Optogenetic reactivation of memory ensembles in the retrosplenial cortex induces systems consolidation

Abstract

The neural circuits underlying memory change over prolonged periods after learning, in a process known as systems consolidation. Postlearning spontaneous reactivation of memory-related neural ensembles is thought to mediate this process, although a causal link has not been established. Here we test this hypothesis in mice by using optogenetics to selectively reactivate neural ensembles representing a contextual fear memory (sometimes referred to as engram neurons). High-frequency stimulation of these ensembles in the retrosplenial cortex 1 day after learning produced a recent memory with features normally observed in consolidated remote memories, including higher engagement of neocortical areas during retrieval, contextual generalization, and decreased hippocampal dependence. Moreover, this effect was only present if memory ensembles were reactivated during sleep or light anesthesia. These results provide direct support for postlearning memory ensemble reactivation as a mechanism of systems consolidation, and show that this process can be accelerated by ensemble reactivation in an unconscious state.

Keywords: engram; fear conditioning; memory consolidation; replay; retrosplenial cortex.

Fig. 1.

Optogenetic reactivation of RSC memory ensembles produces a recent memory with characteristics of a remote memory. (A) Schematic of the cfos-tTA/tetO-ChEF double-transgenic mouse line. (B) Experimental protocol used in RSC ensemble reactivation and HPC inactivation. (C) Freezing levels of TG animals before (recall 1, 24 h after training) and after (recall 2, 48 h after training) light-emitting diode (LED) stimulation and HPC infusions. (saline+LED, n = 6; saline, n = 6; drug + LED, n = 9; drug, n = 9). (D) arc-positive cells in selected brain areas after recall 2 (saline+LED, n = 6; saline, n = 5; drug + LED, n = 5; drug, n = 6). (E) Experimental protocol used in RSC ensemble reactivation and HPC/ACC inactivation. (F) Freezing levels of WT and TG animals after LED stimulation and HPC or ACC drug infusions (WT/ACC⁻, n = 6; WT/HPC⁻, n = 10; WT/ACC⁻/HPC⁻, n = 7; TG/ACC⁻, n = 6; TG/HPC⁻, n = 9; TG/ACC⁻/HPC⁻, n = 6). Drug, CNQX+TTX; Infl, infralimbic cortex; Prl, prelimbic cortex; ACC, anterior cingulate cortex; HPC, hippocampus; CP, caudate putamen. In all panels, bars represent mean ± SEM. *P < 0.05; ** P < 0.01; ***P < 0.001.

Fig. 2.

Optogenetic reactivation of RSC memory ensembles produces context generalization. (A) Experimental protocol used to test memory generalization. (B) Freezing levels of animals in training context (ctxA) or a novel context (ctxC) after RSC stimulation (TG, n = 13; WT, n = 11). (C) Freezing levels of WT animals 30 d after CFC in ctxA or ctxC (n = 12). (D) Discrimination indexes of stimulated WT and TG mice after optogenetic stimulation induced consolidation and natural consolidation (WT, 30 d). In all panels, bars represent mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 3.

The effect of optogenetic memory ensemble reactivation is dependent on brain state. (A) Experimental protocol used in RSC ensemble reactivation and HPC inactivation. (B) Freezing levels of WT and TG animals during retrieval trial after RSC ensemble reactivation during sleep and HPC infusions (WT/CNQX⁻, n = 10; WT/CNQX⁺, n = 10; TG/CNQX⁻, n = 10; TG/CNQX⁺, n = 13). (C) Freezing levels of WT and TG animals during retrieval trial after RSC ensemble reactivation during awake and HPC infusions (WT/CNQX⁻, n = 8; WT/CNQX⁺, n = 7; TG/CNQX⁻, n = 5; TG/CNQX⁺, n = 7). In all panels, bars represent mean ± SEM. Horizontal axis indicates groups that received hippocampal inactivation (CNQX+TTX) before memory retrieval. *P < 0.05; ***P < 0.001.

Fig. 4.

Responses to RSC memory ensemble stimulation are differentially propagated depending on the brain state. (A) Average of power versus frequency plots for the first 40 s of recording during baseline from each brain region across all animals (n = 13). (B) Experimental protocol used for obtaining LFP recordings from the ACC, RSC, and HPC during control recordings performed before (Pre), and test recordings performed after CFC (Post). (C) The response to stimulation averaged across all mice for isoflurane (n = 5), active awake (n = 4), and natural sleep (n = 4). Curve plots represent mean ± SEM. **P < 0.01; ***P < 0.001.