Journal: Nature Communications

Article Title: Prefrontal chandelier cells encode stimulus salience to influence learning in male mice

doi: 10.1038/s41467-026-68959-3

Figure Lengend Snippet: a Schematics of AAV injections and experimental design for habituation (day 0, D0), trace fear conditioning (TFC, D1), and retention testing (Re, D2). CS conditioned stimulus, US unconditioned stimulus. b Quantification and comparison of freezing behavior during TFC and retention across experimental and control groups. BL baseline. Associative learning groups: paired (CS-US) (green, n = 8 mice); control groups: CS only (black, n = 10 mice) and US only (yellow, n = 6 mice). These group sizes apply to all subsequent panels in this figure. (CS-US vs CS only, P = 0.00005; CS-US vs US only, P = 0.0007; CS only vs US only, P = 0.75, Mann–Whitney test (two-sided)). c , d Group-averaged Ca²⁺ responses and quantification of mPFC ChCs to pre-exposure CS during habituation across groups (CS-US: P = 0.000048, paired two-sided t -test; CS only: P = 0.00195, Wilcoxon signed-rank test (two-sided); US only: P = 0.011, paired two-sided t -test). e–g Ca²⁺ responses of mPFC ChCs to CS during TFC and retention, showing learning-dependent enhancement in the paired group ( f F 1,16 = 11.83, P = 0.0034, two-way repeated-measures ANOVA; Sidak’s multiple comparisons test (two-sided): trial #5, P = 0.006; trial #6, P = 0.005. D2 Re: CS-US vs CS only, P = 0.000061; CS-US vs US only, P = 0.0057; CS only vs US only, P = 0.28; unpaired two-sided t -test. g CS-US: P = 0.0037; CS only: P = 0.45, paired two-sided t -test). h Quantification and comparison of ChC Ca²⁺ responses to US during TFC across groups ( h F 1,12 = 8.769, P = 0.012, two-way repeated-measures ANOVA; Sidak’s multiple comparisons test (two-sided): trial #4, P = 0.022; trial #5, P = 0.034; trial #6, P = 0.004. i CS-US: P = 0.84; US only: P = 0.02, paired two-sided t -test). Each line linking two open symbols represented the data from an individual mouse. Data are presented as mean ± SEM, with individual animals shown as open symbols; box plots indicate median, quartiles, and range. Statistics as indicated, *** P < 0.001, ** P < 0.01, * P < 0.05, ns is not significant. Source data are provided as a Source Data file.

Article Snippet: Neutral tone (8 kHz, 60 dB, 2 s) and electric foot shock (0.6 mA, 2 s) were delivered by the standard fear conditioning shock box (UGO BASILE SRL, Italy).

Techniques: Comparison, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Prefrontal chandelier cells encode stimulus salience to influence learning in male mice

doi: 10.1038/s41467-026-68959-3

Figure Lengend Snippet: a , b Schematic of the experimental design and trace fear conditioning paradigm with optogenetic inhibition during conditioning. c Group-averaged Z-scored Ca²⁺ traces (mean ± SEM) of mPFC ChCs aligned to CS or US across habituation (D0), conditioning (D1), and retention (D2) in AI eJaws and mRuby3 groups (left: AI eJaws, n = 8 mice; right: AI mRuby3, n = 8 mice, group sizes apply to all panels). CS, US, and light delivery periods are indicated in the figure. d , e Quantification and comparison of ChCs Ca²⁺ responses to the CS across D0-D2 and to the US during D1 in AI eJaws and AI mRuby3 groups ( d D0 CS, F 1, 14 = 0.2399, P = 0.6319, two-way repeated-measures ANOVA; D1 CS, F 1, 14 = 3.486, P = 0.0830; Sidak’s multiple comparisons test (two-sided): trial6, P = 0.0022. D1 US, F 1, 14 = 0.9478, P = 0.3468, two-way repeated-measures ANOVA. e AI mRuby3: D0 CS, Trial1 vs Trial6, P = 0.0001; D1 CS, Trial1 vs Trial6, P = 0.0007; D1 US, Trial1 vs Trial6, P = 0.12, paired two-sided t -test. AI eJaws: D0 CS, Trial1 vs Trial6, P = 0.00088; D1 CS, Trial1 vs Trial6, P = 0.28, paired two-sided t -test; D1 US, Trial1 vs Trial6, P = 0.012, paired two-sided t -test. AI eJaws vs AI mRuby3: D1 CS trial6, P = 0.00002; D2 CS, P = 0.012; D1 US trial6, P = 0.07, unpaired two-sided t -test). f–h Same as ( c–e ), but for PVT eJaws and PVT mRuby3 groups ( g D0 CS, F 1, 16 = 0.0074, P = 0.9325, two-way repeated-measures ANOVA; D1 CS, F 1, 16 = 1.929, P = 0.1839, two-way repeated-measures ANOVA; Sidak’s multiple comparisons test (two-sided): trial6, P = 0.0087; D1 US, F 1, 16 = 0.4206, P = 0.5259, two-way repeated-measures ANOVA. h PVT mRuby3: D0 CS, Trial1 vs Trial6, P = 0.00007; D1 CS, Trial1 vs Trial6, P = 0.00005, paired two-sided t -test; D1 US, Trial1 vs Trial6, P = 0.30, Wilcoxon signed-rank test (two-sided). PVT eJaws: D0 CS, Trial1 vs Trial6, P = 0.000075; D1 CS, Trial1 vs Trial6, P = 0.53; D1 US, Trial1 vs Trial6, P = 0.033, paired two-sided t -test. PVT eJaws vs PVT mRuby3: D1 CS trial6, P = 0.00045; D2 CS, P = 0.000003, unpaired two-sided t -test; D1 US trial6, P = 0.013, Mann–Whitney test (two-sided); PVT eJaws, n = 9 mice, PVT mRuby3, n = 9 mice). i Behavioral effects of optogenetic inhibition of AI inputs during TFC. Left: Freezing behavior across D1 (conditioning) and D2 (retention. F 1, 14 = 4.296, P = 0.0571, two-way repeated-measures ANOVA; Sidak’s multiple comparison test (two-sided): trial 4, P = 0.0375, trial 5, P = 0.0244, trial 6, P = 0.0089) Right: Comparison of freezing responses to CS during D2 (AI eJaws vs AI mRuby3, P = 0.014, unpaired two-sided t -test). j Same as ( i ), but for PVT inhibition (D1: F 1, 16 = 1.18, P = 0.29, two-way repeated-measures ANOVA; D2, PVT eJaws vs PVT mRuby3, P = 0.006, Mann–Whitney test (two-sided); PVT mRuby3 vs AI mRuby3, P = 0.77, unpaired two-sided t -test). Statistics as indicated, *** P < 0.001, ** P < 0.01, * P < 0.05, ns is not significant. Source data are provided as a Source Data file.

Article Snippet: Neutral tone (8 kHz, 60 dB, 2 s) and electric foot shock (0.6 mA, 2 s) were delivered by the standard fear conditioning shock box (UGO BASILE SRL, Italy).

Techniques: Inhibition, Comparison, MANN-WHITNEY

Journal: Nature Communications

Article Title: Prefrontal chandelier cells encode stimulus salience to influence learning in male mice

doi: 10.1038/s41467-026-68959-3

Figure Lengend Snippet: a Viral injection, fiber implantation, and representative NpHR expression in mPFC ChCs. b , c Behavioral effects of ChC inhibition during conditioning, applied as indicated (NpHR group (orange) vs EYFP group (black): D1 TFC, F 1,17 = 21.57, P = 0.0002, two-way repeated-measures ANOVA; Sidak’s multiple comparison test (two-sided): trial #3, P = 0.0087, trial #4, P = 0.0052, trial #5, P < 0.0001, trial #6, P = 0.0348. D2 Re, NpHR vs EYFP, P = 0.0047, unpaired two-sided t -test. NpHR, n = 11 mice; EYFP, n = 8 mice). c D1 TFC, F 1,16 = 11.69, P = 0.0035, two-way repeated-measures ANOVA; Sidak’s multiple comparison test (two-sided): trial #3, P = 0.0495, trial #6, P = 0.0153. D2 Re, NpHR vs EYFP, P = 0.0038, unpaired two-sided t -test. NpHR, n = 9 mice; EYFP, n = 9 mice). d , e Schematics of AAV injections and experimental design for chemogenetic manipulation of ChC activity, with representative images of GCaMP7s co-expressed with hM3Dq ( n = 7 mice) or hM4Di ( n = 9 mice). f , g Corresponding average traces and quantification of ChC Ca²⁺ responses to tone stimuli at baseline and after CNO ( f hM3Dq, P = 0.0029, paired two-sided t -test, n = 7 mice. g hM4Di, P = 0.0075, paired two-sided t -test, n = 9 mice). h Schematics and representative hM3Dq expression in mPFC ChCs. i , j Behavioral effects of chemogenetic activation or inhibition of ChCs during conditioning ( i CNO(hM3Dq) vs Saline, D1 TFC, F 1,22 = 10.97, P = 0.0032, two-way repeated-measures ANOVA; Sidak’s multiple comparison test (two-sided): trial #4, P = 0.0026, trial #5, P = 0.0011, trial #6, P = 0.0277. D2 Re: P = 0.0052, unpaired two-sided t -test. CNO(hM3Dq), n = 12 mice; Saline, n = 12 mice. j CNO(hM4Di) vs Saline, D1 TFC, F 1, 19 = 6.323, P = 0.0211, two-way repeated-measures ANOVA; Sidak’s multiple comparison test (two-sided): trial #4, P = 0.0194. D2 Re: P = 0.012, unpaired two-sided t -test. CNO(hM4Di), n = 10 mice; Saline, n = 11 mice). Statistics as indicated, ** P < 0.01, * P < 0.05. Source data are provided as a Source Data file.

Article Snippet: Neutral tone (8 kHz, 60 dB, 2 s) and electric foot shock (0.6 mA, 2 s) were delivered by the standard fear conditioning shock box (UGO BASILE SRL, Italy).

Techniques: Injection, Expressing, Inhibition, Comparison, Activity Assay, Activation Assay, Saline

Journal: bioRxiv

Article Title: Dopamine in the basal amygdala signals salient somatosensory events during fear learning

doi: 10.1101/716589

Figure Lengend Snippet: VTA neurons, and amongst them dopamine neurons, respond to footshocks and acquire CS - responsiveness. ( A ) Schematic showing an optrode with 16 recording channels implanted in the VTA of a DAT Cre × ChR2-eYFP mouse. ( B ) Post-hoc histological image showing the placement of an optrode (dashed line) in the VTA in the example mouse of panels B – H (FT7612). The tracks of two tetrodes are visible (arrows). Bottom, scheme of the design of four tetrodes (T1 - T4) around the optical fiber. ( C ) Illustration of optogenetic identification of putative DAT + units. Raster plot for four electrodes (E1-E4) of one tetrode, showing unsorted spikes aligned to the onsets of n = 100, 2 ms – long laser light pulses (blue shading). Spikes at 2-8 ms after the light pulse were collected and subjected to spike clustering (see Materials and Methods). ( D ) Time course of freezing of the example mouse during the three days of the fear learning protocol. ( E ) Spiking activity of a single opto-tagged putative DAT + unit during day 2 (training day). AP frequency (top) and z-score (bottom) are shown in response to the CS (averages over the n = 30 tone presentations for each tone block), as well as in response to footshocks (averages over the n = 6 footshock presentations; right). ( F ) Spiking activity of the same unit as (E), but for responses to CS presentations on day 3 (threat memory retrieval). Right, AP-waveforms for the unit shown in E, F. ( G, H ) Responses of all units in this mouse (FT7612) to tone presentations during day1 - day 3 (G), and to footshocks on day 2 (H). Z-scores were analyzed and plotted as a function of time (number of tone block). Units classified as CS - entrained are shown in pink, and the others in gray. Thick red and black lines show average ± S.E.M. across these groups, respectively. Square symbols connected by lines represent DAT + units identified by optotagging. ( I, J, K ) Venn-type diagrams showing the number of units in the different response classes and their overlap. The data in (I) is from the example mouse shown in panels B-H. Note the overall similar distribution and overlap of response types across n = 3 mice. Two US responsive units in mouse FT6963 showed a reduction in firing upon the footshock (J, dashed areas).

Article Snippet: A classical auditory cued threat memory paradigm was performed in a conditioning chamber of a NIR Video Fear Conditioning Optogenetics Package for Mouse (MED-VFC-OPTO-M, Med Associates Inc., VT, USA).

Techniques: Activity Assay, Blocking Assay

Journal: eNeuro

Article Title: Absence of VGLUT3 Expression Leads to Impaired Fear Memory in Mice

doi: 10.1523/ENEURO.0304-22.2023

Figure Lengend Snippet: Pattern separation of VGLUT3 –/– mice. A , Behavioral protocol. B , VGLUT3 +/+ mice performances. C , VGLUT3 –/– mice performances. D–G , Freezing levels on different days: ( D ) day 0, before conditioning; ( E ) day 1, VGLUT3 –/– mice already show a higher freezing level; ( F ) day 7, VGLUT3 +/+ mice start to discriminate the different contexts. G , On day 10, VGLUT3 –/– mice still do not discriminate the different contexts. Data are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All corresponding statistics are presented in Extended Data .

Article Snippet: The Fear Conditioning Apparatus (BIOSEB) is made of black methacrylate walls, a grid floor and transparent ceiling and front door.

Techniques:

Journal: eNeuro

Article Title: Absence of VGLUT3 Expression Leads to Impaired Fear Memory in Mice

doi: 10.1523/ENEURO.0304-22.2023

Figure Lengend Snippet: Cohorts used

Article Snippet: The Fear Conditioning Apparatus (BIOSEB) is made of black methacrylate walls, a grid floor and transparent ceiling and front door.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome

doi: 10.1016/j.mcpro.2023.100661

Figure Lengend Snippet: Functional validation of Ppm1h in synaptic plasticity . A , Differential regulation of surface expression of AMPAR and NMDAR subunits by Ppm1h overexpression. When Ppm1h was overexpressed, cortical neurons exhibited increased surface expression of GluN1 and GluN2A. In contrast, surface expression of GluA1, A2, GluA1 pS831, pS845 and GluN2B showed a significant decreased following Ppm1h overexpression (n = 12 for GluA1 and GluA2, and six for other glutamate receptors; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.005, and ∗∗∗∗ p ≤ 0.001 by Student’s t test). B and C , regulation of Ppm1h during glycine-induced chemical LTP (GI-cLTP). B , scheme indicating experimental workflow for GI-cLTP. Cultured cortical neurons (DIV18 or older) were treated with 200 μM glycine for 10 min followed by chase with Mg 2+ -containing ACSF for 30 min. C , GI-cLTP increased the expression level of Ppm1h levels after both 10 min stimulation and 30 min chase (n = 9/group; adjusted p -value for Control vs. 10 min and Control vs. 30 min chase, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.005 by 1-way ANOVA). D and E , effect of enriched environment (EE) exposure to the level of Ppm1h in the mouse hippocampus. D , scheme indicating experimental workflow for exposure to EE. Mice were tested for 2 weeks using a 2-day EE exposure paradigm (see ), followed by isolation of subcellular fractions (nuclear, cytosolic, and PSD fractions) from the hippocampus. E , characterization of Ppm1h expression in different subcellular fractions following EE exposure. EE resulted in a significant increase of Ppm1h in the nucleus and PSD while cytosolic Ppm1h levels did not change (n = 18/group for P1 and PS2 fractions, n = 12 for PSD fractions; ∗∗∗∗ p ≤ 0.001 by Student’s t test). F and G , effect of contextual fear conditioning (cFC) to the level of Ppm1h in the mouse hippocampus. F , scheme indicating experimental workflow for cFC. Mice were trained via cFC task (see ), followed by isolation of subcellular fractions (nuclear, cytosolic, and PSD fractions) from the hippocampus. Mice showed a significant increase in time spent freezing after cFC (n = 4/group; adjusted p -value, ∗∗∗ p < 0.005 by 2-way ANOVA). G , characterization of Ppm1h expression in different subcellular fractions following cFC. Ppm1h significantly increased in PSD fractions while cytosolic or nuclear Ppm1h levels showed a significant decrease or no change after cFC training, respectively (n = 4; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.005 by Student’s t test).

Article Snippet: Fear conditioning was performed using Ugo Basile Fear Conditioning System (Stoelting Co) and recorded using ANY-maze behavior tracking software as previously described with slight modifications ( ).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Over Expression, Cell Culture, Control, Isolation

Journal: The Journal of Biological Chemistry

Article Title: An Antibody Biosensor Establishes the Activation of the M 1 Muscarinic Acetylcholine Receptor during Learning and Memory * ^♦

doi: 10.1074/jbc.M115.681726

Figure Lengend Snippet: M 1 mAChR is activated in the hippocampus following drug treatment and memory acquisition. A, C57/BL6/NTAC mice were injected (i.p.) with vehicle or xanomeline (5 mg/kg). After 30 min, tissues were fixed by transcardial perfusion, and sections were obtained and stained with the phospho-specific serine 228 antibody and with DAPI stain to reveal the nuclei. Shown are representative sections through the CA1 region of the hippocampus. B, C57/BL6/NTAC mice ( Wild-Type ) or M 1 mAChR-knock-out mice ( M1-KO ) were injected (intraperitoneally) with BQCA (15 mg/kg) or vehicle, and after 30 min hippocampal membranes were prepared from which the M 1 mAChR was immunoprecipitated. The sample was then processed in Western blots, which were probed with phospho-specific serine 228 antibody or an M 1 mAChR-specific antibody to detect total M 1 mAChR. C, quantification of Western blots from B . The data are presented as means ± S.E. ( n = 3). Statistical analysis uses Student's paired t test. D, C57/BL6/NTAC mice were subjected to a fear conditioning training protocol or to an unpaired immediate foot shock as a control; 30 min later tissue was fixed by transcardial perfusion, and sections obtained and stained with phosphorylated Ser 228 -specific antibody ( upper panel ) or anti-c-FOS antibody ( lower panel ). All the data shown are typical of at least three independent experiments.

Article Snippet: Male C57Bl6/NTAC mice (8–15 weeks old) were placed in the conditioning chamber (Stoelting ANY-maze fear conditioning system), and after a 2-min adaptation period, the mice received three tone/foot shock pairings, where a tone (conditioned stimulus, 2.8 kH, 85 db, 30 s) always co-terminated with the foot shock (unconditioned stimulus, 2 s, 0.4 mA).

Techniques: Injection, Staining, Knock-Out, Immunoprecipitation, Western Blot, Control