Development of a novel viral tool to study early changes in the properties of CA3 engram cells.

(A) Scheme representing the maturation of engram neurons during memory formation. Top panel: during learning, a neuron receives numerous synaptic inputs, resulting in sequences of action potentials. This activity triggers the rapid expression of immediate early genes (IEGs), marking the neuron as part of an engram (yellow). As the engram matures (green), IEGs expression is not detectable anymore, yet the neuron retains established engram properties. Bottom panel: expression function of IEG-based construct to investigate and compare early and late engram properties. A construct with rapid offset (yellow) best captures engrams shortly after learning, whereas a progressively expressing construct best captures more mature engrams. (B) FLEN construct (c-Fos.ZsGreen1-DR) encapsulated in AAV2/9 vector. Bottom panel: representative images showing ZsGreen1 fluorescence in c-Fos+ neurons (arrowhead) and c-Fos-neurons (arrow). Scalebar = 50 µm. (C) Timeline of in vitro experiments to determine FLEN expression and offset time course. After infection (DIV3) and activation (DIV11), samples are washed with a blocking medium and images are acquired at different intervals (downward arrows). (D) Percentage of ZsGreen+ vs. all transduced neurons (mCherry+ cells) at the different intervals following activation. (E) Representative images of activated cultured neurons at 1, 2, 3, 4.5 and 6 hours after activation. Scalebar = 100 µm.

In vivo FLEN labeling and decay time course following a salient experience.

(A) FLEN and an infection marker viral vectors are injected bilaterally into CA3. Mice undergo contextual fear conditioning (CFC), and sections are collected (downward arrows) 1.5, 3, 4.5, 6 and 12 hours following CFC. As a control, sections are obtained from mice left undisturbed in their home cage (HC). (B) Left: one-time CFC experiment scheme. Mice explore an arena for 5 minutes, 3 foot-shocks are given at 180, 210 and 270 seconds after the start of the test (red line). Top-right: paired comparison of the percentage of time mice spent freezing during training and when replaced in the conditioning arena 1 hour after CFC. Bottom-right: comparison of the percentage of time mice spent freezing during training, when placed in a different neutral environment and 24 hours after CFC. These experiments show that the CA3-dependent shock-context association is rapidly learned (within 1 hour), is highly environment-selective and it is retained for 24 hours. (C) Representative images of CA3 neurons expressing the infection marker (mCherry+, indicated by arrow) and neurons expressing both the infection marker and FLEN (ZsGreen1+/mCherry+, indicated by arrowhead). The latter are considered FLEN+ neurons. Scalebar = 50 µm. (D) Percentage of ZsGreen+ CA3 neurons over the total number of mCherry+ neurons at different intervals following activation. (E) Representative sections of CFC-trained mice compared to HC untrained mice at different intervals following CFC. Dashed lines outline the dentate gyrus (DG) cellular layer, while the dashed segment indicates the separation between CA3 and CA2. Scale bar = 100 µm. (F) Left panel: Schematic representation of neuronal position along the proximodistal axis analysis of CA3. The CA3 pyramidal layer was straightened and the x position of FLEN+ cells was normalized on a 0 (closer to DG) to 1 (closer to CA2) scale. Right panel: comparison of normalized proximodistal frequency distribution of CA3 FLEN+ cells placement in HC mice (gray) and 3 hours post-CFC mice (yellow). Bold lines represent the median of the respective distributions. HC median is displayed in the CFC plot to highlight distribution difference.

Electrophysiological analysis of intrinsic properties of CA3 engram neurons.

(A) Experimental outline: mice were injected with either FLEN or RAM construct, subjected to CFC and then sacrificed at either 3- or 24-hours post-conditioning to collect acute hippocampal slices. Right: representative images of FLEN+ and RAM+ cells targeted for patch clamp recordings, with neighboring control FLEN- or RAM-neurons. Scale bar = 20 µm. (B) Membrane potential response. Left panel: representative membrane potential trace in response to incremental injected current in control cells (gray) and engram cells (yellow for FLEN and green for RAM). Right panel: graphs of input resistance for comparison between engram cells and their corresponding control cells. (C) Action potential firing pattern. Left panel: sample traces of long (1 second) above-rheobase current step to analyze the pattern of action potential firing current in control cells (gray) and engram cells (yellow and green). Right panel: average spike frequency graphs. (D) Cumulative spike frequency curve over the 1 second current step shown in (C) showing a similar distribution between FLEN+ and FLEN – neurons (top), and a more sustained firing of RAM+ compared to their RAM-neurons (bottom).

Analysis of EPSCs and IPSCs in CA3 engram neurons

(A) Sample traces and cumulative distributions of sEPSCs recorded from CA3 engram neurons (FLEN in yellow, RAM in green). Top panels: comparison between FLEN⁺ and FLEN⁻ neurons reports no difference in mean sEPSC amplitude cumulative distribution but a higher event frequency in FLEN⁺ neurons, as seen in the inter-event interval (IEI) cumulative distribution. Inset displays the comparison of the average frequency (Hz) of such events. Bottom panels: RAM⁺ vs RAM⁻ neurons show a similar pattern characterized by no change in amplitude and increased frequency in RAM⁺ neurons. (B) Same analysis as in (A), for miniature EPSCs (mEPSCs). The inset in the amplitude plot shows the fraction of mEPSCs exceeding 40 pA, indicative of giant mossy fiber events. (C) As (A) and (B), for miniature IPSCs (mIPSCs), comparing frequency and amplitude in FLEN+ vs FLEN-and RAM+ vs, RAM-neurons.

Properties of Mf-CA3 synapses in CA3 engram neurons

(A) Schematic representation of Mf-CA3 EPSCs and feedfoward inhibition recordings. (B) Left panel: representative traces showing EPSC amplitude in FLEN-(gray) and FLEN+ (yellow) cells following low frequency stimulation (0.1 Hz) and corresponding increase evoked with a higher stimulation frequency (1 Hz), due to a presynaptic form of synaptic plasticity characteristics of Mf-CA3 synapses (frequency facilitation). Center panel: amplitude plot for FLEN-and FLEN+ stimulated at 0.1 Hz and 1 Hz. Right panel: frequency facilitation ratio plot shows no difference between FLEN-and FLEN+ neurons. (C) Left panel: representative traces showing Mf-driven IPSCs (feedforward inhibition) and EPSCs (direct excitatory activity) recorded in individual FLEN-(gray) and FLEN+ (yellow) neurons. Center panel: amplitude plot of EPSCs and IPSCs for FLEN- and FLEN+ neurons. Right panel: graph showing excitation-to-inhibition ratio, comparing FLEN- and FLEN+ neurons.