Selective increase of correlated activity in Arc-positive neurons after chemically induced long-term potentiation in cultured hippocampal neurons.

The activity-dependent expression of immediate-early genes (IEGs) has been utilised to label memory traces. However, their roles in engram specification are incompletely understood. Outstanding questions remain as to whether expression of IEGs can interplay with network properties such as functional connectivity and also if neurons expressing different IEGs are functionally distinct. In order to connect IEG expression at the cellular level with changes in functional-connectivity, we investigated the expression of 2 IEGs, Arc and c-Fos, in cultured hippocampal neurons. Primary neuronal cultures were treated with a chemical cocktail (4-aminopyridine, bicuculline, and forskolin) to increase neuronal activity, IEG expression, and induce chemical long-term potentiation. Neuronal firing is assayed by intracellular calcium imaging using GCaMP6m and expression of IEGs is assessed by immunofluorescence staining. We noted an emergent network property of refinement in network activity, characterized by a global downregulation of correlated activity, together with an increase in correlated activity between subsets of specific neurons. Subsequently, we show that Arc expression correlates with the effects of refinement, as the increase in correlated activity occurs specifically between Arc-positive neurons. The expression patterns of the IEGs c-Fos and Arc strongly overlap, but Arc was more selectively expressed than c-Fos. A subpopulation of neurons positive for both Arc and c-Fos shows increased correlated activity, while correlated firing between Arc+/cFos- neurons is reduced. Our results relate neuronal activity-dependent expression of the IEGs Arc and c-Fos on the individual cellular level to changes in correlated activity of the neuronal network.Establishing a stable long-lasting memory requires neuronal network-level changes in connection strengths in a subset of neurons, which together constitute a memory trace or engram. Two genes, c-Fos and Arc, have been implicated to play critical roles in the formation of the engram. They have been studied extensively at the cellular/molecular level, and have been used as markers of memory traces in mice. We have correlated Arc and c-Fos cellular expression with refinement of correlated neuronal activity following pharmacological activation of networks formed by cultured hippocampal neurons. Whereas there is a global loss of correlated activity, Arc-positive neurons show selectively increased correlated activity. Arc is more selectively expressed than c-Fos, but the two genes act together in encoding information about changes in correlated firing.