One Memory Engram, Two Functionally Distinct Neuronal Populations

Post by Elisa Guma

What's the science?

Engrams are defined as biological changes that take place in the brain to encode specific experiences or memories. Each engram is thought to be made up of a sparse population of neurons that is activated by specific learning experiences, with long-lasting synaptic modifications. However, it is unclear whether there is functional heterogeneity within each memory engram, and whether separate neuronal populations encode distinct aspects of memory. This week in Cell, Sun and colleagues present causal evidence to show neurons within a single memory engram are functionally heterogeneous to allow for different aspects of the memory to be individually represented and regulated.

How did they do it?

Mice underwent contextual fear conditioning: first, they were exposed to foot shocks (fear stimulus) in context A. 24 h later, they were exposed to either 1) the conditioned context A, 2) unconditioned context B similar to A, or 3) very distinct context C. Using a novel technique called Robust Activity Marking (RAM) the authors identified neuronal ensembles in the fear memory engram by the transcription output of immediate early genes (IEGs) as a proxy for neuronal activity, Fos and Npas4 (F-RAM+ and N-RAM+ neuronal ensembles). To confirm that F-RAM+ and N-RAM+ neurons are dependent on Fos and Npas4 gene expression, the authors knocked out the expression of Fos or Npas4 genes in the dentate gyrus of a separate group of mice prior to fear conditioning. They also used a mouse line (FosTRAP) that expresses a fluorescent protein Fos when neurons are active to show colocalization between Fos expression and F-RAM+ neurons. Further, they characterized the synaptic properties of these two populations using patch-clamp electrophysiology experiments.

To assess differential roles in memory discrimination-generalization, the authors exposed mice to either context A, B, or C 24 hours after fear conditioning and measured F-RAM+ and N-RAM+ neuron activity first by immunostaining of IEGs, then by fiber photometry, which measures calcium signals as a proxy for neuronal activity. To assess their role in memory recall in vivo, the authors then manipulated the activity of these neurons using chemogenetics (designer receptors exclusively activated by designer drugs). To identify which circuit inputs were driving F-RAM+ and N-RAM+ dentate gyrus cells they used electrically, chemogenetically, or optogenetically stimulated axonal fibers of different pathways that synapse onto dentate gyrus neurons while measuring activity of the F-RAM+ and N-RAM+ neurons.

What did they find?

The authors observed distinct identities between the two neuronal ensembles: F-RAM+ neurons received stronger excitatory inputs, whereas N-RAM+ neurons received stronger inhibitory inputs. Selective knockout of Fos and Npas4 inhibited the formation of F-RAM+ and N-RAM+ ensembles, respectively. Further, in the FosTRAP mice there was substantial colocalization and similar electrophysiological properties between Fos positive cells and F-RAM+ neurons, but not the N-RAM+ cells.

The authors observed a similar activation of F-RAM+ neurons (as measured by the number of activated cells and calcium imaging) in both contexts A and B, but less in context C, suggesting that this ensemble is not sensitive to small differences in context and favours memory generalization. N-RAM neurons, however, seemed to play a greater role in memory differentiation in similar contexts as they were significantly less activated in context A than B (and not very activated in context C suggesting they were not responding to novelty). The chemogenetic inhibition of F-RAM+ neurons during recall enhanced the discrimination between contexts A and B, suggesting that memory generalization was impaired. Inhibiting N-RAM reduced memory discrimination between similar contexts A and B, but not between A and C. The authors identified the medial entorhinal cortex to be the main pathway innervating F-RAM+ neurons, and that optogenetic inhibition of this pathway decreased the number Fos+ cells in the dentate gyrus specifically and disrupted memory generalization (enhanced discrimination between context A and B).

With a combination of optogenetics and pharmacology, they identified cholecystokinin-expression GABAergic neurons as the main inhibitory drivers to N-RAM+ neurons, and that chemogenetic inhibition of these interneurons impaired memory discrimination (by abolishing discrimination between contexts A and B and reduced discrimination between A and C).

What's the impact?

This thorough study presents compelling, causal evidence for the hypothesis that there is functional heterogeneity within the memory engrams. They identify the synaptic and circuit mechanisms used by two different neuronal ensembles associated with the same fear memory and show their roles in regulating the balance between memory discrimination and generalization. This study sheds important light on some important, and previously poorly understood, cellular and circuit mechanisms underlying memory formation, and is the first to provide evidence for heterogeneity within engram neuronal populations.

Sun et al. Functionally distinct neuronal ensembles within the memory engram. Cell (2020). Access the original scientific publication here.