Early remodeling of the neocortex upon episodic memory encoding

Abstract

Understanding the mechanisms by which long-term memories are formed and stored in the brain represents a central aim of neuroscience. Prevailing theory suggests that long-term memory encoding involves early plasticity within hippocampal circuits, whereas reorganization of the neocortex is thought to occur weeks to months later to subserve remote memory storage. Here we report that long-term memory encoding can elicit early transcriptional, structural, and functional remodeling of the neocortex. Parallel studies using genome-wide RNA sequencing, ultrastructural imaging, and whole-cell recording in wild-type mice suggest that contextual fear conditioning initiates a transcriptional program in the medial prefrontal cortex (mPFC) that is accompanied by rapid expansion of the synaptic active zone and postsynaptic density, enhanced dendritic spine plasticity, and increased synaptic efficacy. To address the real-time contribution of the mPFC to long-term memory encoding, we performed temporally precise optogenetic inhibition of excitatory mPFC neurons during contextual fear conditioning. Using this approach, we found that real-time inhibition of the mPFC inhibited activation of the entorhinal-hippocampal circuit and impaired the formation of long-term associative memory. These findings suggest that encoding of long-term episodic memory is associated with early remodeling of neocortical circuits, identify the prefrontal cortex as a critical regulator of encoding-induced hippocampal activation and long-term memory formation, and have important implications for understanding memory processing in healthy and diseased brain states.

Keywords: hippocampus; learning; neuroplasticity; optogenetics; transcriptome.

Fig. 1.

Associative memory encoding primes the mPFC transcriptome for neuroplasticity. (A) Heatmap depicting 342 differentially expressed genes (DEGs) determined by genome-wide RNA sequencing (RNA-seq) of the mPFC upon control exposure (CTL) or contextual fear conditioning (FC). Rows represent DEGs, columns represent transcriptomic profiles of individual animals. Blue and red represent low and high levels of gene expression, respectively (n = 4 mice per group). (B) Ingenuity Gene Ontology Analysis depicting to which biological processes the DEGs contribute (red, up-regulated upon FC; blue, down-regulated upon FC). (C) Ingenuity Gene Network Analyses of DEGs up-regulated (Cell Signaling; P = 1 × 10⁻⁵⁰) or down-regulated (Immune Cell Trafficking; P = 1 × 10⁻³⁴) in the mPFC upon FC. Colored and uncolored nodes represent DEGs identified by RNA-seq and the Ingenuity network generation algorithm, respectively. (D) Expression of a subset of DEGs revealed by RNA-seq was confirmed in independent mPFC samples using quantitative RT-PCR (n = 5–10 mice per group). Values are normalized to expression levels of Gapdh. *P ≤ 0.05; **P ≤ 0.01. Values represent mean ± SEM.

Fig. 2.

Early remodeling of mPFC synapse ultrastructure upon associative memory encoding. (A) Representative transmission electron micrographs depicting mPFC synapse ultrastructure upon CTL or FC. (Scale bar, 500 nm.) Arrows indicate magnified synapses shown at Right. (Scale bar, 200 nm.) (B and C) Quantification of active zone and postsynaptic density length (B; n = 77–92 synapses from four mice per group) and docked synaptic vesicle number (C; n = 69–92 synapses from four mice per group) in mPFC synapses upon CTL or FC. (D) Active zone length was correlated with docked synaptic vesicle number across treatment groups. Blue and red circles indicate synapses in CTL and FC groups, respectively (Pearson r = 0.7991; P ≤ 0.0001). (E) Representative immunohistochemical images depicting synaptophysin immunoreactivity in the mPFC upon CTL or FC. (Scale bar, 50 μm.) (F) Quantification of E (n = 6 mice per group). **P ≤ 0.01. Values represent mean ± SEM.

Fig. 3.

Associative memory encoding enhances dendritic spine plasticity and synaptic efficacy in the mPFC. (A) Representative Golgi–Cox impregnated dendritic spines present on layer II/III mPFC pyramidal neurons upon CTL or FC. Green and blue arrows indicate thin and mushroom-shaped spines, respectively. (Scale bar, 5 μm.) (B) Quantification of A (n = 716–753 spines from three to four mice per group). (C) Representative traces of miniature excitatory postsynaptic currents (mEPSCs) recorded from layer II/III mPFC pyramidal neurons upon CTL or FC. (Scale bar, 40 pA, 2.5 s. Quantification of mEPSC frequency and amplitude in mPFC pyramidal neurons upon CTL or FC (n = 10–11 neurons per group). *P ≤ 0.05; n.s., not significant. Values represent mean ± SEM.

Fig. 4.

Optogenetic silencing of excitatory mPFC neurons impairs entorhinal–hippocampal circuit activation during associative memory encoding. (A) AAV₅-CaMKIIα-eNpHR3.0-EYFP was injected into bilateral mPFC to selectively transduce mPFC excitatory neurons and permit optogenetic inhibition thereof. (Scale bar, 500 μm.) (B) Representative immunohistochemical images depicting CaMKIIα expression in mPFC neurons transduced with CaMKIIα-eNpHR3.0-EYFP. (Scale bar, 10 μm.) (C) Ex vivo optical (593 nm) inhibition of action potential spiking (50 pA current injection) in a representative mPFC pyramidal neuron expressing eNpHR3.0-EYFP. (Scale bar, 20 mV, 100 ms.) (D) Schematic of the experimental paradigm used for in vivo mPFC optogenetic inhibition experiments shown in E–J. Animals injected with AAV₅-CaMKIIα-eNpHR3.0-EYFP or AAV₅-CaMKIIα-EYFP control virus received continuous optogenetic inhibition of excitatory mPFC neurons during FC. One hour later, tissue was harvested for analysis. (E–J) Representative immunohistochemical images depicting Zif268 and synaptophysin immunoreactivity in the mPFC (E and F), hippocampal area CA1 (CA1; G and H), and entorhinal cortex (EC; I and J) of mice expressing eNpHR3.0-EYFP or EYFP alone following continuous optogenetic inhibition of mPFC during FC. (Scale bars, 50 μm.) (F) Quantification of E (n = 3–4 mice per group). (H) Quantification of G (n = 3–4 mice per group). (J) Quantification of I (n = 3–4 mice per group). *P ≤ 0.05; **P ≤ 0.01. Values represent mean ± SEM.

Fig. 5.

Real-time activity of excitatory mPFC neurons is critical for long-term associative memory formation. (A) Schematic of the experimental paradigm used for in vivo mPFC optogenetic inhibition experiments shown in B–E. Animals injected with AAV₅-CaMKIIα-eNpHR3.0-EYFP or AAV₅-CaMKIIα-EYFP received continuous optogenetic inhibition of excitatory mPFC neurons during FC. Mice were returned to the experimental context 1 d and 30 d later to assess recent and remote long-term memory, respectively. (B and C) Real-time optogenetic inhibition of excitatory mPFC neurons during FC significantly impaired the formation of recent (B) and remote (C) long-term associative memory in mice expressing eNpHR3.0-EYFP compared with EYFP controls (n = 6 mice per group). (D and E) Optogenetic inhibition of the mPFC did not alter basal locomotor activity (D) or response to foot shock (E) during the conditioning period (n = 6 mice per group). *P ≤ 0.05; **P ≤ 0.01. Values represent mean ± SEM.