fear through projections to the PAGdl. These findings may be applied in more general terms to understand memory processing of fear threats entraining hypothalamic circuits (i.e., such as the predator- and conspecific-responsive circuits underlying predatory and social threats, respectively) that engage the AMv>ACA pathway (Gross and Canteras, 2019). Notably, both the predator- and conspecific-responsive hypothalamic circuits comprise the dorsal premammillary nucleus that projects densely to the AMv nucleus (Canteras and Swanson, 1992), and therefore, engage the AMv>ACA pathway. Of relevance, previous studies showed that the anteromedial thalamus' cytotoxic lesions impair contextual fear in a social defeat-associated context (Rangel et al., 2018). Thus, the present results open interesting perspectives for understanding how the ACA is involved in processing contextual fear memory to predator threats as well as other ethologic threatening conditions such as those seen in confrontation with a conspecific aggressor during social disputes.

We observed that male and female mice present similar defensive responses during exposure to the predator or predatory context (Appendix 1—table 1). Here, it is important to note that regardless of similar innate and contextual fear responses seen in males and females, the underlying circuits are not necessarily the same. In fact, in the literate, there is a lack of investigation on the neural circuits mediating anti-predatory responses in female rodents, and this is certainly a limitation of the present investigation. A recent study carried out in male and female mice examining the role of the dorsal premammillary nucleus—a key element of the hypothalamic predator-responsive circuit—on coordination of anti-predatory responses did not report differences between sexes (Wang et al., 2021). However, sex differences are expected in terms of the responsivity of elements of defensive circuits, especially considering that critical sites in these circuits, such as the hypothalamus, hippocampus, and amygdala, are responsive to gonadal steroid hormones (Simerly et al., 1990). At this point, further studies are needed to investigate sex differences in the circuits mediating contextual fear memory to predatory threats.

All data generated and analysed in this study are included in the manuscript and supporting files.

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Author details

1. Miguel Antonio Xavier de Lima

   Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

2. Marcus Vinicius C Baldo

   Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

   Contribution

   Formal analysis, Statiscal analysis, Supervision

   Competing interests

   No competing interests declared

3. Fernando A Oliveira

   Cellular and Molecular Neurobiology Laboratory (LaNeC) - Center for Mathematics, Computing and Cognition (CMCC), Federal University of ABC, São Bernardo do Campo, Brazil

   Contribution

   Data curation, Formal analysis, Methodology, performed and analyzed the patch clamp studies

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1632-4267

4. Newton Sabino Canteras

   Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   newton@icb.usp.br

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7205-5372

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