The role of neuronal excitability, allocation to an engram and memory linking in the behavioral generation of a false memory in mice

Abstract

Memory is a constructive, not reproductive, process that is prone to errors. Errors in memory, though, may originate from normally adaptive memory processes. At the extreme of memory distortion is falsely "remembering" an event that did not occur. False memories are well-studied in cognitive psychology, but have received relatively less attention in neuroscience. Here, we took advantage of mechanistic insights into how neurons are allocated or recruited into an engram (memory trace) to generate a false memory in mice using only behavioral manipulations. At the time of an event, neurons compete for allocation to an engram supporting the memory for this event; neurons with higher excitability win this competition (Han et al., 2007). Even after the event, these allocated "engram neurons" remain temporarily (~6 h) more excitable than neighboring neurons. Should a similar event occur in this 6 h period of heightened engram neuron excitability, an overlapping population of neurons will be co-allocated to this second engram, which serves to functionally link the two memories (Rashid et al., 2016). Here, we applied this principle of co-allocation and found that mice develop a false fear memory to a neutral stimulus if exposed to this stimulus shortly (3 h), but not a longer time (24 h), after cued fear conditioning. Similar to co-allocation, the generation of this false memory depended on the post-training excitability of engram neurons such that these neurons remained more excitable during exposure to the neutral stimulus at 3 h but not 24 h. Optogenetically silencing engram neurons 3 h after cued fear conditioning impaired formation of a false fear memory to the neutral stimulus, while optogenetically activating engram neurons 24 h after cued fear conditioning created a false fear memory. These results suggest that some false memories may originate from normally adaptive mnemonic processes such as neuronal excitability-dependent allocation and memory linking.

Keywords: Allocation; Engram; False memories; Fear; Memory; Mice; Neuronal excitability.

Fig. 1.

Depiction of neuronal allocation, memory linking and false memory creation hypothesis. (A) Memories of two similar events occurring in close temporal proximity become linked by virtue of neuronal allocation to overlapping engrams (termed co-allocation) (from Rashid et al., 2016). Lateral amygdala (LA) neurons with relatively increased excitability (orange outline) during Event1 (CS1 + footshock) become allocated to engram supporting Event1 memory (neurons filled green). These allocated engram neurons remain relatively more excitable than neighbors for ~6 h post-training (orange outline), before becoming less excitable than their neighbors (~24 h post-training, red outline). If a similar Event2 (CS2 + footshock) occurs shortly after Event1 (within 6 h time-period of heightened engram neuron excitability), Event1 engram neurons also become allocated to Event2 engram (co-allocation, neurons filled with blue + green), linking the memories for Event1 and Event2. However, if Event2 occurs > 6 h after Event1, a distinct population of neurons is allocated to Event2 engram (blue neurons), the engrams are dis-allocated and the two memories are remembered separately. (B). We examined whether neuronal allocation mechanisms underlying memory linking could be hijacked to create a false fear memory. Either 3 h or 24 h post-cued fear conditioning (CS1 + footshock), mice were presented with motivationally-neutral tone CS (CS2). We examined whether a false fear memory to this tone CS was created in the 3 h, but not 24 h, group.

Fig. 2.

Behavioral generation of a “false fear memory” in mice using two auditory stimuli. (A) 3 h or 24 h post-auditory fear conditioning (CS1 + footshock), a motivationally-neutral CS2 tone was presented in a new context (without footshock). Freezing to CS1 (real conditioned fear memory) and CS2 (false memory) subsequently tested in novel contexts. (B) Similar freezing to novel auditory CS2 (CS2 Exposure) presented either 3 h (n = 11) or 24 h (n = 14) post-auditory CS1 + footshock fear conditioning. During memory tests, 3 h mice froze more to CS2 than 24 h mice, but both groups froze at similar levels to CS1. (C) During a memory test, mice presented with CS2 24 h after fear conditioning froze at similar levels to mice first exposed to CS2 during the memory test (never exposed, NE; n = 6). All groups froze similarly to original CS1. Data represent mean ± SEM unless otherwise specified.

Fig. 3.

Behavioral generation of a “false fear memory” in mice using visual and auditory stimuli. (A) 3 h or 24 h after light fear conditioning (CS1 + footshock), mice presented with novel motivationally-neutral CS2 tone without footshock. Freezing assessed upon re-exposure to CS1 (real conditioned fear memory) and CS2 (false memory). (B). During the memory tests, 3 h mice (n = 12) froze more to CS2 than 24 h mice (n = 10). No differences in freezing during initial CS2 exposure, to CS1 in memory test, or pre-CS.

Fig. 4.

Verifying the “allocate-and-manipulate” strategy using behavior. (A) HSV-NpACY construct, expressing both blue light (BL)-responsive excitatory opsin channelrhodopsin [ChR2(H134R)-eYFP] and red light (RL)-responsive inhibitory opsin halorhodopsin (eNpHR3.0), allowed optogenetic activation or inhibition of the same population of infected neurons. Robust expression of NpACY in small population of pyramidal neurons (eYFP) in lateral amygdala (LA) but not basal amygdala (BA). (B) Design of behavioral experiment verifying optogenetic biasing of NpACY-expressing neurons to engram supporting auditory fear memory. Mice expressing HSV-NpACY in random LA neurons (neuron outlined in purple) were fear conditioned (CS + footshock) either immediately after BL photostimulation (to activate ChR2, increase the excitability of these neurons [neuron outlined in orange] and bias their allocation to the underlying engram [green filled neuron], BL+ before training, allocated), or not (BL− before training, non-allocated). Memory was assessed by presenting the tone CS either in the absence (RL−) or presence (RL +) of RL to inhibit the activity of NpACY-expressing neurons [neuron outlined in red]. (C) During memory test, both groups (BL+, n = 9, BL−, n = 9) froze similarly to the CS in RL− condition, but BL+ group froze less when NpACY-expressing neurons were inhibited with RL, verifying that optogenetically “priming” neurons biased their allocation to engram supporting conditioned fear memory.

Fig. 5.

Verifying the “allocate-and-manipulate” strategy using activity-dependent gene expression. (A) Effects of optogenetically activating neurons alone or before fear conditioning on neuronal activity were examined using fos expression. Three groups expressing HSV-NpACY (green filled neurons) were used (n = 3). ¹⁾ BL + FC mice received BL immediately before fear conditioning (FC) to increase the excitability of NpACY-expressing neurons (neuron outlined in orange). ²⁾ FC mice were fear conditioned without BL. ³⁾ BL+ mice received BL but were not fear conditioned. (B) Histology showing co-localization between GFP+ (NpACY-expressing) neurons with fos-expressing neurons (red). (C) Similar number of NpACY-infected (GFP+) neurons across groups, but more fos-expressing neurons in BL + FC and FC groups. (D) High overlap of c-fos+ and GFP+ neurons in BL + FC mice, little overlap in BL+ alone mice, indicating BL + FC biased allocation to neurons active during training (proxy of engram) and that BL+ activation of a small population of neurons not sufficient to induce c-fos expression.

Fig. 6.

Verifying the “allocate-and-manipulate” strategy using engram tagging technology. (A) To examine the effects of optogenetically activating a small random number of neurons alone or before fear conditioning on allocation to an engram, engram cells (those active during fear conditioning) were tagged using AAV-RAMGFP (Sørensen et al., 2016). In the absence of doxycycline (DOX), AAV-RAM-GFP labels with GFP active engram neurons during fear conditioning. 3 groups expressing both AAV-RAM-GFP (green outlined neuron, referred to as R-GFP) and HSV-ChR2-mCherry (red filled neuron) were used (n = 3). ¹⁾ BL + FC mice received BL immediately before fear conditioning. ²⁾ FC mice were fear conditioned without BL. ³⁾ BL+ received BL but were not fear conditioned. Control (CTL) mice expressed HSV-ChR2-mCherry and control AAV virus expressing GFP in a non-activity-dependent manner (rather than RAM vector, referred to as GFP). (B) Histology showing overlap of GFP+ and mCherry+ neurons in different experimental groups. Note R-GFP is derived from AAV-RAM-GFP whereas GFP in CTL condition derived from AAV-GFP. (C) Similar number of neurons expressing mCherry across conditions verified similar expression of ChR2-mCherry across groups. High numbers of neurons expressing R-GFP except in mice that were not fear conditioned (BL+) indicated similarly-sized engrams across conditions. (D) Higher overlap (GFP+, mCherry+) in BL + FC mice than in mice that received FC alone, which was not different from the control “floor” condition (CTL). Together, these finding verify this “allocate-and-manipulate” approach to target engram neurons after a training event.

Fig. 7.

Using “allocate-and-manipulate” strategy to examine neuronal mechanisms underlying creation of a false memory. (A) Experimental timeline. 3 h condition (top). We hypothesized a false memory was generated if CS2 was presented 3 h post-fear conditioning because engram neurons remain more excitable for several hours. To test this, we first allocated neurons to Event1 engram (BL before CS1 + footshock training) in mice expressing NpACY. During CS2 exposure 3 h later, at a time when NpACY-expressing engram neurons were hypothesized to be more excitable than their neighbors, NpACY-expressing neurons were inhibited (RL) (3 h-NpACY-RL). Control mice were treated similarly, but received BL (not RL) during CS2 exposure (3 h-NpACY-BL) or expressed TdTomato (not NpACY) (3 h-TdTomato-RL). 24 h condition (bottom). NpACY-expressing neurons were allocated to Event1 engram with BL. During CS2 exposure 24 h later, at a time when infected engram neurons are hypothesized to be less excitable than their neighbors, NpACY-expressing neurons were excited (BL) (24 h-NpACY-BL). Control mice received RL (not BL) during CS2 exposure (24 h-NpACY-RL) or expressed TdTomato (not NpACY) (24 h-TdTomato-BL). (B) CS2 freezing 3 h Condition. Optogenetically inhibiting, but not exciting, engram neurons, impaired creation of false memory to CS2 when presented 3 h post-fear conditioning (TdTomato RL, n = 11; NpACY-RL, n = 11; NpACY-BL, n = 14). 24 h Condition. Optogenetically activating, but not inhibiting, engram neurons, facilitated creation of a false memory to CS2 when presented 24 h post-fear conditioning (TdTomato-BL, n = 10; NpACY-BL, n = 8; NpACY-RL, n = 13). These findings agree with our hypothesis outlined in Fig. 1B. CS1 freezing. All groups froze similarly to the trained CS1 except for 3 h-NpACY-RL condition; inhibiting allocated neurons 3 h post-training disrupted long-term memory formation.

Fig. 8.

False memory formation depends on excitability-mediated neuronal co-allocation. (A) Experimental timeline. (Top) BL+ before training (Allocated). NpACY-expressing neurons were allocated to CS1 + footshock engram via BL. Mice exposed to CS2 3 h later without photostimulation. Memory (CS1 and CS2) was assessed when NpACY-expressing neurons were (and were not) inhibited (RL+ vs. RL−). (Bottom) BL− before training (Non-allocated). Control mice treated similarly, but did not receive BL before CS1 + footshock training, such that infected neurons were not preferentially allocated to the engram. (B) Schematic depicting NpACY-expressing neurons (neurons outlined with purple) being preferentially allocated to CS1 + footshock engram (green filled neurons) via BL stimulation (to increase excitability, orange outline). 3 h later, during CS2 exposure, allocated engram neurons are thought to remain more excitable than their neighbors (orange outlined neurons) and co-allocate CS2 representation to overlapping neurons (green and blue neurons). By virtue of co-allocation, decreasing activity of NpACY-expressing neurons via RL (red outlined neurons) would decrease freezing to both CS1 and CS2 during memory test. (C) During CS1 and CS2 test, mice in experimental allocation group (n = 11) froze less during RL+, than RL−, suggesting NpACY-expressing neurons supported both real and false fear memories. Non-allocated mice (n = 9) showed equivalent freezing in RL+/− conditions, indicating inhibition of a small number of LA neurons failed to impact freezing.