The intrinsic excitability of and autophagy protein expression levels in dentate gyrus ensembles regulate fear generalization

Abstract The overgeneralization of fear is associated with psychiatric disorders and cognitive decline. Recent studies have shown that engram cells in the dorsal dentate gyrus are integrated into functionally heterogeneous ensembles that are involved in contextual fear memory generalization and discrimination. However, the intracellular signals that promote fear generalization remain to be fully elucidated. In this study, we labeled and manipulated the c-Fos+ and Npas4+ ensembles in the dorsal dentate gyrus that are activated by contextual fear conditioning using a robust activity marking system. The results showed that increasing the excitability of Fos-dependent robust activity marking by overexpressing NaChBac or decreasing the excitability of Npas4-dependent robust activity marking by overexpressing Kir2.1 promoted fear memory generalization. Furthermore, CRISPR-mediated downregulation of the autophagy-related Atg5 or Atg7 genes in dorsal dentate gyrus neurons inhibited activation of c-Fos, but not Npas4. Knockdown of Atg5 or Atg7 in the Fos-dependent robust activity marking or Npas4-dependent robust activity marking ensemble led to an increase in neuronal excitability and a decrease in spine density in both ensembles. However, Atg7 knockdown in the Fos-dependent robust activity marking ensemble promoted memory generalization, while knockdown of Atg5 or Atg7 in the Npas4-dependent robust activity marking ensemble increased anxiety levels. These results contribute to our understanding of how the varying plasticity of memory engrams is involved in regulating fear memory generalization and anxiety.