Competition between engrams influences fear memory formation and recall - PubMed (original) (raw)

. 2016 Jul 22;353(6297):383-7.

doi: 10.1126/science.aaf0594.

Chen Yan 1, Valentina Mercaldo 1, Hwa-Lin Liz Hsiang 1, Sungmo Park 1, Christina J Cole 1, Antonietta De Cristofaro 2, Julia Yu 2, Charu Ramakrishnan 3, Soo Yeun Lee 3, Karl Deisseroth 3, Paul W Frankland 4, Sheena A Josselyn 4

Affiliations

Competition between engrams influences fear memory formation and recall

Asim J Rashid et al. Science. 2016.

Abstract

Collections of cells called engrams are thought to represent memories. Although there has been progress in identifying and manipulating single engrams, little is known about how multiple engrams interact to influence memory. In lateral amygdala (LA), neurons with increased excitability during training outcompete their neighbors for allocation to an engram. We examined whether competition based on neuronal excitability also governs the interaction between engrams. Mice received two distinct fear conditioning events separated by different intervals. LA neuron excitability was optogenetically manipulated and revealed a transient competitive process that integrates memories for events occurring closely in time (coallocating overlapping populations of neurons to both engrams) and separates memories for events occurring at distal times (disallocating nonoverlapping populations to each engram).

Copyright © 2016, American Association for the Advancement of Science.

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Figures

Fig. 1.

Fig. 1.. Events occurring closely in time are coallocated to overlapping engrams, and memories become linked.

(A) Mice distinguish conditioned stimuli (CS1, CS2), freezing more in response to trained, than untrained, auditory CS. ANOVA: Train-CS × Test-CS, F(1,12) = 44.29, *P < 0.001. Means ± SEM. n = 7 mice per group. *P < 0.05. (B) Event 2 memory was enhanced if event 1 (CS1+shock) occurred within a short intertraining interval (ITI). Enhancement was not due to CS1 alone or immediate shock (Imm shock) during event 1; F(8,142) = 17.03, *P < 0.001. n = 56 for CS1 alone (see fig. S1A), n = 5 to 16 for other groups. n.s., not statistically different. (C) Engrams for events with a 6-hour (but not 24-hour) ITI coallocated to overlapping LA neurons; F(3,8) = 119.01, *P < 0.001. (Right) Coallocation in 6-hour, but not 24-hour, ITI. arc+ (CS2 reexposure, red), homer1a+ (h1a+, CS1 reexposure, green) neurons, 4´,6-diamidino-2-phenylindole (DAPI; blue, nuclear stain). Scale bar, 20 μm. n = 3 mice per group. (D) Extinguishing event 2 memory also decreased event 1 memory if a 6-hour (but not 24-hour) ITI was used. Group × CS-Freezing, F(2,50) = 12.53, *P < 0.001. n = 12 mice, 6-hour, 24-hour groups; n = 4 mice, control group.

Fig. 2.

Fig. 2.. Engram interaction may be governed by neuronal CREB function and excitability.

(A) (Top) Fear conditioning transiently increases the percentage of LA neurons with activated CREB (pCREB) relative to home-cage (HC), CS alone (6h), or immediate shock (6h, Imm shock); F(6,21) = 12.53, *P < 0.001. (Bottom) Left panel: LA. Scale bar, 400 μm. Right panels: pCREB staining, Scale bar, 25 μm. n = 3 to 5 mice per group. (B) Schematic of hypothesized engram interaction. Event 1 transiently increases CREB function and excitability in a population of LA neurons (purple). If event 2 occurs when these neurons have elevated CREB and excitability (6h), then engrams are coal-located, memories are linked (purple+orange neurons), and event 2 memory is enhanced. If event 2 occurs later (24h), neurons activated by event 1 are no longer more excitable (perhaps they are in a “refractory-like period”), engrams are disallocated to nonoverlapping neurons, and memories are distinct.

Fig. 3.

Fig. 3.. Bidirectionally manipulating excitability to force coallocation or disallocation reveals competition governing engram interaction.

(A) HSV-NpACY was used to excite (ChR2) and inhibit (NpHR3.0) the same neurons. (Bottom) Yellow fluorescent protein (YFP) expression. LA, lateral amygdala; BA, basal amygdala. Scale bars, 400 μm (left), 150 μm (right). (B) Exciting HSV-NpACY neurons immediately (but not 24 hours) before event 1 [blue light (BL+)] allocates them to the engram; inhibiting them [red light (RL+)] during the test decreased CS freezing versus no red light (RL−). There was no effect of inhibiting random neurons (BL−). Group × Test condition, F(3,28) = 17.41, *P < 0.001. n = 7 to 9 mice for all groups. (C) Exciting neurons before event 1 (BL+) allocates them to event 1 engram (*P < 0.001) and coallocates them to event 2, if event 2 occurs 6 hours, but not 24 hours, later. (D) Exciting the same neurons before event 2 (24h/BL+, yellow circle) forces coallocation, F(2,20) = 17.91, *P < 0.001. n = 8 for 6-hour, n = 7 for 24-hour, and n = 8 for 24-hour/BL+ groups. (E) Exciting neurons before event 1, but inhibiting them during event 2 (RL+, yellow circle), impairs event 2 memory, if event 2 occurred 6 hours, but not 24 hours, later. F(1,16) = 5.63, *P < 0.05. n = 7 for 24-hour and n = 11 for 6-hour groups.

Fig. 4.

Fig. 4.. Competition-mediating engram interaction depends on excitation-inhibition.

(A) Fear conditioning transiently increases perisomatic PV immunolabeling (green) surrounding LA principal neurons relative to home-cage (HC), CS alone (6h), or immediate shock (6h, Imm shock); *P < 0.001. (Right) High or low perisomatic PV, with or without DAPI-labeled nuclei (blue, perisomatic region outlined). Scale bar, 10 μm. _n_ = 3 mice per group. Arrows indicate perisomatic PV immunolabel in example cells designated “high PV”or “low PV.”(**B**) Event 2 memory is impaired (Fig. 3E) if events are separated by 6 hours and neurons allocated to event 1 are silenced (RL+) during event 2. Inhibiting PV interneurons (hM4Di+CNO) permits event 2 memory formation, group difference only, _F_(3,23) = 22.61, *_P_ < 0.001. (Right) HSV-NpACY (YFP, green), AAV-hM4Di (mCherry, red). Scale bar, 200 μm. _n_ = 4 to 8 mice for all groups. (**C**) HSV-infected neurons inhibited (RL+) during event 1 are not allocated to event 1 engram; *_P_ > 0.05. Exciting these neurons before event 2 (BL+) allocates them to event 2 engram, F(1,7) = 51.02, *P < 0.001. n = 8 mice. (D) Event 2 memory is enhanced if CS1 is recalled 6 hours, but not 24 hours, before event 2; F(2,29) = 5.28, *P < 0.05. n = 8 to 12 mice for all groups.

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