Understanding whole-brain circuits mediating fear memory attenuation

Project Information

TaggingMemory

Grant agreement ID: 101107833

DOI

10.3030/101107833

EC signature date 5 May 2023

Start date 1 October 2023

End date 30 November 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 288 859,20

Coordinated by

LABORATORIO EUROPEO DI SPETTROSCOPIE NON LINEARI
Italy

Periodic Reporting for period 1 - TaggingMemory (Understanding whole-brain circuits mediating fear memory attenuation)

Reporting period: 2023-10-01 to 2025-11-30

Summary of the context and overall objectives of the project

Post-traumatic Stress Disorder (PTSD) is a severe mental health condition that affects millions of people worldwide and represents a major societal and clinical challenge in Europe and beyond. A defining feature of PTSD is the persistence and maladaptive generalisation of fear, whereby individuals experience fear responses even in situations that should be perceived as safe. Although current treatments can reduce symptoms, the brain-wide mechanisms that drive fear extinction and fear discrimination are still poorly understood, limiting the development of more precise and effective interventions.
This project addresses this gap by investigating how fear memories are formed, modified, and disrupted across whole-brain neural circuits. By combining behavioural paradigms with brain-wide cellular mapping, the project aims to identify the networks that support adaptive fear regulation and those that underlie pathological fear generalisation. Importantly, the project moves beyond extinction alone by explicitly including fear discrimination, a process that is often impaired in PTSD and closely linked to fear generalisation.
To achieve these goals, the project developed an innovative multiple Arc (mArc) tagging system that enables simultaneous tracking of different memory-related neuronal ensembles within the same brain. This approach allows researchers to follow how fear memories evolve across learning, recall, discrimination, and extinction within individual subjects. When combined with whole-brain imaging and computational analysis, this strategy generates detailed, single-cell-resolution maps of memory-related brain activity.
In parallel, the project examined how psychedelic and psychedelic-like compounds influence fear regulation. By testing pharmacological interventions in preclinical models, the project explored how these compounds affect brain-wide fear networks and whether they can reduce maladaptive fear generalisation. The identification of circuit-level changes linked to improved fear discrimination provides a mechanistic basis that may guide future PTSD therapies.
Overall, the project advances understanding of how fear memories become dysregulated in pathological states and identifies the brain networks involved in PTSD-like behaviours, with long-term relevance for mental health research and European innovation priorities.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the project, an integrated experimental and analytical framework was established to explore how fear memories are formed, altered, and disrupted across whole-brain neural circuits. The work brought together behavioural tasks, genetic and viral tools, brain-wide imaging, and computational analysis to better understand fear extinction and fear discrimination.
A central achievement was the development and validation of mArc tagging system, which allows the tagging of different memory-related neuronal populations within the same brain. By combining an Arc-based genetic strategy with an Arc-RAM viral approach, the system avoids complex breeding schemes and offers greater experimental flexibility. In vivo experiments confirmed reliable tagging of neurons activated during fear learning and recall, making mArc a powerful tool to follow memory dynamics over time.
The project also optimised behavioural paradigms relevant to PTSD, including contextual fear conditioning for fear extinction, and pattern separation for discrimination. These paradigms were paired with whole-brain tissue clearing and immunolabelling, enabling single-cell-resolution visualisation of memory-related activity. The large datasets generated by high-resolution imaging required the implementation of a scalable computational pipeline such as the SMARTTr workflow, which supports consistent whole-brain registration and quantitative comparisons across individuals and conditions.
In parallel, pharmacological interventions were tested. Prophylactic ketamine enhanced early fear discrimination in a sex-dependent manner, suggesting a possible strategy to reduce maladaptive fear generalisation. Brain-wide analyses of treated animals are currently ongoing.
Overall, the project delivered new tools, data, and concepts to study fear memories as dynamic, brain-wide processes, laying the groundwork for future research on PTSD-like behaviours and targeted interventions.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project produced results that go beyond current approaches used to study fear and stress-related disorders. Until now, research on fear and PTSD has often focused on small parts of the brain, making it difficult to understand how fear is processed as a whole. This project takes a different approach by studying fear memories across the entire brain, at the level of individual cells, within the same subject. This provides a more complete and realistic picture of how memories related to fear can change over time.
A major innovation of the project is the development of the mArc tagging system. This method allows scientists to track different groups of brain cells involved in memory without relying on complex genetic breeding. By combining genetic tools with a viral approach, the system is easy to use and flexible. As a result, it becomes possible to follow how fear-related brain cells are activated during learning, recall, discrimination between safe and dangerous situations, and extinction.
Another important advance is the shift from studying fear extinction to fear discrimination. In PTSD, people often respond with fear even when they are safe. By focusing on this problem, the project offers a more realistic way to study pathological fear. Instead of pointing to a single brain area, the results show that PTSD-like behaviour is linked to changes across entire brain networks.
The project also combined advanced brain imaging, tissue clearing, and computer-based analysis to handle very large datasets in a reliable and reproducible way. Finally, the project explored how drugs can influence fear-related brain networks. The finding that ketamine improves early fear discrimination suggests new directions for preventing harmful fear generalisation.
Overall, the project introduces new tools and ideas that help explain how fear memories work across the whole brain, supporting future research on PTSD and mental health.