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Jackson Laboratory rhop23h mice b6 129 s6 cg rhotm1 1 kpal j
Rhop23h Mice B6 129 S6 Cg Rhotm1 1 Kpal J, supplied by Jackson Laboratory, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rhop23h mice b6 129 s6 cg rhotm1 1 kpal j - by Bioz Stars, 2026-07

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IFN-γ suppresses type 3 inflammation through STAT1. (A) Schematic of experimental model used. (B) Total cell number, (C) Neutrophil, (D) TRAMs (E) Ly6C+ Monocytes, and (F) MoAMs in BALs of female and male WT and STAT1 KO mice 8 hours post treatment as enumerated using flow cytometry. Fold changes in aggregated data were calculated relative to the -IFN-γ treatment group within each sex. Unpaired t test. (G) Principal component analysis (PCA) plot of compiled BAL cellularity data from C57BL/6J WT and STAT1 KO mice based on total cell numbers and 11 distinct cell types identified by flow cytometry. (H) PCA loadings indicating the magnitude and direction of each variable’s contribution to a principal component. p value: *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤ 0.0001. All data have n≥3 mice, 2 experiments, mean ± SEM.

Journal: Frontiers in Immunology

Article Title: Second-order regulation: IFN-γ suppresses IL-17A-mediated type 3 inflammation

doi: 10.3389/fimmu.2026.1744476

Figure Lengend Snippet: IFN-γ suppresses type 3 inflammation through STAT1. (A) Schematic of experimental model used. (B) Total cell number, (C) Neutrophil, (D) TRAMs (E) Ly6C+ Monocytes, and (F) MoAMs in BALs of female and male WT and STAT1 KO mice 8 hours post treatment as enumerated using flow cytometry. Fold changes in aggregated data were calculated relative to the -IFN-γ treatment group within each sex. Unpaired t test. (G) Principal component analysis (PCA) plot of compiled BAL cellularity data from C57BL/6J WT and STAT1 KO mice based on total cell numbers and 11 distinct cell types identified by flow cytometry. (H) PCA loadings indicating the magnitude and direction of each variable’s contribution to a principal component. p value: *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤ 0.0001. All data have n≥3 mice, 2 experiments, mean ± SEM.

Article Snippet: 6-week-old C57BL/6J (stock# 000664), IFN-γ KO (B6.129S7-Ifng tm1Ts /J, Stock# 002287), and STAT1 KO (B6.129S(Cg)-Stat1 tm1Dlv /J, Stock# 012606) mice were obtained from Jackson labs (USA) and subsequently bred in-house to ensure acclimation to local conditions.

Techniques: Flow Cytometry

$(A) Ensemble-tagging strategy in the mPFC (coronal sections centered near Bregma +1.53) of CX3CR1FosTRAP2::Ai14;;CX3CR1-GFP mice. For fear ensembles, 4-OHT was administered immediately after auditory fear conditioning (Cond.; CS–US pairings) to TRAP-label fear-learning–activated neurons (tdTomato + ), followed by a retrieval test on day 7 (Fear Retr.). For extinction ensembles, a separate cohort underwent extinction training (Ext.; days 2–4) and received 4-OHT immediately after the third extinction session to TRAP-label extinction-learning–activated neurons, followed by an extinction retrieval test on day 10 (Ext. Retr.). Brains were collected after the retrieval/CS-only session for imaging. (B) Freezing during CS presentations across conditioning and retrieval sessions (left) or conditioning, extinction training (4-trial blocks), and extinction retrieval (right). CS-only control groups received the same CS exposure without shock. Data are mean ± s.e.m. Fear CS only, n = 11 mice; Fear memory, n = 7 mice; Ext. CS only, n = 11 mice; Ext. memory, n = 11 mice. (C) Schematic of compartment-resolved quantification of microglia–ensemble interfaces (soma, neurites, and spines) in mPFC. (D) Representative 3D reconstructions of CX3CR1-GFP + microglia (green) and corresponding Imaris Filament reconstructions (cyan). White outline denotes the surveillance territory defined by the outermost process tips. Scale bar, 10 μm. (E to H) Quantification of microglial morphology from 3D reconstructions: soma area and total process area (E), number of bulbous endings (F), surveillance territory area (G), and arbor complexity by Sholl analysis (branch points and intersections) (H). Extinction retrieval increased process elaboration (process area, bulbous endings, surveillance territory, and branching) without changing soma size, whereas fear retrieval did not induce comparable process expansion and reduced branching complexity. Each dot represents one microglial cell; n = 29–72 cells from 7 mice per group (exact n varies by metric). (I) Leica Lightning super-resolution confocal z-stacks (effective lateral resolution ∼120 nm) showing tdTomato + dendrites/spines (tdTomato, red) and CX3CR1-GFP + microglial processes (green); arrows indicate microglia–spine appositions. Scale bar, 5 μm. (J) Spine-level engagement quantified from 3D reconstructions: tdTomato + spine density, number of microglial processes apposed to tdTomato + spines, and total microglia–spine contact area. Fear retrieval reduced microglial process apposition to tdTomato+ dendrites/spines without increasing contact area, whereas extinction retrieval increased both process apposition and contact area. (K) Operational classification of microglia–spine interactions based on 3D coverage: no contact, contact (<50% surface coverage), enwrapped (≥50% coverage), or engulfed. Scale bar, 0.5 μm. (L) Distribution of tdTomato + spines across interaction categories for fear (left) and extinction (right) group. Extinction retrieval shifted tdTomato + spines from the “no contact” to the “contact” category, with no detectable change in enwrapped or engulfed fractions; fear retrieval did not measurably redistribute spine categories. n = 42–58 dendritic segments (or equivalent analyzed units) from 7 mice per group (Fear CS-only, 58; Fear memory, 57; Ext. CS-only, 42; Ext. memory, 55). N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Statistics: two-way repeated-measures ANOVA for conditioning/extinction time courses (B) and two-tailed unpaired Student’s t tests for pairwise comparisons versus the corresponding CS-only controls unless otherwise indicated.$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Ensemble-tagging strategy in the mPFC (coronal sections centered near Bregma +1.53) of CX3CR1FosTRAP2::Ai14;;CX3CR1-GFP mice. For fear ensembles, 4-OHT was administered immediately after auditory fear conditioning (Cond.; CS–US pairings) to TRAP-label fear-learning–activated neurons (tdTomato + ), followed by a retrieval test on day 7 (Fear Retr.). For extinction ensembles, a separate cohort underwent extinction training (Ext.; days 2–4) and received 4-OHT immediately after the third extinction session to TRAP-label extinction-learning–activated neurons, followed by an extinction retrieval test on day 10 (Ext. Retr.). Brains were collected after the retrieval/CS-only session for imaging. (B) Freezing during CS presentations across conditioning and retrieval sessions (left) or conditioning, extinction training (4-trial blocks), and extinction retrieval (right). CS-only control groups received the same CS exposure without shock. Data are mean ± s.e.m. Fear CS only, n = 11 mice; Fear memory, n = 7 mice; Ext. CS only, n = 11 mice; Ext. memory, n = 11 mice. (C) Schematic of compartment-resolved quantification of microglia–ensemble interfaces (soma, neurites, and spines) in mPFC. (D) Representative 3D reconstructions of CX3CR1-GFP + microglia (green) and corresponding Imaris Filament reconstructions (cyan). White outline denotes the surveillance territory defined by the outermost process tips. Scale bar, 10 μm. (E to H) Quantification of microglial morphology from 3D reconstructions: soma area and total process area (E), number of bulbous endings (F), surveillance territory area (G), and arbor complexity by Sholl analysis (branch points and intersections) (H). Extinction retrieval increased process elaboration (process area, bulbous endings, surveillance territory, and branching) without changing soma size, whereas fear retrieval did not induce comparable process expansion and reduced branching complexity. Each dot represents one microglial cell; n = 29–72 cells from 7 mice per group (exact n varies by metric). (I) Leica Lightning super-resolution confocal z-stacks (effective lateral resolution ∼120 nm) showing tdTomato + dendrites/spines (tdTomato, red) and CX3CR1-GFP + microglial processes (green); arrows indicate microglia–spine appositions. Scale bar, 5 μm. (J) Spine-level engagement quantified from 3D reconstructions: tdTomato + spine density, number of microglial processes apposed to tdTomato + spines, and total microglia–spine contact area. Fear retrieval reduced microglial process apposition to tdTomato+ dendrites/spines without increasing contact area, whereas extinction retrieval increased both process apposition and contact area. (K) Operational classification of microglia–spine interactions based on 3D coverage: no contact, contact (<50% surface coverage), enwrapped (≥50% coverage), or engulfed. Scale bar, 0.5 μm. (L) Distribution of tdTomato + spines across interaction categories for fear (left) and extinction (right) group. Extinction retrieval shifted tdTomato + spines from the “no contact” to the “contact” category, with no detectable change in enwrapped or engulfed fractions; fear retrieval did not measurably redistribute spine categories. n = 42–58 dendritic segments (or equivalent analyzed units) from 7 mice per group (Fear CS-only, 58; Fear memory, 57; Ext. CS-only, 42; Ext. memory, 55). N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001. Statistics: two-way repeated-measures ANOVA for conditioning/extinction time courses (B) and two-tailed unpaired Student’s t tests for pairwise comparisons versus the corresponding CS-only controls unless otherwise indicated.

Article Snippet: The following C57BL/6-background transgenic lines were used: CX3CR1-GFP (B6.129P2(Cg)-Cx3cr1 tm1Litt/J ; The Jackson Laboratory, stock 005582); CX3CR1-CreER (B6.129P2(C)-Cx3cr1 tm2.1(cre/ERT2)Jung/J ; The Jackson Laboratory, stock 020940); Ai14 (B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J ; The Jackson Laboratory, stock 007914); Mertk fl/fl (C57BL/6JCya- Mertk em1flox /Cya, Cyagen, stock S-CKO-03713); R26-CAG-LSL-tdTomato-2A-DTR (C57BL/6Smoc- Gt(ROSA)26Sor em1(CAG-LSL-R-tdTomato-2A-DTR)Smoc , Shanghai Model Organisms Center, stock NM-KI-210094); P2ry12 fl/fl (kindly provided by Prof. Jiyun Peng, Nanchang University); LSL-hM4Di-YFP (B6.129-Gt(ROSA)26Sor tm1(CAG-CHRM4*-mCitrine)Ute/J ; The Jackson Laboratory, stock 026219); FosTRAP2 (Fos-2A-iCreER; The Jackson Laboratory, stock 030323); Rosa26-CAG-LSL-Cas9-GFP (B6J.129(Cg)-Gt(ROSA)26Sor tm1.1(CAG-cas9*,-EGFP)Fezh/J ; The Jackson Laboratory, stock 024857).

Techniques: Imaging, Control, Two Tailed Test

(A) Representative 3D renderings from FosTRAP2::Ai14;;CX3CR1 mPFC showing single CX3CR1-GFP + microglia (green) apposed to tdTomato + neuronal bodies (tdTomato, red) in Fear CS only, Fear memory, Ext. CS only, and Ext. memory groups. Coloc panels depict microglia–TRAP interface voxels mapped to the tdTomato + body surface (white; arrows). Scale bars, 10 μm. (B) Representative high-magnification views of microglia (green) and tdTomato + structures (red) with corresponding Coloc maps (white; arrows) illustrating group differences in microglia–ensemble apposition. Scale bars, 10 μm. (C) Somatic interface quantification. tdTomato + body area is unchanged across groups (left). Microglial coverage of tdTomato + bodies (absolute interface area, middle; percent coverage, right) is reduced in Fear memory relative to Fear CS only, but increased in Ext. memory relative to Ext. CS only. Fear CS only, n = 229 cells (7 mice); Fear memory, n = 135 cells (7 mice); Ext. CS only, n = 188 cells (7 mice); Ext. memory, n = 269 cells (7 mice). (D) Neurite interface quantification. Microglial coverage of tdTomato + neurites (absolute area, left; percent coverage, right) decreases after fear retrieval and increases after extinction retrieval relative to the corresponding CS-only controls. Fear CS only, n = 26 sections (7 mice); Fear memory, n = 21 sections (7 mice); Ext. CS only, n = 27 sections (7 mice); Ext. memory, n = 25 sections (7 mice). (E) Number of microglial processes apposed to each tdTomato + neuronal body, showing fewer contacts in Fear memory and more contacts in Ext. memory relative to their respective controls. Same sections as in (D). N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test (memory versus corresponding CS-only control).

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Representative 3D renderings from FosTRAP2::Ai14;;CX3CR1 mPFC showing single CX3CR1-GFP + microglia (green) apposed to tdTomato + neuronal bodies (tdTomato, red) in Fear CS only, Fear memory, Ext. CS only, and Ext. memory groups. Coloc panels depict microglia–TRAP interface voxels mapped to the tdTomato + body surface (white; arrows). Scale bars, 10 μm. (B) Representative high-magnification views of microglia (green) and tdTomato + structures (red) with corresponding Coloc maps (white; arrows) illustrating group differences in microglia–ensemble apposition. Scale bars, 10 μm. (C) Somatic interface quantification. tdTomato + body area is unchanged across groups (left). Microglial coverage of tdTomato + bodies (absolute interface area, middle; percent coverage, right) is reduced in Fear memory relative to Fear CS only, but increased in Ext. memory relative to Ext. CS only. Fear CS only, n = 229 cells (7 mice); Fear memory, n = 135 cells (7 mice); Ext. CS only, n = 188 cells (7 mice); Ext. memory, n = 269 cells (7 mice). (D) Neurite interface quantification. Microglial coverage of tdTomato + neurites (absolute area, left; percent coverage, right) decreases after fear retrieval and increases after extinction retrieval relative to the corresponding CS-only controls. Fear CS only, n = 26 sections (7 mice); Fear memory, n = 21 sections (7 mice); Ext. CS only, n = 27 sections (7 mice); Ext. memory, n = 25 sections (7 mice). (E) Number of microglial processes apposed to each tdTomato + neuronal body, showing fewer contacts in Fear memory and more contacts in Ext. memory relative to their respective controls. Same sections as in (D). N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test (memory versus corresponding CS-only control).

Techniques: Control

(A) Workflow for CLEM with near-infrared branding (NIRB) in FosTRAP2::Ai14;; CX3CR1-GFP mice. Fear engrams were TRAP-labeled by 4-OHT delivery after conditioning (Cond.) and examined after fear retrieval (Fear Retr., day 7). Extinction engrams were TRAP-labeled by 4-OHT delivery after the last extinction session (Ext., days 2–4) and examined after extinction retrieval (Ext. Retr., day 10). After retrieval, acute mPFC slices were imaged by two-photon microscopy to identify candidate CX3CR1-GFP + microglia processes contacting tdTomato + engram bodies/neurites; the region of interest was then marked in situ by NIRB, followed by EM sample preparation and relocation of the branded site for 3D-SEM acquisition and alignment across modalities. Scale bars, 10 μm (two-photon), 10 μm (EM relocation view), and 5 μm (burn mark zoom). (B and C) Representative CLEM examples of microglial processes apposed to fear (top) or extinction (bottom) engram somata or dendrites. For each condition, two-photon images (left) show GFP + microglia (green) contacting tdTomato + engram neurons (red); corresponding 3D surface reconstructions (second panels) and zoomed views (third panels) highlight the contact region (boxed). Serial block-face SEM sections (right) depict the ultrastructural interface between the same microglial process (false-colored green) and ensemble neuronal bodies (B) or dendrites (C) (false-colored red). Scale bars, 10 μm (two-photon), 10 μm (3D-reconstruction Surfaces), 1 μm (3D-reconstruction Zoom) and 1 μm (SEM).

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Workflow for CLEM with near-infrared branding (NIRB) in FosTRAP2::Ai14;; CX3CR1-GFP mice. Fear engrams were TRAP-labeled by 4-OHT delivery after conditioning (Cond.) and examined after fear retrieval (Fear Retr., day 7). Extinction engrams were TRAP-labeled by 4-OHT delivery after the last extinction session (Ext., days 2–4) and examined after extinction retrieval (Ext. Retr., day 10). After retrieval, acute mPFC slices were imaged by two-photon microscopy to identify candidate CX3CR1-GFP + microglia processes contacting tdTomato + engram bodies/neurites; the region of interest was then marked in situ by NIRB, followed by EM sample preparation and relocation of the branded site for 3D-SEM acquisition and alignment across modalities. Scale bars, 10 μm (two-photon), 10 μm (EM relocation view), and 5 μm (burn mark zoom). (B and C) Representative CLEM examples of microglial processes apposed to fear (top) or extinction (bottom) engram somata or dendrites. For each condition, two-photon images (left) show GFP + microglia (green) contacting tdTomato + engram neurons (red); corresponding 3D surface reconstructions (second panels) and zoomed views (third panels) highlight the contact region (boxed). Serial block-face SEM sections (right) depict the ultrastructural interface between the same microglial process (false-colored green) and ensemble neuronal bodies (B) or dendrites (C) (false-colored red). Scale bars, 10 μm (two-photon), 10 μm (3D-reconstruction Surfaces), 1 μm (3D-reconstruction Zoom) and 1 μm (SEM).

Techniques: Labeling, Microscopy, In Situ, Sample Prep, Blocking Assay

(A) Two-photon imaging schedule in CX3CR1-GFP mice. Animals underwent auditory fear conditioning (Cond.), and extinction training (Ext.), followed by GRIN lens implantation over mPFC. The same fields were imaged at baseline (Days 0–1), during re-extinction (Re-Ext., Day 2), and at extinction retrieval (Ext. Retr., Day 3). (B) Representative microglial cell imaged on each day. Overlays show the same cell at 0 min (red) and 25 min (green) within a session; overlap appears yellow. Scale bar, 10 μm. (C–E) Quantification of within-session process dynamics from paired images (0 and 25 min), expressed relative to Day 0 for each cell. (C) Moving area, area swept by motile processes. (D) Surveillance area, total territory explored by processes over the imaging interval. (E) Bulbous endings, number of distal bulbous process termini. Moving area remains stable across days, whereas surveillance area increases selectively at extinction retrieval and bulbous endings are elevated during re-extinction and retrieval. n = 23 cells (4 mice). Bars, mean ± s.e.m.; dots, individual cells. Statistics: one-way repeated-measures ANOVA with Dunnett’s multiple comparisons versus Day 0 and planned paired comparisons between days, as indicated. N.S., not significant; * P < 0.05, **** P < 0.0001, paired Student’s t test. ## P < 0.01, #### P < 0.0001, one-way ANOVA with Dunnett’s multiple-comparisons test.

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Two-photon imaging schedule in CX3CR1-GFP mice. Animals underwent auditory fear conditioning (Cond.), and extinction training (Ext.), followed by GRIN lens implantation over mPFC. The same fields were imaged at baseline (Days 0–1), during re-extinction (Re-Ext., Day 2), and at extinction retrieval (Ext. Retr., Day 3). (B) Representative microglial cell imaged on each day. Overlays show the same cell at 0 min (red) and 25 min (green) within a session; overlap appears yellow. Scale bar, 10 μm. (C–E) Quantification of within-session process dynamics from paired images (0 and 25 min), expressed relative to Day 0 for each cell. (C) Moving area, area swept by motile processes. (D) Surveillance area, total territory explored by processes over the imaging interval. (E) Bulbous endings, number of distal bulbous process termini. Moving area remains stable across days, whereas surveillance area increases selectively at extinction retrieval and bulbous endings are elevated during re-extinction and retrieval. n = 23 cells (4 mice). Bars, mean ± s.e.m.; dots, individual cells. Statistics: one-way repeated-measures ANOVA with Dunnett’s multiple comparisons versus Day 0 and planned paired comparisons between days, as indicated. N.S., not significant; * P < 0.05, **** P < 0.0001, paired Student’s t test. ## P < 0.01, #### P < 0.0001, one-way ANOVA with Dunnett’s multiple-comparisons test.

Techniques: Imaging

(A) Conceptual schematic illustrating that, across baseline, re-extinction (Re-Ext.), and extinction retrieval (Ext. Retr.), microglial processes can expand their contact interfaces with extinction-tagged neurites and engage phagocytic programs. (B) Experimental timeline for longitudinal two-photon imaging through a GRIN lens in FosTRAP2::Ai14;;CX3CR1-GFP mice. Mice underwent fear conditioning (Cond.) followed extinction training (Ext.); 4-OHT was administered after the third extinction session to TRAP-label extinction ensembles (tdTomato + ). After GRIN-lens implantation over mPFC and recovery (∼4 weeks; allowing robust tdTomato expression and spontaneous recovery of extinguished fear), the same fields were imaged at baseline (Days 0–1), during re-extinction (Day 2), and at extinction retrieval (Day 3). (C) Representative z-projections showing CX3CR1-GFP + microglia (green) and tdTomato + extinction-ensemble neurites (red) across days (top row). Middle row, 3D surface renderings of the same microglia. Bottom row, voxels assigned to the microglia–tdTomato + neurite interface (Coloc). Scale bar, 5 μm. (D to G) Longitudinal quantification of microglial morphology from the tracked cells, soma area remained stable (D, left), whereas process area (D, right), the number of bulbous endings (E), surveillance territory (F), and branching complexity (G; branch points and Sholl intersections) increased across sessions and peaked at extinction retrieval. (H) Microglial engagement of extinction ensembles quantified as microglial coverage of tdTomato + neurites (left, absolute covered area; right, percent coverage). Coverage increased during re-extinction and was further elevated at extinction retrieval. n = 19 microglia (2 mice). Data are mean ± s.e.m. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparisons test versus Day 0).

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Conceptual schematic illustrating that, across baseline, re-extinction (Re-Ext.), and extinction retrieval (Ext. Retr.), microglial processes can expand their contact interfaces with extinction-tagged neurites and engage phagocytic programs. (B) Experimental timeline for longitudinal two-photon imaging through a GRIN lens in FosTRAP2::Ai14;;CX3CR1-GFP mice. Mice underwent fear conditioning (Cond.) followed extinction training (Ext.); 4-OHT was administered after the third extinction session to TRAP-label extinction ensembles (tdTomato + ). After GRIN-lens implantation over mPFC and recovery (∼4 weeks; allowing robust tdTomato expression and spontaneous recovery of extinguished fear), the same fields were imaged at baseline (Days 0–1), during re-extinction (Day 2), and at extinction retrieval (Day 3). (C) Representative z-projections showing CX3CR1-GFP + microglia (green) and tdTomato + extinction-ensemble neurites (red) across days (top row). Middle row, 3D surface renderings of the same microglia. Bottom row, voxels assigned to the microglia–tdTomato + neurite interface (Coloc). Scale bar, 5 μm. (D to G) Longitudinal quantification of microglial morphology from the tracked cells, soma area remained stable (D, left), whereas process area (D, right), the number of bulbous endings (E), surveillance territory (F), and branching complexity (G; branch points and Sholl intersections) increased across sessions and peaked at extinction retrieval. (H) Microglial engagement of extinction ensembles quantified as microglial coverage of tdTomato + neurites (left, absolute covered area; right, percent coverage). Coverage increased during re-extinction and was further elevated at extinction retrieval. n = 19 microglia (2 mice). Data are mean ± s.e.m. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparisons test versus Day 0).

Techniques: Imaging, Expressing

$(A) Analysis framework. Microglia contact fear- or extinction-tagged (tdTomato + ) neuronal ensembles in mPFC and internalize ensemble-derived puncta into CD68 + phagolysosomes; engulfed excitatory postsynaptic cargo was identified by PSD-95. (B) Representative 3D confocal reconstructions from mPFC of FosTRAP2::Ai14;;CX3CR1-GFP mice showing CX3CR1-GFP + microglia (green), TRAP-labeled ensemble material (tdTomato, red), and CD68 (magenta) in Fear CS-only, Fear memory, Ext. CS-only, and Ext. memory groups. Insets show higher-magnification views and colocalization masks (Coloc) used to score internalized tdTomato + puncta. Scale bar, 10 μm. (C) CD68 + compartment size per microglia, expressed as absolute area (left) and as fraction of microglial area (right). Fear retrieval reduced CD68 burden relative to Fear CS-only controls, whereas extinction retrieval increased CD68 burden relative to Ext. CS-only controls. Fear CS only, n = 36 cells (7 mice); Fear memory, n = 38 cells (7 mice); Extinction CS only, n = 38 cells (7 mice); Extinction memory, n = 46 cells (7 mice). (D) Ensemble-derived cargo quantified as tdTomato + puncta enclosed within microglial surfaces, shown as total puncta per microglia (left) and partitioned to soma (middle) or processes (right). Extinction retrieval increased tdTomato + puncta in both somatic and process compartments relative to Ext. CS-only, whereas fear retrieval did not increase tdTomato + puncta relative to Fear CS-only. Fear CS only, n = 40 cells (7 mice); Fear memory, n = 40 cells (7 mice); Extinction CS only, n = 46 cells (7 mice); Extinction memory, n = 59 cells (7 mice). (E) tdTomato + puncta within CD68 + microglial compartments. tdTomato + cargo within CD68⁺ volumes decreased after fear retrieval and increased after extinction retrieval relative to matched CS-only controls. Fear CS only, n = 40 cells (7 mice); Fear memory, n = 40 cells (7 mice); Extinction CS only, n = 38 cells (7 mice); Extinction memory, n = 49 cells (7 mice). (F) Representative reconstructions showing tdTomato + PSD-95 + puncta (cyan) within CD68 + microglial compartments (magenta) in Ext. CS-only (top) and Ext. memory (bottom). Arrows indicate tdTomato + PSD-95 + puncta contained within CD68 + volumes. Scale bars, 10 μm (main) and 2 μm (zoom). (G) Quantification of tdTomato + PSD-95 + puncta within CD68 + microglia in Ext. CS-only versus Ext. memory groups: summed puncta area per microglia (left) and puncta number per microglia (right). Extinction retrieval increased both measures, consistent with preferential uptake of extinction-ensemble excitatory postsynaptic cargo. Ext. CS only, n = 34 cells (5 mice); Ext. memory, n = 49 cells (5 mice). (H) Timeline for longitudinal two-photon imaging of extinction-ensemble spines in FosTRAP2::Ai14;;CX3CR1-GFP mice. Mice underwent conditioning (Cond.) and extinction training (Ext.); extinction ensembles were labeled by 4-hydroxytamoxifen (4-OHT) after the final extinction session. After surgery, the same dendritic fields were imaged at baseline (Days 0–1), during re-extinction (Re-Ext., Day 2), and at extinction retrieval (Ext. Retr., Day 3) in mice maintained on control diet (CD) or PLX5622 chow to deplete microglia. (I) Representative longitudinal images of tdTomato-labeled extinction-ensemble dendritic segments across Days 0–3 in CD and PLX5622 groups. Filled arrowheads indicate newly formed spines; open arrowheads indicate eliminated spines. Scale bar, 2 μm. (J) Spine density and turnover on extinction-ensemble dendrites with or without microglia. Relative spine density was normalized to Day 0; daily changes, formation fraction, and elimination fraction were quantified across imaging intervals. During re-extinction (Day 2), spine density increased in CD mice and showed a similar upward trend in PLX5622-treated mice. At extinction retrieval (Day 3), spine density declined in CD controls due to increased elimination with little additional formation, whereas it remained elevated in PLX5622-treated mice because formation persisted and elimination was reduced. CD, n = 10 dendritic segments (2 mice); PLX5622, n = 10 dendritic segments (3 mice). Data are mean ± s.e.m. N.S., not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-tailed unpaired Student’s t tests comparing each memory group with its matched CS-only control (C–E) or Ext. memory versus Ext. CS-only (G). Statistical significance is indicated in the panels (tests as described in Methods); in (J), * indicates comparisons versus Day 0 within a diet group, # indicates within-group comparisons across days, and & indicates CD versus PLX5622 at the same time point.$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Analysis framework. Microglia contact fear- or extinction-tagged (tdTomato + ) neuronal ensembles in mPFC and internalize ensemble-derived puncta into CD68 + phagolysosomes; engulfed excitatory postsynaptic cargo was identified by PSD-95. (B) Representative 3D confocal reconstructions from mPFC of FosTRAP2::Ai14;;CX3CR1-GFP mice showing CX3CR1-GFP + microglia (green), TRAP-labeled ensemble material (tdTomato, red), and CD68 (magenta) in Fear CS-only, Fear memory, Ext. CS-only, and Ext. memory groups. Insets show higher-magnification views and colocalization masks (Coloc) used to score internalized tdTomato + puncta. Scale bar, 10 μm. (C) CD68 + compartment size per microglia, expressed as absolute area (left) and as fraction of microglial area (right). Fear retrieval reduced CD68 burden relative to Fear CS-only controls, whereas extinction retrieval increased CD68 burden relative to Ext. CS-only controls. Fear CS only, n = 36 cells (7 mice); Fear memory, n = 38 cells (7 mice); Extinction CS only, n = 38 cells (7 mice); Extinction memory, n = 46 cells (7 mice). (D) Ensemble-derived cargo quantified as tdTomato + puncta enclosed within microglial surfaces, shown as total puncta per microglia (left) and partitioned to soma (middle) or processes (right). Extinction retrieval increased tdTomato + puncta in both somatic and process compartments relative to Ext. CS-only, whereas fear retrieval did not increase tdTomato + puncta relative to Fear CS-only. Fear CS only, n = 40 cells (7 mice); Fear memory, n = 40 cells (7 mice); Extinction CS only, n = 46 cells (7 mice); Extinction memory, n = 59 cells (7 mice). (E) tdTomato + puncta within CD68 + microglial compartments. tdTomato + cargo within CD68⁺ volumes decreased after fear retrieval and increased after extinction retrieval relative to matched CS-only controls. Fear CS only, n = 40 cells (7 mice); Fear memory, n = 40 cells (7 mice); Extinction CS only, n = 38 cells (7 mice); Extinction memory, n = 49 cells (7 mice). (F) Representative reconstructions showing tdTomato + PSD-95 + puncta (cyan) within CD68 + microglial compartments (magenta) in Ext. CS-only (top) and Ext. memory (bottom). Arrows indicate tdTomato + PSD-95 + puncta contained within CD68 + volumes. Scale bars, 10 μm (main) and 2 μm (zoom). (G) Quantification of tdTomato + PSD-95 + puncta within CD68 + microglia in Ext. CS-only versus Ext. memory groups: summed puncta area per microglia (left) and puncta number per microglia (right). Extinction retrieval increased both measures, consistent with preferential uptake of extinction-ensemble excitatory postsynaptic cargo. Ext. CS only, n = 34 cells (5 mice); Ext. memory, n = 49 cells (5 mice). (H) Timeline for longitudinal two-photon imaging of extinction-ensemble spines in FosTRAP2::Ai14;;CX3CR1-GFP mice. Mice underwent conditioning (Cond.) and extinction training (Ext.); extinction ensembles were labeled by 4-hydroxytamoxifen (4-OHT) after the final extinction session. After surgery, the same dendritic fields were imaged at baseline (Days 0–1), during re-extinction (Re-Ext., Day 2), and at extinction retrieval (Ext. Retr., Day 3) in mice maintained on control diet (CD) or PLX5622 chow to deplete microglia. (I) Representative longitudinal images of tdTomato-labeled extinction-ensemble dendritic segments across Days 0–3 in CD and PLX5622 groups. Filled arrowheads indicate newly formed spines; open arrowheads indicate eliminated spines. Scale bar, 2 μm. (J) Spine density and turnover on extinction-ensemble dendrites with or without microglia. Relative spine density was normalized to Day 0; daily changes, formation fraction, and elimination fraction were quantified across imaging intervals. During re-extinction (Day 2), spine density increased in CD mice and showed a similar upward trend in PLX5622-treated mice. At extinction retrieval (Day 3), spine density declined in CD controls due to increased elimination with little additional formation, whereas it remained elevated in PLX5622-treated mice because formation persisted and elimination was reduced. CD, n = 10 dendritic segments (2 mice); PLX5622, n = 10 dendritic segments (3 mice). Data are mean ± s.e.m. N.S., not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-tailed unpaired Student’s t tests comparing each memory group with its matched CS-only control (C–E) or Ext. memory versus Ext. CS-only (G). Statistical significance is indicated in the panels (tests as described in Methods); in (J), * indicates comparisons versus Day 0 within a diet group, # indicates within-group comparisons across days, and & indicates CD versus PLX5622 at the same time point.

Techniques: Derivative Assay, Labeling, Imaging, Control, Two Tailed Test

(A) Representative 3D confocal reconstructions from mPFC of FosTRAP2::Ai14;;CX3CR1-GFP mice showing microglia (CX3CR1-GFP, green), extinction-tagged ensemble signal (tdTomato, red), and PSD-95 (cyan) in Ext. CS only and Ext memory (extinction retrieval) groups. Insets show a higher-magnification view (Zoom) and a colocalization map (Coloc) highlighting tdTomato + synaptic puncta assigned to microglial contact/engulfment (arrows). Scale bars, 10 μm (3D) and 2 μm (zoom). (B and C) Quantification of PSD-95 engagement. (B) Contact area between microglia and PSD95 + puncta (left) and the subset attributable to tdTomato + PSD-95 + puncta (right). (C) Engulfment quantified as the number of tdTomato + PSD-95 + puncta enclosed within microglial volumes per cell. Ext. CS only, n = 40 cells (7 mice); Ext. memory, n = 40 cells (7 mice). (D) As in (A), but for vGLUT1 (cyan). (E and F) vGLUT1 engagement. (E) Contact area with all vGLUT1 + puncta (left) and with tdTomato + vGLUT1 + puncta (right). (F) Number of engulfed tdTomato + vGLUT1 + puncta per microglia. Ext. CS only, n = 38 microglia (7 mice); Ext. memory, n = 41 microglia (7 mice). (H) As in (A), but for Gephyrin (cyan). (I and J) Gephyrin engagement. (I) Contact area with all Gephyrin + puncta (left) and with tdTomato + Gephyrin + puncta (right). (J) Number of engulfed tdTomato + Gephyrin + puncta per microglia. Ext. CS only, n = 42 microglia (7 mice); Ext. memory, n = 42 microglia (7 mice). (K) As in (A), but for vGAT (cyan). (L and M) vGAT engagement. (L) Contact area with all vGAT + puncta (left) and with tdTomato + vGAT + puncta (right). (M) Number of engulfed tdTomato + vGAT + puncta per microglia. Ext. CS only, n = 42 microglia (7 mice); Ext. memory, n = 42 microglia (7 mice). Across markers, extinction retrieval increased contact with—and engulfment of—tdTomato + PSD-95 + postsynaptic puncta, did not increase engulfment of tdTomato + vGLUT1 + or tdTomato + Gephyrin + puncta, and reduced both contact and engulfment of tdTomato + VGAT + puncta. N.S., not significant; ** P < 0.01, *** P < 0.001, two-sided unpaired Student’s t test.

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Representative 3D confocal reconstructions from mPFC of FosTRAP2::Ai14;;CX3CR1-GFP mice showing microglia (CX3CR1-GFP, green), extinction-tagged ensemble signal (tdTomato, red), and PSD-95 (cyan) in Ext. CS only and Ext memory (extinction retrieval) groups. Insets show a higher-magnification view (Zoom) and a colocalization map (Coloc) highlighting tdTomato + synaptic puncta assigned to microglial contact/engulfment (arrows). Scale bars, 10 μm (3D) and 2 μm (zoom). (B and C) Quantification of PSD-95 engagement. (B) Contact area between microglia and PSD95 + puncta (left) and the subset attributable to tdTomato + PSD-95 + puncta (right). (C) Engulfment quantified as the number of tdTomato + PSD-95 + puncta enclosed within microglial volumes per cell. Ext. CS only, n = 40 cells (7 mice); Ext. memory, n = 40 cells (7 mice). (D) As in (A), but for vGLUT1 (cyan). (E and F) vGLUT1 engagement. (E) Contact area with all vGLUT1 + puncta (left) and with tdTomato + vGLUT1 + puncta (right). (F) Number of engulfed tdTomato + vGLUT1 + puncta per microglia. Ext. CS only, n = 38 microglia (7 mice); Ext. memory, n = 41 microglia (7 mice). (H) As in (A), but for Gephyrin (cyan). (I and J) Gephyrin engagement. (I) Contact area with all Gephyrin + puncta (left) and with tdTomato + Gephyrin + puncta (right). (J) Number of engulfed tdTomato + Gephyrin + puncta per microglia. Ext. CS only, n = 42 microglia (7 mice); Ext. memory, n = 42 microglia (7 mice). (K) As in (A), but for vGAT (cyan). (L and M) vGAT engagement. (L) Contact area with all vGAT + puncta (left) and with tdTomato + vGAT + puncta (right). (M) Number of engulfed tdTomato + vGAT + puncta per microglia. Ext. CS only, n = 42 microglia (7 mice); Ext. memory, n = 42 microglia (7 mice). Across markers, extinction retrieval increased contact with—and engulfment of—tdTomato + PSD-95 + postsynaptic puncta, did not increase engulfment of tdTomato + vGLUT1 + or tdTomato + Gephyrin + puncta, and reduced both contact and engulfment of tdTomato + VGAT + puncta. N.S., not significant; ** P < 0.01, *** P < 0.001, two-sided unpaired Student’s t test.

Techniques:

$(A) Experimental timeline in FosTRAP2::Ai14;;CX3CR1-GFP mice. Minocycline (Mino) or vehicle was provided in drinking water starting from day −6. Mice underwent fear conditioning (Cond., day 1) followed by extinction training (Ext., days 2–4); 4-OHT was administered after the third extinction session to TRAP-label extinction-ensemble neurons. Extinction retrieval (Ext. Retr.) was tested on day 10. (B) Freezing during Cond., Ext., and Ext. Retr. Minocycline spared fear acquisition but accelerated extinction and reduced freezing at retrieval. Vehicle, n = 14 mice; Mino, n = 12 mice. (C) Synaptic physiology in TRAP-labeled extinction-ensemble neurons after Ext. Retr. Left, schematic and representative traces; right, quantification of the NMDAR/AMPAR eEPSC ratio. Vehicle, n = 30 cells (6 mice); Mino, n = 26 cells (6 mice). (D) Representative confocal images showing CX3CR1-GFP + microglia (green) and tdTomato + extinction-ensemble neurons (red) after Ext. Retr. Coloc indicates microglia–ensemble apposition (arrows). Scale bar, 10 μm. (E to G) Minocycline reduces structural coupling between microglia and extinction ensembles. (E) Ensemble body (left), microglial coverage of ensemble body (middle; absolute area); and somatic coverage expressed as a percentage (right). (F) Number of microglial processes contacting each ensemble body. (G) Microglial coverage of ensemble neurites (left; absolute area) and neurite coverage as a percentage (right). Vehicle, n = 66 cells from 10 sections (5 mice); Mino, n = 74 cells from 10 sections (5 mice). (H) Representative 3D renderings of microglia (green) with lysosomes (CD68, cyan) and postsynaptic puncta (PSD-95, magenta) illustrating reduced microglial lysosomal loading and reduced tdTomato + PSD-95 + cargo after minocycline. Insets show zoom views; arrows indicate engulfed puncta. Scale bars, 7 μm (overview), 2 μm (zoom). (I–K) Minocycline blunts retrieval-associated microglial state changes. (I) Total microglial area (left), soma area (middle), and process area (right). (J) CD68 + lysosomal area per microglia (left) and as a fraction of microglial area (right). (K) Surveillance territory per microglia. Vehicle, n = 64 cells (5 mice); Mino, n = 55 cells (5 mice); surveillance territory: vehicle, n = 48 cells (5 mice); Mino, n = 59 cells (5 mice). (L–O) Minocycline suppresses internalization of extinction-ensemble material and excitatory postsynaptic cargo. (L) Total tdTomato + puncta per microglia. (M) tdTomato + puncta within CD68 + compartments. (N) tdTomato + PSD-95 + puncta per microglia (left) and within CD68 + compartments (right). Vehicle, n = 48 cells (5 mice); Mino, n = 47 cells (5 mice). Data are mean ± s.e.m. (B and C) or shown as individual cells with summary statistics as indicated. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test.$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Experimental timeline in FosTRAP2::Ai14;;CX3CR1-GFP mice. Minocycline (Mino) or vehicle was provided in drinking water starting from day −6. Mice underwent fear conditioning (Cond., day 1) followed by extinction training (Ext., days 2–4); 4-OHT was administered after the third extinction session to TRAP-label extinction-ensemble neurons. Extinction retrieval (Ext. Retr.) was tested on day 10. (B) Freezing during Cond., Ext., and Ext. Retr. Minocycline spared fear acquisition but accelerated extinction and reduced freezing at retrieval. Vehicle, n = 14 mice; Mino, n = 12 mice. (C) Synaptic physiology in TRAP-labeled extinction-ensemble neurons after Ext. Retr. Left, schematic and representative traces; right, quantification of the NMDAR/AMPAR eEPSC ratio. Vehicle, n = 30 cells (6 mice); Mino, n = 26 cells (6 mice). (D) Representative confocal images showing CX3CR1-GFP + microglia (green) and tdTomato + extinction-ensemble neurons (red) after Ext. Retr. Coloc indicates microglia–ensemble apposition (arrows). Scale bar, 10 μm. (E to G) Minocycline reduces structural coupling between microglia and extinction ensembles. (E) Ensemble body (left), microglial coverage of ensemble body (middle; absolute area); and somatic coverage expressed as a percentage (right). (F) Number of microglial processes contacting each ensemble body. (G) Microglial coverage of ensemble neurites (left; absolute area) and neurite coverage as a percentage (right). Vehicle, n = 66 cells from 10 sections (5 mice); Mino, n = 74 cells from 10 sections (5 mice). (H) Representative 3D renderings of microglia (green) with lysosomes (CD68, cyan) and postsynaptic puncta (PSD-95, magenta) illustrating reduced microglial lysosomal loading and reduced tdTomato + PSD-95 + cargo after minocycline. Insets show zoom views; arrows indicate engulfed puncta. Scale bars, 7 μm (overview), 2 μm (zoom). (I–K) Minocycline blunts retrieval-associated microglial state changes. (I) Total microglial area (left), soma area (middle), and process area (right). (J) CD68 + lysosomal area per microglia (left) and as a fraction of microglial area (right). (K) Surveillance territory per microglia. Vehicle, n = 64 cells (5 mice); Mino, n = 55 cells (5 mice); surveillance territory: vehicle, n = 48 cells (5 mice); Mino, n = 59 cells (5 mice). (L–O) Minocycline suppresses internalization of extinction-ensemble material and excitatory postsynaptic cargo. (L) Total tdTomato + puncta per microglia. (M) tdTomato + puncta within CD68 + compartments. (N) tdTomato + PSD-95 + puncta per microglia (left) and within CD68 + compartments (right). Vehicle, n = 48 cells (5 mice); Mino, n = 47 cells (5 mice). Data are mean ± s.e.m. (B and C) or shown as individual cells with summary statistics as indicated. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test.

Techniques: Labeling

$(A) Representative 3D reconstructions of FosTRAP2::Ai14;;CX3CR1-GFP mice immunolabeled for vesicular nucleotide transporter vNUT (cyan). Microglia (CX3CR1-GFP, green) contact TRAP-labeled ensemble bodies (tdTomato, red). Surfaces , 3D surface renderings of microglia and TRAP⁺ somata; Coloc , vNUT signal restricted to the microglia-apposed somatic surface (puncta color-coded by area as indicated). Scale bar, 3 μm. (B) Microglia coverage of vNUT + somatic microdomains on tdTomato + ensemble neurons, quantified as absolute coverage area (left) and fractional coverage (right). Fear retrieval increases microglial apposition to vNUT + domains, whereas extinction retrieval does not. Fear CS only, n = 82 cells (6 mice); fear memory, n = 91 cells (6 mice); Ext. CS only, n = 54 cells (7 mice); Ext. memory, n = 102 cells (7 mice). (C) As in (A), but immunolabeled for K V 2.1 (cyan). Scale bar, 3 μm. (D) Microglial coverage of K V 2.1 + somatic microdomains on tdTomato + ensemble neurons (absolute coverage area, left; fractional coverage, right). Fear retrieval reduces microglial engagement of Kv2.1⁺ domains, whereas extinction retrieval selectively increases fractional coverage (with no significant change in absolute covered area). Fear CS only, n = 203 cells (7 mice); fear memory, n = 111 cells (7 mice); Ext. CS only, n = 238 cells (7 mice); Ext. memory, n = 224 cells (7 mice). N.S., not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test (memory versus corresponding CS-only control).$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Representative 3D reconstructions of FosTRAP2::Ai14;;CX3CR1-GFP mice immunolabeled for vesicular nucleotide transporter vNUT (cyan). Microglia (CX3CR1-GFP, green) contact TRAP-labeled ensemble bodies (tdTomato, red). Surfaces , 3D surface renderings of microglia and TRAP⁺ somata; Coloc , vNUT signal restricted to the microglia-apposed somatic surface (puncta color-coded by area as indicated). Scale bar, 3 μm. (B) Microglia coverage of vNUT + somatic microdomains on tdTomato + ensemble neurons, quantified as absolute coverage area (left) and fractional coverage (right). Fear retrieval increases microglial apposition to vNUT + domains, whereas extinction retrieval does not. Fear CS only, n = 82 cells (6 mice); fear memory, n = 91 cells (6 mice); Ext. CS only, n = 54 cells (7 mice); Ext. memory, n = 102 cells (7 mice). (C) As in (A), but immunolabeled for K V 2.1 (cyan). Scale bar, 3 μm. (D) Microglial coverage of K V 2.1 + somatic microdomains on tdTomato + ensemble neurons (absolute coverage area, left; fractional coverage, right). Fear retrieval reduces microglial engagement of Kv2.1⁺ domains, whereas extinction retrieval selectively increases fractional coverage (with no significant change in absolute covered area). Fear CS only, n = 203 cells (7 mice); fear memory, n = 111 cells (7 mice); Ext. CS only, n = 238 cells (7 mice); Ext. memory, n = 224 cells (7 mice). N.S., not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test (memory versus corresponding CS-only control).

Techniques: Immunolabeling, Labeling, Control

$(A) Continuous intracerebroventricular (ICV) infusion of the P2Y12 antagonist clopidogrel or vehicle control via osmotic minipumps in FosTRAP2::Ai14;;CX3CR1-GFP mice (pump implantation, Day – 6; conditioning, Cond., Day 1; extinction training, Ext., Days 2–4, with 4-OHT after the third session; extinction retrieval, Ext. Retr., Day 10). Right, freezing during conditioning, extinction (4-trial blocks), and extinction retrieval (mean ± s.e.m.). Vehicle, n = 10 mice; clopidogrel, n = 9 mice. (B) Representative mPFC reconstructions at extinction retrieval showing CX3CR1-GFP + microglia (green), extinction-tagged neurons (tdTomato, red), and P2Y12 (cyan). Coloc , P2Y12 signal within the microglia–TRAP contact shell (arrows). Scale bar, 10 μm. (C and D) Gross microglia–ensemble apposition is preserved after clopidogrel treatment. Microglial coverage of tdTomato + neurons (C; tdTomato + surface) and tdTomato + bodies (D) quantified as absolute contact area (left) and percent surface coverage (right). (E and F) P2Y12 + area within microglia-apposed tdTomato + surfaces (E) and within microglia-apposed tdTomato + neuronal bodies (F). Vehicle, n = 99 cells from 15 sections (5 mice); clopidogrel, n = 96 cells from 18 sections (5 mice). (G) Representative single-cell 3D reconstructions illustrating microglial morphology and intracellular cargo after vehicle- and clopidogrel. Channels show GFP (microglia, green), P2Y12 (cyan), CD68 (magenta), and TRAP (red). Coloc , tdTomato + puncta within microglia (arrows). Scale bar, 10 μm. (H and I) Microglial state metrics. Total area, soma area, and process area (H), and CD68⁺ lysosomal area per microglia and fraction of CD68 + microglia (I). Vehicle, n = 43 cells (5 mice); clopidogrel, n = 35 cells (5 mice). (J and K) Engulfment of extinction-ensemble material. Total tdTomato + puncta within microglia, partitioned into somatic versus process compartments (J), and tdTomato + puncta within CD68 + compartments (K). Vehicle, n = 48 cells (5 mice); clopidogrel, n = 47 cells (5 mice). N.S., not significant; * P < 0.05, *** P < 0.001, two-way repeated-measures ANOVA or two-tailed Student’s t test as appropriate.$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Continuous intracerebroventricular (ICV) infusion of the P2Y12 antagonist clopidogrel or vehicle control via osmotic minipumps in FosTRAP2::Ai14;;CX3CR1-GFP mice (pump implantation, Day – 6; conditioning, Cond., Day 1; extinction training, Ext., Days 2–4, with 4-OHT after the third session; extinction retrieval, Ext. Retr., Day 10). Right, freezing during conditioning, extinction (4-trial blocks), and extinction retrieval (mean ± s.e.m.). Vehicle, n = 10 mice; clopidogrel, n = 9 mice. (B) Representative mPFC reconstructions at extinction retrieval showing CX3CR1-GFP + microglia (green), extinction-tagged neurons (tdTomato, red), and P2Y12 (cyan). Coloc , P2Y12 signal within the microglia–TRAP contact shell (arrows). Scale bar, 10 μm. (C and D) Gross microglia–ensemble apposition is preserved after clopidogrel treatment. Microglial coverage of tdTomato + neurons (C; tdTomato + surface) and tdTomato + bodies (D) quantified as absolute contact area (left) and percent surface coverage (right). (E and F) P2Y12 + area within microglia-apposed tdTomato + surfaces (E) and within microglia-apposed tdTomato + neuronal bodies (F). Vehicle, n = 99 cells from 15 sections (5 mice); clopidogrel, n = 96 cells from 18 sections (5 mice). (G) Representative single-cell 3D reconstructions illustrating microglial morphology and intracellular cargo after vehicle- and clopidogrel. Channels show GFP (microglia, green), P2Y12 (cyan), CD68 (magenta), and TRAP (red). Coloc , tdTomato + puncta within microglia (arrows). Scale bar, 10 μm. (H and I) Microglial state metrics. Total area, soma area, and process area (H), and CD68⁺ lysosomal area per microglia and fraction of CD68 + microglia (I). Vehicle, n = 43 cells (5 mice); clopidogrel, n = 35 cells (5 mice). (J and K) Engulfment of extinction-ensemble material. Total tdTomato + puncta within microglia, partitioned into somatic versus process compartments (J), and tdTomato + puncta within CD68 + compartments (K). Vehicle, n = 48 cells (5 mice); clopidogrel, n = 47 cells (5 mice). N.S., not significant; * P < 0.05, *** P < 0.001, two-way repeated-measures ANOVA or two-tailed Student’s t test as appropriate.

Techniques: Control, Single Cell, Two Tailed Test

$(A) Schematic of an “eat-me” pathway in which extracellularly exposed phosphatidylserine (PS) on ensemble membranes is bridged by Gas6 to the microglial phagocytic receptor MERTK, promoting engulfment. (B) Timeline for labeling extracellular PS with PSVue-647 (a membrane-impermeant PS probe delivered intracerebroventricularly through an implanted cannula) and assessing fear or extinction retrieval in FosTRAP2::Ai14;;CX3CR1-GFP mice (ensemble tagging as in ). (C) Representative mPFC confocal images showing microglia (CX3CR1-GFP, green), TRAP-labeled ensemble neurons (tdTomato, red), and PSVue signal (cyan). Right, “Coloc” indicates PSVue puncta segmented within microglial surfaces (i.e., PSVue-labeled PS-bearing material internalized by microglia). Scale bar, 10 μm. (D) Total PSVue signal per field (left) and fraction of PSVue signal associated with TRAP structures (right). Fear CS only, n = 12 sections (4 mice); Fear memory, n = 22 sections (6 mice); Ext. CS only, n = 29 sections (7 mice); Ext. memory, n = 26 sections (7 mice). N.S., not significant; *** P < 0.001, unpaired Student’s t test. (E) Quantification of PSVue + puncta detected within microglia (left) and TRAP⁺PSVue⁺ puncta within microglia (right). Fear CS only, n = 8 sections (3 mice); Fear memory, n = 16 sections (6 mice); Ext. CS only, n = 29 sections (7 mice); Ext. memory, n = 26 sections (7 mice). (F) Ensemble-targeted CRISPR–Cas9 strategy to disrupt Tmem16f selectively in extinction-tagged neurons in FosTRAP2::Cas9 mice using AAV-sgTmem16f-CAG-FLEX-mCherry or AAV-sgCtrl-CAG-FLEX-mCherry, with 4-OHT administered after extinction training to restrict editing to extinction ensembles. (G) Representative mPFC images showing mCherry-labeled extinction ensemble neurons (red) and TMEM16F immunoreactivity (cyan); “Coloc” indicates TMEM16F signal within mCherry⁺ surfaces. Scale bar, 5 μm. (H) TMEM16F knockdown efficiency in tdTomato + neurons (fraction of tdTomato + puncta positive for TMEM16F). sgCtrl, n = 20 sections (5 mice); sgTmem16f, n = 20 sections (5 mice). (I) Freezing during conditioning, extinction, re-extinction, and extinction retrieval in sgCtrl and sgTmem16f groups. sgCtrl, n = 12 mice; sgTmem16f, n = 14 mice. (J) Scheme for tamoxifen-induced microglia-specific deletion of Mertk (CX3CR1 +/CreER :: Mertk fl/fl ). (K) Representative mPFC images showing microglia (IBA1, red) and MERTK (green); “Coloc” indicates MERTK signal within IBA1 + microglia. Scale bar, 10 μm. (L) Fraction of IBA1 + microglia that are MERTK + . (M) Freezing during conditioning, extinction, and extinction retrieval in control and microglia-specific Mertk conditional knockout mice. CX3CR1 +/CreER , n = 12 mice; and CX3CR1 +/CreER :: Mertk fl/fl , n = 11 mice. Data are mean ± s.e.m. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test for two-group comparisons; two-way repeated-measures ANOVA for behavioral time courses, with post hoc tests as appropriate.$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Schematic of an “eat-me” pathway in which extracellularly exposed phosphatidylserine (PS) on ensemble membranes is bridged by Gas6 to the microglial phagocytic receptor MERTK, promoting engulfment. (B) Timeline for labeling extracellular PS with PSVue-647 (a membrane-impermeant PS probe delivered intracerebroventricularly through an implanted cannula) and assessing fear or extinction retrieval in FosTRAP2::Ai14;;CX3CR1-GFP mice (ensemble tagging as in ). (C) Representative mPFC confocal images showing microglia (CX3CR1-GFP, green), TRAP-labeled ensemble neurons (tdTomato, red), and PSVue signal (cyan). Right, “Coloc” indicates PSVue puncta segmented within microglial surfaces (i.e., PSVue-labeled PS-bearing material internalized by microglia). Scale bar, 10 μm. (D) Total PSVue signal per field (left) and fraction of PSVue signal associated with TRAP structures (right). Fear CS only, n = 12 sections (4 mice); Fear memory, n = 22 sections (6 mice); Ext. CS only, n = 29 sections (7 mice); Ext. memory, n = 26 sections (7 mice). N.S., not significant; *** P < 0.001, unpaired Student’s t test. (E) Quantification of PSVue + puncta detected within microglia (left) and TRAP⁺PSVue⁺ puncta within microglia (right). Fear CS only, n = 8 sections (3 mice); Fear memory, n = 16 sections (6 mice); Ext. CS only, n = 29 sections (7 mice); Ext. memory, n = 26 sections (7 mice). (F) Ensemble-targeted CRISPR–Cas9 strategy to disrupt Tmem16f selectively in extinction-tagged neurons in FosTRAP2::Cas9 mice using AAV-sgTmem16f-CAG-FLEX-mCherry or AAV-sgCtrl-CAG-FLEX-mCherry, with 4-OHT administered after extinction training to restrict editing to extinction ensembles. (G) Representative mPFC images showing mCherry-labeled extinction ensemble neurons (red) and TMEM16F immunoreactivity (cyan); “Coloc” indicates TMEM16F signal within mCherry⁺ surfaces. Scale bar, 5 μm. (H) TMEM16F knockdown efficiency in tdTomato + neurons (fraction of tdTomato + puncta positive for TMEM16F). sgCtrl, n = 20 sections (5 mice); sgTmem16f, n = 20 sections (5 mice). (I) Freezing during conditioning, extinction, re-extinction, and extinction retrieval in sgCtrl and sgTmem16f groups. sgCtrl, n = 12 mice; sgTmem16f, n = 14 mice. (J) Scheme for tamoxifen-induced microglia-specific deletion of Mertk (CX3CR1 +/CreER :: Mertk fl/fl ). (K) Representative mPFC images showing microglia (IBA1, red) and MERTK (green); “Coloc” indicates MERTK signal within IBA1 + microglia. Scale bar, 10 μm. (L) Fraction of IBA1 + microglia that are MERTK + . (M) Freezing during conditioning, extinction, and extinction retrieval in control and microglia-specific Mertk conditional knockout mice. CX3CR1 +/CreER , n = 12 mice; and CX3CR1 +/CreER :: Mertk fl/fl , n = 11 mice. Data are mean ± s.e.m. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test for two-group comparisons; two-way repeated-measures ANOVA for behavioral time courses, with post hoc tests as appropriate.

Techniques: Labeling, Membrane, CRISPR, Knockdown, Control, Knock-Out

$(A) Representative confocal images of TMEM16F immunoreactivity (cyan) and TRAP-labeled ensemble neurons (tdTomato, red) in mPFC from fear CS-only, fear retrieval (Fear memory), extinction CS-only, and extinction retrieval (Ext. memory) groups. Left, merged view; middle, TMEM16F channel; right, TMEM16F puncta overlapping TRAP signal ( Coloc ; heat map). Scale bar, 10 μm. (B) Quantification of TMEM16F signal. Left, total TMEM16F + area per field. Right, fraction of TMEM16F + puncta colocalized with TRAP⁺ elements. TMEM16F levels and TRAP association were unchanged by fear or extinction retrieval relative to the corresponding CS-only controls. Fear CS only, n = 20 sections (5 mice); Fear memory, n = 19 sections (5 mice); Ext. CS only, n = 20 sections (5 mice); Ext. memory, n = 20 sections (5 mice). (C) Representative confocal images of Gas6 immunoreactivity (cyan) and TRAP labeling (tdTomato, red) across the same behavioral conditions. Left, merged view; middle, 3D surface rendering of Gas6 signal ( Surfaces ); right, Gas6 puncta overlapping tdTomato + elements ( Coloc ; heat map). Scale bar, 10 μm. (D) Quantification of Gas6 signal. Left, total Gas6 + area per field. Right, fraction of Gas6 + puncta colocalized with tdTomato⁺ elements. Gas6 immunoreactivity decreased after fear retrieval but increased after extinction retrieval relative to the corresponding CS-only controls, whereas Gas6-TRAP overlap remained unchanged. Fear CS only, n = 11 sections (5 mice); Fear memory, n = 10 sections (5 mice); Ext. CS only, n = 10 sections (5 mice); Ext. memory, n = 10 sections (5 mice). (E) Representative confocal images of microglia (CX3CR1-GFP, green) and MERTK (cyan; heat map) in mPFC across conditions. Left, merged view; middle, microglial surface reconstructions ( Surfaces ); right, MERTK signal within microglial volumes ( Coloc ; heat map). Scale bar, 5 μm. (F) Quantification of MERTK signal. Left, total MERTK + area per field. Right, fraction of MERTK signal contained within microglia (MERTK colocalized with CX3CR1-GFP microglial volume / total MERTK). Both fear and extinction retrieval increased MERTK immunoreactivity and its enrichment in microglia relative to the corresponding CS-only controls. Fear CS only, n = 18 sections (7 mice); Fear memory, n = 20 sections (7 mice); Ext. CS only, n = 19 sections (7 mice); Ext. memory, n = 21 sections (7 mice). ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test (retrieval versus the corresponding CS-only control).$

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Representative confocal images of TMEM16F immunoreactivity (cyan) and TRAP-labeled ensemble neurons (tdTomato, red) in mPFC from fear CS-only, fear retrieval (Fear memory), extinction CS-only, and extinction retrieval (Ext. memory) groups. Left, merged view; middle, TMEM16F channel; right, TMEM16F puncta overlapping TRAP signal ( Coloc ; heat map). Scale bar, 10 μm. (B) Quantification of TMEM16F signal. Left, total TMEM16F + area per field. Right, fraction of TMEM16F + puncta colocalized with TRAP⁺ elements. TMEM16F levels and TRAP association were unchanged by fear or extinction retrieval relative to the corresponding CS-only controls. Fear CS only, n = 20 sections (5 mice); Fear memory, n = 19 sections (5 mice); Ext. CS only, n = 20 sections (5 mice); Ext. memory, n = 20 sections (5 mice). (C) Representative confocal images of Gas6 immunoreactivity (cyan) and TRAP labeling (tdTomato, red) across the same behavioral conditions. Left, merged view; middle, 3D surface rendering of Gas6 signal ( Surfaces ); right, Gas6 puncta overlapping tdTomato + elements ( Coloc ; heat map). Scale bar, 10 μm. (D) Quantification of Gas6 signal. Left, total Gas6 + area per field. Right, fraction of Gas6 + puncta colocalized with tdTomato⁺ elements. Gas6 immunoreactivity decreased after fear retrieval but increased after extinction retrieval relative to the corresponding CS-only controls, whereas Gas6-TRAP overlap remained unchanged. Fear CS only, n = 11 sections (5 mice); Fear memory, n = 10 sections (5 mice); Ext. CS only, n = 10 sections (5 mice); Ext. memory, n = 10 sections (5 mice). (E) Representative confocal images of microglia (CX3CR1-GFP, green) and MERTK (cyan; heat map) in mPFC across conditions. Left, merged view; middle, microglial surface reconstructions ( Surfaces ); right, MERTK signal within microglial volumes ( Coloc ; heat map). Scale bar, 5 μm. (F) Quantification of MERTK signal. Left, total MERTK + area per field. Right, fraction of MERTK signal contained within microglia (MERTK colocalized with CX3CR1-GFP microglial volume / total MERTK). Both fear and extinction retrieval increased MERTK immunoreactivity and its enrichment in microglia relative to the corresponding CS-only controls. Fear CS only, n = 18 sections (7 mice); Fear memory, n = 20 sections (7 mice); Ext. CS only, n = 19 sections (7 mice); Ext. memory, n = 21 sections (7 mice). ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired Student’s t test (retrieval versus the corresponding CS-only control).

Techniques: Labeling, Control

(A) Representative confocal images showing TRAP-labeled ensemble neurons (tdTomato, red) and CD47 immunoreactivity (cyan) in Fear CS-only, Fear memory (fear retrieval), Ext. CS-only, and Ext. memory (extinction retrieval) groups. Surfaces, 3D reconstructions of tdTomato + neuronal membranes; Coloc, CD47 signal associated with TRAP⁺ membranes. Scale bar, 10 µm. (B) Quantification of CD47 signal on tdTomato + membranes (left, CD47 + area per section) and CD47 enrichment on ensemble membranes (right; percentage of tdTomato + puncta that are CD47 + ). Fear CS only, n = 50 sections (5 mice); fear memory, n = 50 sections (5 mice); Ext. CS only, n = 60 sections (5 mice); Ext. memory, n = 40 sections (5 mice). (C) Representative images of microglia (CX3CR1-GFP, green) and SIRPα (cyan) in the four behavioral groups. Surfaces, 3D reconstructions of individual microglia; Coloc, SIRPα signal within microglial volumes. Scale bar, 5 µm. (D) Quantification of microglial SIRPα (left; SIRPα + area within microglia per section; right, SIRPα + area normalized to microglial area). Fear CS only, n = 19 sections (5 mice); fear memory, n = 20 sections (mice); Ext. CS only, n = 20 sections (5 mice); Ext. memory, n = 18 sections (5 mice). (E) Strategy for fear-ensemble–restricted Cd47 disruption. FosTRAP2::Cas9 mice received intra-mPFC microinjection of AAV-sgCD47-CAG-FLEX-mCherry or AAV-sgCtrl-CAG-FLEX-mCherry; 4-OHT after conditioning restricted editing to fear-activated (tdTomato + ) neurons. (F) Representative mPFC images after extinction retrieval showing reduced CD47 signal in mCherry-labeled fear ensembles in sgCd47 mice. Coloc, CD47 signal associated with tdTomato + neuronal volumes. Scale bar, 10 µm. (G) Cd47 disruption efficiency, quantified as the percentage of tdTomato + puncta that are CD47 + . sgCtrl, n = 28 sections (7 mice); sgCd47, n = 24 sections (6 mice). * P < 0.05, unpaired Student’s t test. (H) Behavioral effect of fear-ensemble CD47 disruption: accelerated extinction training and reduced freezing at extinction retrieval. sgCtrl, n = 7 mice; sgCd47, n = 6 mice. (I) Timeline for continuous intra-mPFC microinjection of infusion of a SIRPα-blocking antibody or IgG control (osmotic pump) in FosTRAP2::Ai14;;CX3CR1-GFP mice across conditioning, extinction training, and extinction retrieval. (J) Representative images after extinction retrieval showing microglia (CX3CR1-GFP, green) and fear ensembles (tdTomato, red) under IgG control or anti-SIRPα treatment. Surfaces , microglial reconstructions; Coloc , tdTomato + puncta detected within microglial volumes. Scale bar, 10 µm. (K) SIRPα blockade increased microglial engagement of fear ensembles (left, tdTomato + puncta per microglia; right, percent microglial coverage of tdTomato + neuronal bodies). IgG, n = 48 microglia (6 mice); anti-SIRPα, n = 55 microglia (6 mice). (L) Behavioral effect of SIRPα blockade: facilitated extinction and reduced freezing at extinction retrieval. IgG, n = 13 mice; anti-SIRPα, n = 13 mice. (M) Model: fear ensembles up-regulate CD47, engaging microglial SIRPα to inhibit phagocytosis (“don’t-eat-me” signaling). Data are mean ± s.e.m. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-sided unpaired Student’s t test for single comparisons; two-way repeated-measures ANOVA with appropriate post hoc tests for learning curves, and unpaired Student’s t test for retrieval comparisons, as applicable.

Journal: bioRxiv

Article Title: Microglial pruning of extinction-ensemble synapses preserves fear memory

doi: 10.64898/2026.05.05.722833

Figure Lengend Snippet: (A) Representative confocal images showing TRAP-labeled ensemble neurons (tdTomato, red) and CD47 immunoreactivity (cyan) in Fear CS-only, Fear memory (fear retrieval), Ext. CS-only, and Ext. memory (extinction retrieval) groups. Surfaces, 3D reconstructions of tdTomato + neuronal membranes; Coloc, CD47 signal associated with TRAP⁺ membranes. Scale bar, 10 µm. (B) Quantification of CD47 signal on tdTomato + membranes (left, CD47 + area per section) and CD47 enrichment on ensemble membranes (right; percentage of tdTomato + puncta that are CD47 + ). Fear CS only, n = 50 sections (5 mice); fear memory, n = 50 sections (5 mice); Ext. CS only, n = 60 sections (5 mice); Ext. memory, n = 40 sections (5 mice). (C) Representative images of microglia (CX3CR1-GFP, green) and SIRPα (cyan) in the four behavioral groups. Surfaces, 3D reconstructions of individual microglia; Coloc, SIRPα signal within microglial volumes. Scale bar, 5 µm. (D) Quantification of microglial SIRPα (left; SIRPα + area within microglia per section; right, SIRPα + area normalized to microglial area). Fear CS only, n = 19 sections (5 mice); fear memory, n = 20 sections (mice); Ext. CS only, n = 20 sections (5 mice); Ext. memory, n = 18 sections (5 mice). (E) Strategy for fear-ensemble–restricted Cd47 disruption. FosTRAP2::Cas9 mice received intra-mPFC microinjection of AAV-sgCD47-CAG-FLEX-mCherry or AAV-sgCtrl-CAG-FLEX-mCherry; 4-OHT after conditioning restricted editing to fear-activated (tdTomato + ) neurons. (F) Representative mPFC images after extinction retrieval showing reduced CD47 signal in mCherry-labeled fear ensembles in sgCd47 mice. Coloc, CD47 signal associated with tdTomato + neuronal volumes. Scale bar, 10 µm. (G) Cd47 disruption efficiency, quantified as the percentage of tdTomato + puncta that are CD47 + . sgCtrl, n = 28 sections (7 mice); sgCd47, n = 24 sections (6 mice). * P < 0.05, unpaired Student’s t test. (H) Behavioral effect of fear-ensemble CD47 disruption: accelerated extinction training and reduced freezing at extinction retrieval. sgCtrl, n = 7 mice; sgCd47, n = 6 mice. (I) Timeline for continuous intra-mPFC microinjection of infusion of a SIRPα-blocking antibody or IgG control (osmotic pump) in FosTRAP2::Ai14;;CX3CR1-GFP mice across conditioning, extinction training, and extinction retrieval. (J) Representative images after extinction retrieval showing microglia (CX3CR1-GFP, green) and fear ensembles (tdTomato, red) under IgG control or anti-SIRPα treatment. Surfaces , microglial reconstructions; Coloc , tdTomato + puncta detected within microglial volumes. Scale bar, 10 µm. (K) SIRPα blockade increased microglial engagement of fear ensembles (left, tdTomato + puncta per microglia; right, percent microglial coverage of tdTomato + neuronal bodies). IgG, n = 48 microglia (6 mice); anti-SIRPα, n = 55 microglia (6 mice). (L) Behavioral effect of SIRPα blockade: facilitated extinction and reduced freezing at extinction retrieval. IgG, n = 13 mice; anti-SIRPα, n = 13 mice. (M) Model: fear ensembles up-regulate CD47, engaging microglial SIRPα to inhibit phagocytosis (“don’t-eat-me” signaling). Data are mean ± s.e.m. N.S., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-sided unpaired Student’s t test for single comparisons; two-way repeated-measures ANOVA with appropriate post hoc tests for learning curves, and unpaired Student’s t test for retrieval comparisons, as applicable.

Techniques: Labeling, Disruption, Microinjection, Blocking Assay, Control

Fibrin abrogates microglia motility in 5XFAD mice. (A) Representative of in vivo 2P time-lapse imaging of microglia surveillance in Cx3CR1 GFP/+ mice. ( B-E ) Quantification of microglia motility surrounding amyloid plaque. Mean process motility(B), mean tip motility (C), protraction (D) and retraction (E). Data represents mean ± s.e.m from n = 6 5XFAD, n = 4 5XFAD: Fgg γ390-396A . *P <0.05, by student t-test. ( F-I ) Analysis of the morphology of amyloid plaque in 5XFAD and 5XFAD: Fgg γ390-396A mice by 3DMorph. Representative image (F) and quantification of ramification index (G), skeleton (H) and branch point (I) of microglia cluster surrounding amyloid plaque analyzed by 3DMorph. Data represents mean ± s.e.m from n = 10 5XFAD, n=8 5XFAD: Fgg γ390-396A . *P <0.05, **P <0.01, by student t-test. ( J ) Quantification of average entry per trial over 4 days in the Cost-Benefit Conflict (CBC) test. Data represents mean ± s.e.m from n = 13 NTG, n = 21 5XFAD, and n = 17 5XFAD: Fgg γ390-396A female mice and n = 4 NTG, n = 3 5XFAD, and n = 5 5XFAD: Fgg γ390-396A male mice, ns. ns, not significant. ( K ) Light/Dark preference determined by the percentage of time spent in the light side in the CBC test. Data represents mean ± s.e.m from n = 13 NTG, n = 21 5XFAD, and n = 17 5XFAD: Fgg γ390-396A female mice and n = 4 NTG, n = 3 5XFAD, and n = 5 5XFAD: Fgg γ390-396A male mice, ns. ns, not significant.

Journal: bioRxiv

Article Title: Resilience to neuronal hyperactivity and restoration of the neuroimmune interactome and decision-making by blocking fibrin in a model of Alzheimer’s disease

doi: 10.64898/2026.05.01.722077

Figure Lengend Snippet: Fibrin abrogates microglia motility in 5XFAD mice. (A) Representative of in vivo 2P time-lapse imaging of microglia surveillance in Cx3CR1 GFP/+ mice. ( B-E ) Quantification of microglia motility surrounding amyloid plaque. Mean process motility(B), mean tip motility (C), protraction (D) and retraction (E). Data represents mean ± s.e.m from n = 6 5XFAD, n = 4 5XFAD: Fgg γ390-396A . *P <0.05, by student t-test. ( F-I ) Analysis of the morphology of amyloid plaque in 5XFAD and 5XFAD: Fgg γ390-396A mice by 3DMorph. Representative image (F) and quantification of ramification index (G), skeleton (H) and branch point (I) of microglia cluster surrounding amyloid plaque analyzed by 3DMorph. Data represents mean ± s.e.m from n = 10 5XFAD, n=8 5XFAD: Fgg γ390-396A . *P <0.05, **P <0.01, by student t-test. ( J ) Quantification of average entry per trial over 4 days in the Cost-Benefit Conflict (CBC) test. Data represents mean ± s.e.m from n = 13 NTG, n = 21 5XFAD, and n = 17 5XFAD: Fgg γ390-396A female mice and n = 4 NTG, n = 3 5XFAD, and n = 5 5XFAD: Fgg γ390-396A male mice, ns. ns, not significant. ( K ) Light/Dark preference determined by the percentage of time spent in the light side in the CBC test. Data represents mean ± s.e.m from n = 13 NTG, n = 21 5XFAD, and n = 17 5XFAD: Fgg γ390-396A female mice and n = 4 NTG, n = 3 5XFAD, and n = 5 5XFAD: Fgg γ390-396A male mice, ns. ns, not significant.

Article Snippet: B6.129P2(Cg)-Cx3cr1tm1Litt/J( Cx3cr1 GFP/GFP ) and B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax(5XFAD) mice were obtained from the Jackson Laboratory.

Techniques: In Vivo, Imaging

Blockade of the fibrin inflammatory domain restores microglia contacts with neurons in AD mice (A) Microglia-neuron contact analysis. 3D reconstruction of microglia (GFP), active neurons (jRCaMP1b) and Aβ plaque (methoxy-X04) by Imaris and quantification of microglia-neuron contacts and distance of plaque-associated microglia to nearby active neuron. Data represents mean ± s.e.m. n = 8 5XFAD: Cx3cr1 GFP/+ and n = 5 5XFAD: Fgg γ390-396A : Cx3cr1 GFP/+ mice. * P < 0.05 by unpaired two-sample t-test. (B) Microglia response to spontaneous neuronal activity in awake mice. Average microglia voxels around each active neuron near Aβ plaque (within 50 μm). Data represents mean ± s.e.m. n = 9 5XFAD: Cx3cr1 GFP/+ and n = 6 5XFAD: Fgg γ390-396A : Cx3cr1 GFP/+ mice. * P < 0.05 by unpaired two-sample t-test. (C-D) SBEM (C) and 3D reconstruction (D) of microglial cell contacts with surrounding neurons and Aβ in 5XFAD and 5XFAD: Fgg γ390-396A mice. (C): Representative CLEM of microglia-neuron interactions in 5XFAD and 5XFAD: Fgg γ390-396A mice. Confocal images (left) show GFP-labeled microglia (green) and surrounding neurons labeled with DRAQ5 (white) near Aβ plaques labeled with methoxy-X04 (red). Corresponding SBEM image planes (right panels) illustrate ultrastructural details of microglial (micro) interactions with neurons (Neu) and Aβ plaques (Aβ) in each condition. Scale bars: 10 μm for confocal images, 1 μm for electron micrographs. (D) 3D reconstruction of microglia-neuron interactions within the neuronal connectome. Whole-body segmentation of microglia (green) and Aβ plaques (red) was performed using semi-automatic segmentation using CDeep3M, a machine-learning algorithm for large-scale EM data. Additionally, all neurons within the SBEM volume of each condition that physically interact with microglia were fully segmented (movie 2,3), including two representative neurons (yellow) and adjacent neurons (grey). Insets provide zoomed-in views of Aβ plaques and associated microglial fine processes, highlighting differences in microglial morphology and engagement between genotypes. Scale: 5XFAD, x = 97 µm, y = 172 µm, z = 38 µm. 5XFAD: Fgg γ390-396A , x = 102 µm, y = 173 µm, z = 42 µm.

Journal: bioRxiv

Article Title: Resilience to neuronal hyperactivity and restoration of the neuroimmune interactome and decision-making by blocking fibrin in a model of Alzheimer’s disease

doi: 10.64898/2026.05.01.722077

Figure Lengend Snippet: Blockade of the fibrin inflammatory domain restores microglia contacts with neurons in AD mice (A) Microglia-neuron contact analysis. 3D reconstruction of microglia (GFP), active neurons (jRCaMP1b) and Aβ plaque (methoxy-X04) by Imaris and quantification of microglia-neuron contacts and distance of plaque-associated microglia to nearby active neuron. Data represents mean ± s.e.m. n = 8 5XFAD: Cx3cr1 GFP/+ and n = 5 5XFAD: Fgg γ390-396A : Cx3cr1 GFP/+ mice. * P < 0.05 by unpaired two-sample t-test. (B) Microglia response to spontaneous neuronal activity in awake mice. Average microglia voxels around each active neuron near Aβ plaque (within 50 μm). Data represents mean ± s.e.m. n = 9 5XFAD: Cx3cr1 GFP/+ and n = 6 5XFAD: Fgg γ390-396A : Cx3cr1 GFP/+ mice. * P < 0.05 by unpaired two-sample t-test. (C-D) SBEM (C) and 3D reconstruction (D) of microglial cell contacts with surrounding neurons and Aβ in 5XFAD and 5XFAD: Fgg γ390-396A mice. (C): Representative CLEM of microglia-neuron interactions in 5XFAD and 5XFAD: Fgg γ390-396A mice. Confocal images (left) show GFP-labeled microglia (green) and surrounding neurons labeled with DRAQ5 (white) near Aβ plaques labeled with methoxy-X04 (red). Corresponding SBEM image planes (right panels) illustrate ultrastructural details of microglial (micro) interactions with neurons (Neu) and Aβ plaques (Aβ) in each condition. Scale bars: 10 μm for confocal images, 1 μm for electron micrographs. (D) 3D reconstruction of microglia-neuron interactions within the neuronal connectome. Whole-body segmentation of microglia (green) and Aβ plaques (red) was performed using semi-automatic segmentation using CDeep3M, a machine-learning algorithm for large-scale EM data. Additionally, all neurons within the SBEM volume of each condition that physically interact with microglia were fully segmented (movie 2,3), including two representative neurons (yellow) and adjacent neurons (grey). Insets provide zoomed-in views of Aβ plaques and associated microglial fine processes, highlighting differences in microglial morphology and engagement between genotypes. Scale: 5XFAD, x = 97 µm, y = 172 µm, z = 38 µm. 5XFAD: Fgg γ390-396A , x = 102 µm, y = 173 µm, z = 42 µm.

Article Snippet: B6.129P2(Cg)-Cx3cr1tm1Litt/J( Cx3cr1 GFP/GFP ) and B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax(5XFAD) mice were obtained from the Jackson Laboratory.

Techniques: Activity Assay, Labeling

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