Journal: Frontiers in Behavioral Neuroscience

Article Title: Arc-driven mGRASP highlights CA1 to CA3 synaptic engrams

doi: 10.3389/fnbeh.2022.1072571

Figure Lengend Snippet: cFostTA-dependent expression of mGRASP in dCA1 upon exposure to an Enriched Environment or Trace Fear Conditioning. Schematic description of the viral injection sites in WT animals (A) , the viral constructs injected (B) and the experimental design (C) to determine the expression kinetics of post-mGRASP in dCA1 under the control of cFos upon exposure to EE or HC. (D) Confocal pictures, single Z-planes, of dCA1 at different time points after induction of post-mGRASP expression. Red, dTomato; blue, DAPI. White triangles indicate RFP-positive cells. D, Dorsal, V, Ventral. Scale bar = 20 μm. (E) Percentage of dTomato-expressing over DAPI-positive cells in dCA1 after exposure to EE or HC. The levels of dTomato reached a peak at 3 days ( p 3d–24h = 0.01, p 3d–5d = 0.7, p 3d–7d = 0.3, p 3d–10d = 0.037, p 3d–HC = 0.048; all n = 5; Kruskal–Wallis test after Dunn’s correction for multiple comparisons). (F) Schematic description of the experimental design to determine the dCA1 expression levels of post-mGRASP under the control of cFos upon exposure to TFC, EE, or HC. (G) Confocal pictures, single Z-planes, of dCA1 at different time points after induction of post-mGRASP. Red, dTomato; blue, DAPI. White triangles indicate dTomato-positive cells. D, Dorsal, V, Ventral. Scale bar = 20 μm. (H) The percentage of dTomato-expressing over DAPI-positive cells in dCA1 was not significantly different after exposure to TFC, EE or HC ( p TFC–HC = 0.41 and p EE–HC = 0.26, all n = 6; Kruskal–Wallis test after Dunn’s correction for multiple comparisons). (I) Percentage of freezing time during the context probe 3 days after TFC training ( p = 0.03, n = 6; Wilcoxon Signed-rank test). * p < 0.05.

Article Snippet: On the training day mice were put into a square conditioning chamber (19 cm × 19 cm, black metal walls, stainless steel grid floor, white light illumination, and ethanol odor) (Panlab) which we defined as Context A.

Techniques: Expressing, Injection, Construct, Control

Journal: Frontiers in Behavioral Neuroscience

Article Title: Arc-driven mGRASP highlights CA1 to CA3 synaptic engrams

doi: 10.3389/fnbeh.2022.1072571

Figure Lengend Snippet: ArcCre ERT2 -dependent expression of mGRASP in dCA1 upon exposure to an Enriched Environment or Trace Fear Conditioning. Schematic description of the viral injection sites in ArcCre ERT2 transgenic animals (A) and the viral constructs injected (B) . (C) Experimental design to determine the expression levels of post-mGRASP in dCA1 under the control of Arc at two different time points after exposure to EE. (D) Confocal pictures, single Z-planes, of dCA1 at different time points after induction of post-mGRASP expression. Red, dTomato; blue, DAPI. White triangles indicate dTomato-positive cells. D, Dorsal, V, Ventral. Scale bar = 20 μm. (E) Percentage of dTomato-expressing over DAPI-positive cells in dCA1 3 and 7 days after exposure to EE. The level of dTomato at 7 days was significantly higher than at 3 days ( p = 0.0095; n 3 = 4, n 7 = 6; Mann–Whitney U-test). (F) Percentage of dTomato-expressing over DAPI-positive cells in dCA1 3 and 7 days after exposure to EE in the cFostTA (striped) or ArcCre ERT2 (solid) systems. The level of dTomato at 7 days was significantly higher in the ArcCre ERT2 system than at 3 and 7 days in the cFostTA system ( p 7dArc7dFOS = 0.0005, p 7dArc3dFOS = 0.017, all other p > 0.31; n 7dArc = 6, n 7dFOS = 5, n 3dArc = 4, n 3dFOS = 10; Kruskal–Wallis test after Dunn’s correction for multiple comparisons). (G) Confocal pictures, single Z-planes, of apical (right) and basal (left) dendritic segments of dCA1 pyramidal neurons at 7 days after induction of post-mGRASP expression showing GFP-fluorescent puncta colocalizing with dTomato-positive dendritic segments. Red, dTomato; green, GFP. White triangles indicate synaptic GFP reconstitution. D, Dorsal, V, Ventral. Scale bars = 5 μm. (H) Experimental design to determine the dCA1 and dCA3 expression levels of post-mGRASP under the control of Arc after TFC. (I) Confocal picture, single Z-plane, of dCA1 7 days after induction of post-mGRASP expression. Red, dTomato; blue, DAPI. White triangles indicate dTomato-positive cells. D, Dorsal, V, Ventral. Scale bar = 20 μm. (J) Percentage of dTomato- (dCA1) or mCerulean- (dCA3) expressing over DAPI- (dCA1) or Syto60- (dCA3) positive cells 7 days after exposure to FC. (K) Percentage of freezing time during the context probe 7 days after TFC training ( p = 0.015, n = 7; Wilcoxon Signed-rank test). (L) Confocal pictures, single Z-planes, of a dCA1 pyramidal neuron and dendritic segments (insets) 7 days after induction of post-mGRASP expression showing GFP-fluorescent puncta colocalizing with dTomato-positive dendritic segments. Red, dTomato; green, GFP. White triangles indicate synaptic GFP reconstitution. Inset number 2 is rotated 90 degrees from the original orientation. Scale bars: left = 10 μm, middle and right = 5 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: On the training day mice were put into a square conditioning chamber (19 cm × 19 cm, black metal walls, stainless steel grid floor, white light illumination, and ethanol odor) (Panlab) which we defined as Context A.

Techniques: Expressing, Injection, Transgenic Assay, Construct, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Stability of excitatory structural connectivity predicts the probability of CA1 pyramidal neurons to become engram neurons

doi: 10.1101/759225

Figure Lengend Snippet: (A) Schematic description of the preparation and of the experiment‘s rationale. S.O., Stratum Oriens, S.P., Stratum Pyramidale; S.R., Stratum Radiatum; S.L.M., Stratum Lacunosum-Molecolare. EE, exposure to an Enriched Environment; TAM, Tamoxifen injection. Green, eGFP + neurons; red, tdTomato + neurons; yellow, eGFP / tdTomato double + neurons. (B) Experimental timeline. FC, trace fear conditioning memory task. (C) In vivo two-photon (2P) time-lapse of the same dendritic branch and spines over 14 days. Maximum intensity projections (MIPs) of up to 15 z-planes (Z step, 1 µm). Scale bar, 1 µm. (D) 2P images (MIPs of up to 5 z-planes, 2 µm z-step) of the dorsal CA1 of Arc-Cre ERT2 ; Ai9; Thy1-eGFP live mice. Scale bar, 20 µm. (E) Nex-Cre ERT2 ; Ai9; Thy1-eGFP; mice underwent the same experimental procedure as in A and B but were never exposed to EE. (F) 2P images (MIPs of up to 5 z-planes, 2 µm z-step) of the dorsal CA1 of Nex-Cre ERT2 ; Ai9; Thy1-eGFP live mice. Scale bar, 20 µm. ( G, H ) Spine densities of (G) ArcTom +, ArcTom -and (H) NexTom + and NexTom - neurons. Data points are mean density per neuron ( Table S2 for number of dendrites per neuron). Red: prospective or actual td-Tomato + neurons; grey: prospective or actual td-Tomato - neurons; solid circles: Arc-Cre ERT2 ; Ai9; Thy1-eGFP mice; empty circles: Nex-Cre ERT2 ; Ai9 Thy1-eGFP mice; black bars: means ± S.E.M; horizontal lines: median baseline densities per each group. Table S1 for p values. Blue vertical line, time point of EE+TAM injection; grey vertical line, time point of TAM injection.

Article Snippet: On the training day (Day 15) mice were put into a square conditioning chamber (19 cm x 19 cm, black metal walls, stainless steel grid floor, white light illumination, and ethanol odor) (Panlab) which we defined as Context A.

Techniques: Injection, In Vivo

Journal: bioRxiv

Article Title: Stability of excitatory structural connectivity predicts the probability of CA1 pyramidal neurons to become engram neurons

doi: 10.1101/759225

Figure Lengend Snippet: (A) Schematic description of the trace fear conditioning learning task. ( B, C ) Arc-Cre ERT2 ; Ai9; Thy1-eGFP (Arc, solid circles) and Nex-Cre ERT2 ; Ai9; Thy1-eGFP (Nex, open circles) mice displayed increased freezing during the context (B) and tone (C) probe trials. Circles are percentage freezing for each mouse during: 3 min habituation (day 15) and 3 min context probe (day 16)(B) or during 3 min baseline (day 16) and 4 min tone probe (day 16)(C); horizontal bars: means ± S.E.M. ( D - I ) Correlations between densities (D, E), time constants of the survival (F, G) and gain (H, I) rates of spines during baseline of Arc-Cre ERT2 ; Ai9; Thy1-eGFP (solid circles) and Nex-Cre ERT2 ; Ai9; Thy1-eGFP (open circles) and freezing to the context (D - F) and the tone (G - I). ( B - I ) See Table S1 for p values.

Article Snippet: On the training day (Day 15) mice were put into a square conditioning chamber (19 cm x 19 cm, black metal walls, stainless steel grid floor, white light illumination, and ethanol odor) (Panlab) which we defined as Context A.

Techniques: