Memory. Engram cells retain memory under retrograde amnesia

Abstract

Memory consolidation is the process by which a newly formed and unstable memory transforms into a stable long-term memory. It is unknown whether the process of memory consolidation occurs exclusively through the stabilization of memory engrams. By using learning-dependent cell labeling, we identified an increase of synaptic strength and dendritic spine density specifically in consolidated memory engram cells. Although these properties are lacking in engram cells under protein synthesis inhibitor-induced amnesia, direct optogenetic activation of these cells results in memory retrieval, and this correlates with retained engram cell-specific connectivity. We propose that a specific pattern of connectivity of engram cells may be crucial for memory information storage and that strengthened synapses in these cells critically contribute to the memory retrieval process.

Figure 1. Synaptic Plasticity and Connectivity of Engram Cells

(A) Mice taken Off DOX 24 hrs before contextual fear conditioning (CFC) and dispatched 24 hrs post training. Saline (SAL) or anisomycin (ANI) administered immediately after training. (B) AAV₈-CaMKIIα-ChR2-EYFP and AAV₉-TRE-mCherry viruses injected into the entorhinal cortex and dentate gyrus, respectively, of c-fos-tTA mice. (C) Paired recordings of engram (red) and non-engram (grey) DG cells during optogenetic stimulation of ChR2⁺ perforant path (PP) axons. (D) Representative image of a pair of recorded biocytin-labeled engram (mCherry⁺) and non-engram (mCherry⁻) DG cells. Note ChR2⁺ PP axons in green. (E) (Top) Example traces of AMPA and NMDA receptor-dependent postsynaptic currents in mCherry⁺ and mCherry⁻ cells, evoked by light activation of ChR2⁺ PP axons. (Bottom) EPSC amplitudes and AMPA/NMDA current ratios of mCherry⁺ and mCherry⁻ cells of the two groups are displayed as means (columns) and individual paired data points (grey lines). Paired t-test * p < 0.05, ** p < 0.001. SAL group compared with the ANI group, unpaired t-test * p < 0.05. (F) (Left) Representative confocal images of biocytin filled dendritic fragments derived from SAL and ANI groups for ChR2⁺ and ChR2⁻ cells (arrow heads: dendritic spines). (Right) Average dendritic spine density showing an increase occurring exclusively in ChR2⁺ fragments. Data are represented as mean ± SEM. Unpaired t tests ** p < 0.01, *** p < 0.001. (G) Engram Connectivity. (Top left) AAV₉-TRE-ChR2-EYFP and AAV₉-TRE-mCherry viruses, injected into the DG and CA3, respectively, of c-fos-tTA mice. (Bottom left) Example of mCherry⁺ (1) and mCherry⁻ (2) biocytin-filled CA3 pyramidal cells. Note ChR2⁺ mossy fibers (MF) in green. (Top Right) mCherry⁺ cell but not mCherry⁻ cell displayed EPSPs in response to optogenetic stimulation of MF. (Bottom Right) Probability of connection of DG ChR2⁺ engram axons and CA3 mCherry⁺ and mCherry⁻ cells. Error bars are approximated by binomial distribution. Fisher’s exact test: * p < 0.05.

Figure 2. Optogenetic Stimulation of DG Engram Cells Restores Fear Memory in Retrograde Amnesia

(A) Behavioral schedule. Beige shading signifies that subjects are On DOX, precluding ChR2 expression. Mice taken off DOX 24–30 hrs before CFC in Context B. SAL or ANI was injected into the mice after training. (B) Habituation to Context A with Light-Off and Light-On epochs. Blue light stimulation of the DG did not cause freezing behavior in naïve, unlabelled mice of the pre-SAL (n = 10) or pre-ANI (n = 8) groups. (C) Memory recall in Context B 1 day post-training (Test 1). ANI group displayed significantly less freezing than SAL group (p < 0.005). No-shock groups with SAL (n = 4) or ANI (n = 4) did not display freezing upon re-exposure to Context B. (D) Memory recall in Context A 2 days post-training (Engram Activation) with Light-Off and Light-On epochs. Freezing for the two Light-Off and Light-On epochs are further averaged in the inset. Significant freezing due to light stimulation was observed in both the SAL (p < 0.01) and ANI groups (p < 0.05). Freezing levels did not differ between groups. SAL and ANI-treated no-shock control groups did not freeze in response to light stimulation of context B engram cells. (E) Memory recall in Context B 3 days post-training (Test 2). ANI group displayed significantly less freezing than SAL group (p < 0.05). (F, G) Images showing DG sections from c-fos-tTA mice 24 hrs after SAL or ANI treatment. (H) ChR2-EYFP cell counts from DG sections of SAL (n = 3) and ANI (n = 4) groups. (I) In vivo anesthetized recordings (see Materials and Methods). (J, K) Light pulses induced spikes in DG neurons recorded from head-fixed anesthetized c-fos-tTA mice 24 hrs after treatment with either SAL or ANI. Data presented as mean ± SEM.

Figure 3. Recovery of Memory from Amnesia under a Variety of Conditions

(A) DG engram activation and optogenetic place avoidance (OptoPA). During habituation neither group displayed significant avoidance of target zone. For Natural Recall the ANI group (n = 10) displayed significantly less freezing than SAL group (n = 12) in Context B (p < 0.005). SAL and ANI displayed similar levels of OptoPA. (B) CA1 engram activation and CFC. 1 day post-CFC (Test 1) ANI group (n = 9) displayed significantly less freezing than SAL group (n = 10) in Context B (p < 0.01). 2 days post-training (Engram Activation), light-activation of CA1 engrams elicited freezing in both SAL (p < 0.01) and ANI groups (p < 0.001). 3 days post-training (Test 2) ANI group froze less than SAL group in Context B (p < 0.01). (C) Lateral amygdala (LA) engram activation and tone fear conditioning (TFC). The behavioral schedule was identical to that in Fig. 3B, except that context tests were replaced with tone tests in Context C (Materials and Methods). (Left) example image of ChR2-mCherry labeling of LA neurons. 2% of DAPI cells were labeled by ChR2. (Right) 1 day post-training (Test 1), ANI group (n = 9) displayed significantly less freezing to tone than SAL group (n = 9) (p < 0.05). 2 days post-training (Engram Activation), significant light-induced freezing was observed for both SAL (p < 0.005) and ANI groups (p < 0.005). 3 days post-training (Test 2) ANI group froze less to tone than SAL group (p < 0.05). (D) DG engram activation and CFC reconsolidation. ANI (n = 11) and SAL (n = 11) groups showed similar levels of ChR2 labeling. Both groups showed light-induced freezing behavior 1 day post-training (Engram Activation 1), pre-SAL (P < 0.001), pre-ANI (P < 0.02). 2 days post training, (Test 1) the fear memory was reactivated by exposure to Context B, and SAL or ANI injected. 3 days post-training, (Test 2) the ANI group froze significantly less than SAL to Context B (p < 0.01). 4 days post-training, (Engram Activation 2) significant light-induced freezing was observed for the SAL (p < 0.001) and ANI (p < 0.003) groups. (E) DG Inception (Materials and Methods) following contextual memory amnesia. Context-only engram was labeled for target Context A, followed by injection of SAL (n = 11) or ANI (n = 11). Amnesia demonstrated in ANI group by decreased ChR2⁺/c-Fos⁺ co-labeling following Context A re-exposure 1 day post labeling. Following fear inception, neither SAL nor ANI groups displayed freezing behavior in Context B, while both groups displayed significant freezing in Context A, with no significant difference between groups. No-light inception SAL (n = 7) and ANI (n = 6) controls displayed no freezing to Context A or B. Statistical comparison are performed by using unpaired t tests, *** p < 0.001. Data presented as mean ± SEM.

Figure 4. Amygdala Activation and Functional Connectivity in Amnesia by Light Activation of DG Engram

(A) Schedule for cell counting experiments. Mice were either given a natural recall session in Context B, or a light-induced recall session in Context A. Mice were perfused 1 hr post recall. (B) Representative image showing c-Fos expression in the basolateral amygdala (BLA) and central amygdala (CeA).(C) c-Fos⁺ cell counts in the BLA and CeA of mice following natural or light-induced recall (n = 3–4 per group). (D) Schedule for cell counting experiments. c-fos-tTA mice with AAV₉-TRE-ChR2-EYFP injected into the DG and AAV₉-TRE-mCherry injected into both CA3 and BLA were fear conditioned off DOX, and 1 day later were given a natural recall session in Context B, or a light-induced recall session in Context A. Mice were perfused 1 hr post recall. (E – G) Representative images showing mCherry engram cell labeling, c-Fos expression, mCherry⁺/c-Fos⁺ overlap in CA3. (H – J) Representative images showing mCherry engram cell labeling, c-Fos expression, mCherry/c-Fos overlap in BLA. (K) c-Fos⁺/mCherry⁺ overlap cell counts in CA3 and BLA of mice following natural or light-induced recall (n = 3 – 4 per group). Chance levels were estimated at 0.76 (CA3) and 0.42 (BLA). Data are presented as mean ± SEM. Statistical comparison are performed by using unpaired t tests, * p < 0.05, ** p < 0.01.