The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats
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Evaluation Summary:
The manuscript used a naturalistic task where mice were fear conditioned to a context using a live predator (cat) and a variety of behavioural measures including freezing, risk assessment, and exploration. The identification of anterior cingulate cortex and its input and outputs in contextual fear acquisition and expression to predator threat is an important contribution to our understanding of neural mechanism related to fear processing. The paper will be of interest to researchers interested in using naturalistic threats in the lab, and to a more broad audience interested in learning and the related fear circuits.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Predator exposure is a life-threatening experience and elicits learned fear responses to the context in which the predator was encountered. The anterior cingulate area (ACA) occupies a pivotal position in a cortical network responsive to predatory threats, and it exerts a critical role in processing fear memory. The experiments were made in mice and revealed that the ACA is involved in both the acquisition and expression of contextual fear to predatory threat. Overall, the ACA can provide predictive relationships between the context and the predator threat and influences fear memory acquisition through projections to the basolateral amygdala and perirhinal region and the expression of contextual fear through projections to the dorsolateral periaqueductal gray. Our results expand previous studies based on classical fear conditioning and open interesting perspectives for understanding how the ACA is involved in processing contextual fear memory to ethologic threatening conditions that entrain specific medial hypothalamic fear circuits.
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Author Response:
Reviewer #1 (Public Review):
[...] Some weaknesses of the manuscript design include the prolonged continuous optical inhibition, lack of optical control and the possibility of a state-dependent effect on neural manipulation.
The prolonged optical inhibition would conceivably produce tissue damage at the optic fiber tip. Accordingly, controls for the optic stimulation experiments comprised the experimental groups injected with AAV5-hSyn-mCherry—not coding halorhodopsin (HR-; controls)—that were continuously stimulated for 5 min with a yellow laser (589 nm) during either the acquisition or the expression phase. Moreover, histological analysis of the tissue at the optic fiber’s tip after the behavioral procedure showed no tissue damage in the areas surrounding the optic fiber’s tips (see Appendix Figure 11).
The …
Author Response:
Reviewer #1 (Public Review):
[...] Some weaknesses of the manuscript design include the prolonged continuous optical inhibition, lack of optical control and the possibility of a state-dependent effect on neural manipulation.
The prolonged optical inhibition would conceivably produce tissue damage at the optic fiber tip. Accordingly, controls for the optic stimulation experiments comprised the experimental groups injected with AAV5-hSyn-mCherry—not coding halorhodopsin (HR-; controls)—that were continuously stimulated for 5 min with a yellow laser (589 nm) during either the acquisition or the expression phase. Moreover, histological analysis of the tissue at the optic fiber’s tip after the behavioral procedure showed no tissue damage in the areas surrounding the optic fiber’s tips (see Appendix Figure 11).
The possibility of a state-dependent effect on neural manipulation was also discussed. It is important to note that for a given pathway to generate a condition to produce a state-dependent effect on memory, the pathway would be expected to be activated to the same degree during both encoding and retrieval. However, in our case, all ACA paths involved in the acquisition or expression of contextual fear responses showed a differential activation between acquisition and expression of fear memory and are unlikely to provide a condition to produce a state-dependent effect on contextual fear memory (see Discussion).
It is unclear if the similarity in the effects obtained in some cases are due to similarity in the underlying behavioral process that is disrupted (e.g. failing to acquire an accurate representation of the context vs. associating the context with the threat).
Our findings collectively support the idea that the ACA integrates contextual and predator-related cues during the acquisition phase to provide predictive relationships between the context and the threatening stimuli and influence memory storage. As discussed in the manuscript, silencing the AM > ACA pathway during the acquisition phase of contextual fear memory would disrupt ACA integration of the predator-related cues and impair the ability of this cortical field to provide predictive relationships between the context and the predatory threat. Conversely, if the ACA outputs were silenced, this cortical region would nonetheless be able to generate predictive relationships between predatory threats and contextual landmarks but would be unable to influence memory storage sites in the BLA and ventral hippocampus or in brain regions involved in the expression of contextual fear responses, such as the PAGdl.
Reviewer #2 (Public Review):
The authors' main goals were to identify key circuits for the acquisition and expression of contextual fear conditioning in a paradigm that uses a live cat as the unconditioned stimulus. They used neuroanatomical tracing, opto-, and chemo-genetic techniques to observe and manipulate activity in anterior cingulate area (ACA) afferents and efferents at either the acquisition or retrieval stages. The strengths include a thorough characterization of multiple circuits and robust behavioral effects. Weaknesses include the confound of the experimenter being in the room for the acquisition, but not retrieval phase, a lack of characterization of escape-like behaviors, and the exclusive use of male animals, which reduces the potential impact.
In the revised version, we clarified that the experimenter was present in the experimental room during the habituation phase, cat exposure, and the context phase; in all conditions, the experimenter’s position in the experimental room remained consistent (see Methods).
We also clarified that our protocol conditions yielded only occasional escape responses. Thus, only a few escape episodes were noted during cat exposure—mostly when the animals noticed the cat and fled back to Box 1—and during exposure to the predatory context, the animals presented only occasional escape episodes (see Appendix Behavioral Protocol).
Finally, we discussed that the basic circuits organizing these responses should be similar in both genders. However, gender differences are expected in terms of the responsivity of the components within these circuits, and further studies are needed to investigate gender differences in the responsivity of circuits mediating contextual fear memory to predatory threat (see Discussion).
Reviewer #3 (Public Review):
In this study, de Lima et. al. examined the function of ACA in acquisition and expression of contextual fear memory to predator threat. The authors found that ACA is necessary for both processes. Using optogenetic terminal inactivation, the authors further demonstrate a necessary role of AM input to ACA in the contextual fear acquisition phase. At the output level, the projections from ACA to BLA and PERI are necessary for contextual fear acquisition while the projection from ACA to PAG is essential for contextual fear expression. Overall, the study is interesting and the results are straightforward. The presented data largely support the conclusions. The paper will provide new insight into the neural circuit underlying contextual fear learning and expression to predator threat. Some limitations of the study include the lack of controls to demonstrate how specific is the ACA response to threat and threat paired context is and validation of the terminal inhibition. Further characterization of the projections would also help to understand these results.
In the revised manuscript, to demonstrate how specific the ACA response is to threat and a threat-paired context, we performed in the ACA, DAPI and Fos quantification in four conditions: exposure to the cat, the predatory context, the novel non-threat stimulus (plush cat), and the context paired with non-threat stimulus. We were able to show 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 in Appendix, Comparison of ACA activity and Figure 3; and Discussion).
We have also validated the terminal inhibition and performed patch clamp experiments that tested the efficiency of pre-synaptic inhibitions and showed clear inhibition of EPSC of postsynaptic cells after illumination at 585 nm light on halorhodopsin positive fibers, and no rebound excitation after halorhodopsin activation (see Appendix Figure 6).
Finally, to further characterize the ACA projections, first we performed the quantification of ACA terminal fields in the BLA, PERI, PAGdl and POST, revealing the densest ACA projection field in the BLA, followed by the projections to the PERI, PAGdl, and POST, which contained the weakest projections from the ACA in all cases examined (see in Appendix, Quantification of ACA terminal fields and Figure 8).
Next, we performed triple retrograde tracing in the same animal to investigate the distribution of ACA neurons projecting to the BLA, PERI and PAGdl, the ACA projections involved in the acquisition or expression of contextual fear memory (see in Appendix, Triple retrograde tracing and Figure 9). The results revealed a layering pattern of the ACA neurons projecting to BLA, PERI and PAGdl. ACA projecting neurons to PAGdl are located in infragranular layers (layers V and VI) and do not overlap with the neurons projecting to the BLA or the PERI. Conversely, neurons projecting to PERI and BLA are located in the supragranular layers (layers II and III) and present a nearly 50% overlap.
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Evaluation Summary:
The manuscript used a naturalistic task where mice were fear conditioned to a context using a live predator (cat) and a variety of behavioural measures including freezing, risk assessment, and exploration. The identification of anterior cingulate cortex and its input and outputs in contextual fear acquisition and expression to predator threat is an important contribution to our understanding of neural mechanism related to fear processing. The paper will be of interest to researchers interested in using naturalistic threats in the lab, and to a more broad audience interested in learning and the related fear circuits.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to …
Evaluation Summary:
The manuscript used a naturalistic task where mice were fear conditioned to a context using a live predator (cat) and a variety of behavioural measures including freezing, risk assessment, and exploration. The identification of anterior cingulate cortex and its input and outputs in contextual fear acquisition and expression to predator threat is an important contribution to our understanding of neural mechanism related to fear processing. The paper will be of interest to researchers interested in using naturalistic threats in the lab, and to a more broad audience interested in learning and the related fear circuits.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript assessed the role of the anterior cingulate area (ACA) in fear acquisition and expression in a naturalist task using a live cat as the unconditioned stimulus where mice were the experimental subjects. Silencing of the ACA using DREADDs showed that the ACA is important for contextual fear acquisition and expression. Using the retrograde tracer Fluoro Gold in conjunction with Fos immunohistochemistry, the authors provide evidence for the involvement of RSPv, VISam, CL, AMv inputs to the ACA in during cat exposure, and primarily CL input during threat retrieval in the context. The authors then examined the role of some of these input structures to the ACA in the task using continuous 5min optogenetic inhibition. Silencing AM>ACA input during cat exposure (i.e., acquisition) impaired in contextual …
Reviewer #1 (Public Review):
The manuscript assessed the role of the anterior cingulate area (ACA) in fear acquisition and expression in a naturalist task using a live cat as the unconditioned stimulus where mice were the experimental subjects. Silencing of the ACA using DREADDs showed that the ACA is important for contextual fear acquisition and expression. Using the retrograde tracer Fluoro Gold in conjunction with Fos immunohistochemistry, the authors provide evidence for the involvement of RSPv, VISam, CL, AMv inputs to the ACA in during cat exposure, and primarily CL input during threat retrieval in the context. The authors then examined the role of some of these input structures to the ACA in the task using continuous 5min optogenetic inhibition. Silencing AM>ACA input during cat exposure (i.e., acquisition) impaired in contextual fear retrieval. Individual ACA>BLA and ACA>PERI pathway inhibition during cat exposure but not during disrupted contextual fear retrieval in line with greater FG+Fos double labeling in the former compared to the latter condition. ACA>POST pathway inhibition was without effect. ACA>PAGdl pathway inhibition during cat exposure had no effect on contextual fear retrieval whereas inhibition of this pathway during test disrupted retrieval, again in line with greater FG+Fos double labeling in the latter compared to the former condition.
One of the greatest strengths of the manuscript are the detailed analyses of the ACA input and particularly output pathways in the task. Further, the manuscript examined the effects at both acquisition and retrieval, providing a thorough examination of the effects of ACA and related structures at key memory phases. The complementary analysis of FG+Fos is most welcome, showing what level of overlap is necessary for a behavioural effect of pathway inhibition.
Some weaknesses of the manuscript design include the prolonged continuous optical inhibition, lack of optical control and the possibility of a state-dependent effect on neural manipulation. It is unclear if the similarity in the effects obtained in some cases are due to similarity in the underlying behavioral process that is disrupted (e.g. failing to acquire an accurate representation of the context vs. associating the context with the threat).
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Reviewer #2 (Public Review):
The authors' main goals were to identify key circuits for the acquisition and expression of contextual fear conditioning in a paradigm that uses a live cat as the unconditioned stimulus. They used neuroanatomical tracing, opto-, and chemo-genetic techniques to observe and manipulate activity in anterior cingulate area (ACA) afferents and efferents at either the acquisition or retrieval stages. The strengths include a thorough characterization of multiple circuits and robust behavioral effects. Weaknesses include the confound of the experimenter being in the room for the acquisition, but not retrieval phase, a lack of characterization of escape-like behaviors, and the exclusive use of male animals, which reduces the potential impact.
-
Reviewer #3 (Public Review):
In this study, de Lima et. al. examined the function of ACA in acquisition and expression of contextual fear memory to predator threat. The authors found that ACA is necessary for both processes. Using optogenetic terminal inactivation, the authors further demonstrate a necessary role of AM input to ACA in the contextual fear acquisition phase. At the output level, the projections from ACA to BLA and PERI are necessary for contextual fear acquisition while the projection from ACA to PAG is essential for contextual fear expression. Overall, the study is interesting and the results are straightforward. The presented data largely support the conclusions. The paper will provide new insight into the neural circuit underlying contextual fear learning and expression to predator threat. Some limitations of the study …
Reviewer #3 (Public Review):
In this study, de Lima et. al. examined the function of ACA in acquisition and expression of contextual fear memory to predator threat. The authors found that ACA is necessary for both processes. Using optogenetic terminal inactivation, the authors further demonstrate a necessary role of AM input to ACA in the contextual fear acquisition phase. At the output level, the projections from ACA to BLA and PERI are necessary for contextual fear acquisition while the projection from ACA to PAG is essential for contextual fear expression. Overall, the study is interesting and the results are straightforward. The presented data largely support the conclusions. The paper will provide new insight into the neural circuit underlying contextual fear learning and expression to predator threat. Some limitations of the study include the lack of controls to demonstrate how specific is the ACA response to threat and threat paired context is and validation of the terminal inhibition. Further characterization of the projections would also help to understand these results.
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