Excitability mediates allocation of pre-configured ensembles to a hippocampal engram supporting contextual conditioned threat in mice

Abstract

Little is understood about how engrams, sparse groups of neurons that store memories, are formed endogenously. Here, we combined calcium imaging, activity tagging, and optogenetics to examine the role of neuronal excitability and pre-existing functional connectivity on the allocation of mouse cornu ammonis area 1 (CA1) hippocampal neurons to an engram ensemble supporting a contextual threat memory. Engram neurons (high activity during recall or TRAP2-tagged during training) were more active than non-engram neurons 3 h (but not 24 h to 5 days) before training. Consistent with this, optogenetically inhibiting scFLARE2-tagged neurons active in homecage 3 h, but not 24 h, before conditioning disrupted memory retrieval, indicating that neurons with higher pre-training excitability were allocated to the engram. We also observed stable pre-configured functionally connected sub-ensembles of neurons whose activity cycled over days. Sub-ensembles that were more active before training were allocated to the engram, and their functional connectivity increased at training. Therefore, both neuronal excitability and pre-configured functional connectivity mediate allocation to an engram ensemble.

Keywords: activity tagging; allocation; calcium imaging; contextual fear conditioning; engram; excitability; hippocampus; memory; miniature endoscope.

Figure 1.. Optogenetically increasing the excitability of a sparse population of dCA1 pyramidal neurons biases their allocation to an engram supporting contextual threat memory

(A) Schematic of allocate-and-silence strategy. Sparse subset of dCA1 pyramidal neurons express NpACY construct (red) containing both a blue light (BL)-responsive excitatory opsin (ChR2(H134R)) and a red light (RL)-responsive inhibitory opsin (eNpHR3.0). In Allocated group (Alloc), NpACY+ neurons excited with BL immediately before contextual threat conditioning, biasing NpACY+ neurons to become Engram neurons. In Control group, activity of NpACY+ neurons not experimentally manipulated before conditioning. Memory recall (freezing) assessed with No Light and when NpACY+ cells are inhibited with RL. (B) Inhibiting NpACY+ neurons disrupted freezing only in Allocated (BL before Training), not Control, mice. Two-way ANOVA, BL before Training × RL during Test, F(3, 24) = 6.74, p = 0.0019, N = 5-7 per group. **: p < 0.01, by Tukey HSD post-hoc. Data are mean ± SEM unless otherwise specified. Dots represent individual mice. (C) Schematic of scFLARE2 tag-and-silence strategy. Mice expressing scFLARE2 and TRE-eNpHR3.0 such that in the presence of BL active neurons are tagged and express eNpHR3.0. BL delivered either during Training (Training) or in Homecage 10min (10m), 3h or 24h before Training. Control mice did not receive BL (BL−). Memory recall assessed with No Light and when tagged neurons optogenetically inhibited with RL. (D) Inhibiting highly active neurons tagged by scFLARE2 during Training or in the minutes (10m and 3h), but not 24h, before Training disrupts memory recall. RL does not disrupt freezing in Control (BL−) mice. (E) Quantification of (D). Difference in percent freezing between No Light and RL Inhibition conditions for each experimental group (24h, 3h, 10m, Training, -BL). Each dot represents change in freezing per mouse. One-way ANOVA, F(4, 15) = 6.44, p = 0.0032, N = 7 per group. *: p < 0.05, one-sample t-test, Holm-Sidak correction for multiple comparisons. Red line indicates no change in freezing with RL. (F) Schematic of allocate-and-record strategy. Sparse subset of dCA1 pyramidal neurons express both RL-responsive excitatory opsin (ChRmine) and calcium indicator (GCaMP6m). In Allocated mice, GCaMP+ neurons are excited with RL immediately before Training, biasing them to become Engram neurons. In Control mice, GCaMP+ neurons are not experimentally manipulated before training. During memory recall test, freezing behavior measured and GCaMP fluorescence recorded from Allocated or Control neurons. (G) Example GCaMP traces relative to freezing bout onset (dashed line) during memory test in Allocated (RL+) and Control (RL−) mice. (H) Time-course of normalized GCaMP6 fluorescence in Allocated (Alloc) and Control neurons relative to mouse freezing bout onset. Number of freezing bouts = 142 and 144 in Allocated and Control mice, respectively. (I) Fluorescence (arbitrary units, a. u.) higher in Allocated than Control neurons in 2 sec before freezing bout onset, but not during freezing bouts, p < 0.05, Mann-Whitney U test.

Figure 2.. The activity of stably functionally connected neurons changes over days and those with highest activity on the day of conditioning are allocated to become Engram neurons as assessed by 2P imaging

(A) Schematic of strategy to image activity of TRAPed (tagged) Engram neurons before contextual threat Training using 2P imaging in head-fixed mice. TRAP2 transgenic mice express GCaMP7f in dCA1 neurons and mCherry in neurons active during Training (via 4-OHT injection). Neuronal activity of GCaMP+ neurons [either Engram (mCherry+) or Non-Engram (mCherry−)] imaged for 5 days before Training. (B) Examples of tagged (mCherry+) neurons. Top row, raw mCherry signal after 4-OHT treatment (left), background subtracted mCherry signal (middle), and spatial footprints of tagged Engram neurons. Middle and bottom rows, examples of spatial footprints of neurons classified as tagged with background-subtracted mCherry signal shown below. (C) Fluorescence AUC (area under curve) of Engram (blue) and Non-Engram (gray) neurons across baseline days, normalized to each neuron’s average AUC across all baseline sessions. Linear mixed effects model, Engram × Day, p = 0.015, N = 4 mice. ***: p < 0.001, linear mixed effects model between Engram and Non-Engram neurons, Holm-Sidak correction. (D) Relative functional correlation strength between Engram and Non-Engram neurons across baseline days. Linear mixed effects model, Engram × Day, p = 0.97, Engram main effect, p < 0.001. N = 56-71 Engram, 752-1003 Non-Engram neurons per session, N = 4 mice. *: p < 0.05, **: p < 0.01, linear mixed effects model between Engram and Non-Engram neurons, Holm-Sidak correction.

Figure 3.. Neurons show above-chance levels of functional connectivity and those with high activity in the homecage 3h, but not 24h, before training are allocated to Engram ensemble as assessed by 1P imaging

(A) Schematic of strategy to image activity of Engram and Non-Engram neurons in Homecage before and during Training and in Memory Tests using 1P imaging in freely-behaving mice. Top panel. Transgenic mice expressing GCaMP6f in dCA1 hippocampal neurons imaged in Homecage 24h (24h-Pre), 3h (3h-Pre), and Immediately (Imm-Pre) before contextual threat training (5 min context exposure followed by 3 footshocks spaced 1-min apart). Memory assessed in Training context (Ctx A Test) and novel context (Ctx B). Bottom panel. Examples of calcium activity traces. (B) Mice showed contextual threat memory, freezing at higher levels when replaced in the training context during Test than during Training [both before (Pre-S. Training) or after (Post-S. Training) shock delivery] or when tested in novel Ctx B. One-way repeated measures ANOVA, F(3, 21) = 29.4, p < 0.001. *: p < 0.05, **: p < 0.01, Tukey HSD post-hoc test. N = 8 mice. Data are mean ± SEM unless otherwise specified. Dots represent individual mice. (C) Classification of neurons detected in the Test as Engram vs. Non-Engram neurons based on average transient rate during Test. Classified as Engram neurons if average transient rate in Test exceeds 50th percentile (z-score > 0), and Non-Engram if not. (D) Proportion of all imaged neurons Detected and Not detected in Test. Of neurons Detected in Test, 46% categorized as Engram neurons. Neurons with lower activity in Test (z-score < 0) and neurons Not detected in Test categorized as Non-Engram neurons. Engram neurons constitute 9% of total neurons detected throughout experiment. (E) Average time-course of transient probability in Engram (blue) and Non-Engram (gray) neurons relative to freezing bout onset during Ctx A memory Test (all freezing bouts aligned to Time 0). n = 135 Engram neurons, 149 Non-Engram neurons, N = 6 mice. Error bars represent +/− SEM. (F) During training, Engram neurons show greater shock information than Non-Engram neurons. Left, example firing fields of shock-coding neurons. Right, average normalized shock information during Training of Engram and Non-Engram neurons. Linear mixed effects model, p = 0.06, n = 135 Engram neurons, 149 Non-Engram neurons, N = 6 mice. (G) During training, Engram neurons show greater spatial information than Non-Engram neurons. Left, example firing fields of place-coding neurons. Right, Engram neurons show higher average normalized spatial information than Non-Engram neurons. Linear mixed effects model, p = 0.02, n = 135 Engram neurons, 149 Non-Engram neurons, N = 6 mice, data are mean ± SEM. (H) Engram neurons show higher relative transient rates than Non-Engram neurons in Training, immediately and 3h before Training (Imm-Pre, 3h-Pre), but not 24h before Training (24h-Pre) in homecage. Linear mixed effects model, Engram × Session, p = 0.011, n = 81-149 Engram neurons, 510-983 Non-Engram neurons per session, N = 6 mice. *: p < 0.05, **: p < 0.01, ***: p < 0.001, by pairwise linear mixed effects model with Holm-Sidak post-hoc correction. (I) Test patterns of pairwise functional connectivity in both Engram and Non-Engram neurons greater than chance (gray line, circular shuffle) across sessions. For all neurons detected in Test, correlation computed between pattern of pairwise functional connectivity for that neuron in Test and session of interest. In all sessions, both Engram and Non-Engram neuronal functional connectivity showed higher than chance similarity to Test session, ***: p < 0.001 different than circular shuffle in a linear mixed effects model, n = 81-149 Engram neurons, 510-983 Non-Engram neurons per session, N = 6 mice. No difference between Engram and Non-Engram neurons for any session.

Figure 4.. Selective stabilization of Engram ensembles by training

(A) Schematic of strategy to examine functional neuronal connectivity in high dimensions. Activity of neuronal population imaged during Test decomposed into sub-ensembles of functionally-connected neurons (sub-ensemble vector) and their corresponding time courses of activation (sub-ensemble activation) using non-negative matrix factorization (NMF). Sub-ensembles classified as either Engram or Non-Engram based on average transient rate during Test. Activity pattern of Engram and Non-Engram sub-ensembles observed during Test compared to other imaging sessions before (24h-Pre, 3h-Pre, Imm-Pre) Training, during Training and novel Ctx B Test. (B) Test neuronal population activity for one mouse decomposed into sub-ensembles as described above. For each sub-ensemble, activity of each significant member neuron is plotted. Each row separated by dashed lines represents a single sub-ensemble. Time bins with significant sub-ensemble activation are colored. (C) Engram sub-ensembles identified in Test (blue) showed greater activation during Training, but not in Homecage or Ctx B, than Non-Engram sub-ensembles (gray), suggesting structure of Engram sub-ensembles specifically stabilized during Training. n = 12-22 Engram sub-ensembles, 69-102 Non-Engram sub-ensembles per session, N = 6 mice. **: p < 0.01, by pairwise linear mixed effects model with Holm-Sidak post-hoc correction. (D) Time-course of Engram and Non-Engram sub-ensemble activation during Training in 1-min time bins. Highest activation of Engram sub-ensembles identified during Test occurred after footshock delivery. n = 22 Engram sub-ensembles, 102 Non-Engram sub-ensembles per session, N = 6 mice. *: p < 0.05, ***: p < 0.001, by pairwise linear mixed effects model with Holm-Sidak post-hoc correction. (E) Model of sub-ensemble allocation during contextual threat memory acquisition and recall. Average excitability of pre-existing neuronal sub-ensembles changes over days. During training, sub-ensembles that happen to be highly excitable, either by chance or previous memory recall, are allocated to the Engram ensemble supporting that memory. Functional connectivity of neurons in an Engram ensemble observed during Training is selectively stabilized by synaptic plasticity, such that the pattern of activity in Engram ensembles is reinstated by retrieval cues during memory recall Test.