Conference Agenda

How are consolidated memories modified on the basis of experience? In this project we aimed to unravel the neural mechanisms at the basis of memory update. Understanding this biological process allows us to decipher how new information is constantly incorporated into existing memory, how a newly formed memory is integrated into previous knowledge and how the fine balance between memory stability and memory flexibility is maintained.
By using fear memory extinction as a model of memory update, we combined neuronal circuit mapping, fiber photometry, chemogenetic and closed-loop optogenetic manipulations in mice, and showed that the extinction of remote (30-day old) fear memories depends on thalamic nucleus reuniens (NRe) inputs to the basolateral amygdala (BLA). We find that remote, but not recent (1-day old), fear extinction activates NRe to BLA inputs, which become potentiated upon fear reduction. Both monosynaptic NRe to BLA, and total NRe activity increase shortly before freezing cessation, suggesting that the NRe registers and transmits safety signals to the BLA. Accordingly, pan-NRe and pathway-specific NRe to BLA inhibition impairs, while their activation facilitates fear extinction.
These findings identify the NRe as a crucial BLA regulator for extinction, and provide the first functional description of the circuits underlying the experience-based modification of consolidated fear memories.

Fear responses are functionally adaptive behaviors that are strengthened as memories. Indeed, a detailed knowledge of the neural circuitry modulating fear and fear memory could be the turning point for the comprehension of this emotion and its pathological states.

A comprehensive understading of the neural circuits mediating memory encoding, consolidation, and retrieval over time presents the fundamental technological challenge of analyzing activity in the entire brain with single-neuron resolution. In this context, we developed the BRAin-wide Neuron quantification Toolkit (BRANT) for mapping whole-brain neuronal activation at micron-scale resolution, combining tissue clearing, high-resolution light-sheet microscopy, and automated 3D image analysis. The robustness and scalability of this method allowed us to quantify the evolution of neuronal activation patterns across multiple phases of memory in mice. This approach highlighted a strong sexual dimorphism in the circuits recruited during memory recall, which had no counterpart in the behaviour. The methodology presented here paves the way for a comprehensive functional characterization of the evolution of fear memory.

Memory allocation refers to the process by which neurons are recruited into the encoding ensemble to store mnemonic information. In the past, several studies have revealed that this recruitment depends on neuronal selection based on varying degrees of intrinsic excitability (IE) and the expression of the transcription factor CREB. However, whether and to what extent other domains of intrinsic plasticity influence memory allocation remains unknown. Here, we show that chromatin accessibility and histone acetylation in principal neurons of the mouse lateral amygdala display a high degree of heterogeneity, a prerequisite for neuronal selection. Consequently, when we manipulated histone acetylation by lentivirus-mediated overexpression of histone acetyl transferases (HATs), we find that increasing histone acetylation favored memory allocation and retention, while optogenetic silencing of the epigenetically altered neurons prevented memory expression. Mechanistically, using patch-clamp recordings and single-nucleus multi-ome sequencing, we observed that HAT overexpression increased IE and epitranscriptomic changes favoring synaptic plasticity. Lastly, by merging FRET-based molecular beacons with calcium indicators that allow for the simultaneous recording of histone acetylation and calcium dynamics in single neurons in real time, we show that epigenetic heterogeneity underlies IE in a cell-autonomous manner. These findings identify chromatin-templated plasticity as a key factor catalyzing memory allocation.

The Zona Incerta (ZI), one of the most intriguing yet mysterious brain regions, has sparked the interest of research given its involvement in numerous functions and the clinical perspectives emerged by the application of the Deep Brain Stimulation on this nucleus to treat the motor symptoms of parkinsonian patients. To characterize the relationship existing between the ZI and the other brain areas, which is still nowadays an open issue, we applied the algorithm for Cell Assembly Detection by Russo and Durstewitz (2019) to identify the cell assemblies spanning the ZI and approximately 20 external brain regions. Cell assemblies are sets of neurons that repeatedly coordinate their activity in defined temporal patterns and have been defined as the brain's functional units, being able to encode memories or perceptual stimuli. The first step of our analysis, that considered the assemblies made up of two neurons, revealed that the ZI coordinates its activity with multiple areas with different levels of strength: hippocampal, septal and motor-related areas stand out as the ones possessing the highest level of coordination with the ZI, while prefrontal and thalamic areas, together with the basal ganglia, showed lower levels of coordination. The second step of analysis considered assemblies made up of three neurons distributed among two areas in patterns that we defined as loop-like assemblies and showed that the ZI establishes these specific motifs of coordination independently of the capacity of forming assemblies of two elements. Furthermore, a characterization of the coordinative ability of single incertal neurons revealed that neurons entering loop-like assemblies have the highest coordinating power, raising the possibility that such motifs represent a specific form of integration-oriented coordination between neurons belonging to different areas. Lastly, we extended the analytical pipeline developed for the ZI to open the possibility to investigate brain-wide coordination on a large scale.