Journal: bioRxiv

Article Title: Microglia Depletion Selectively Eliminates a Singular Form of Hippocampal Long-Term Potentiation

doi: 10.1101/2022.07.29.501926

Figure Lengend Snippet: A , A’) Images of the same hippocampal cross section viewed in different color channels showing DAPI-labeling of cellular nuclei (A, inverse contrast image) and Iba1 immunoreactivity (A’) in a slice from a vehicle-treated mouse fixed promptly after preparation (SG: dentate gyrus stratum granulosum; CA1 stratum pyramidale at right). Microglia appear white in A’-C. The dashed rectangle in A indicates the placement of the higher magnification images from which the Iba1-immunoreactive cells were quantified. B, C) Representative images of the quantification field showing Iba1 immunoreactivity in slices from vehicle- (B) and PLX5622- (C) treated mice: a severe depletion of Iba1-immunoreactive cells is evident in the PLX5622 case (arrow in C indicates the single immunolabeled cell in the field). Calibration bar in C indicates 200 μm for A and A’, 100 μm for B and C.

Article Snippet: To deplete brain microglia, mice were provided chow containing PLX5622 (1200 ppm) (Cayman Chemical Company, Ann Arbor, MI, USA), an antagonist of the CSF1R which is critical for microglial survival ( ; ).

Techniques: Labeling, Immunolabeling

Journal: bioRxiv

Article Title: Microglia Depletion Selectively Eliminates a Singular Form of Hippocampal Long-Term Potentiation

doi: 10.1101/2022.07.29.501926

Figure Lengend Snippet: Acute hippocampal slices from mice fed either control (VEH) or PLX5622- (PLX) chow for 7-12 days were assessed for treatment effects on LPP transmission and LTP. A ) Input-output curves from the outer molecular layer of the DG for VEH and PLX treated animals; VEH n=8, PLX n=5. B) The response profile to a ten-pulse, 40 Hz train of stimulation was comparable between VEH and PLX groups, both showing the typical response facilitation followed by depression (F 9,135 =0.3653, p=0.9497, VEH n=7; PLX n=10; two-way RM ANOVA). C ) After recording stable baseline responses, a single 100 Hz HFS train was used to induce LTP (at arrow). Both VEH and PLX groups exhibited an initial short-term potentiation and decline to a stable potentiated response, but the magnitude of potentiation was markedly reduced in the PLX group relative to VEH-controls. Representative traces show responses before (solid) and after (dashed) induction of LTP. Scale bars: x=1 mV, y=10 ms. D) Plot of the mean potentiation at 55-60 min post-induction showed mice treated with PLX had significantly reduced LPP-DG LTP relative to VEH mice (**p=0.0029, VEH n=9; PLX n=9; two-tailed unpaired t-test).

Article Snippet: To deplete brain microglia, mice were provided chow containing PLX5622 (1200 ppm) (Cayman Chemical Company, Ann Arbor, MI, USA), an antagonist of the CSF1R which is critical for microglial survival ( ; ).

Techniques: Transmission Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Microglia Depletion Selectively Eliminates a Singular Form of Hippocampal Long-Term Potentiation

doi: 10.1101/2022.07.29.501926

Figure Lengend Snippet: A) Input/output curves for the MPP were comparable in slices from VEH- and PLX5622- (PLX) chow treated mice. B) Plot shows MPP-fEPSP responses to a ten pulse, 40 Hz train were comparable in VEH and PLX mice: in both groups responses were depressed by about 30% over the course of the train (F 9,108 = 1.09, p= 0.379; VEH n=5, PLX n=9; two-way RM ANOVA). C) After recording stable baseline responses for 20 min, MPP potentiation was induced using three, 500ms high frequency stimulation trains (100Hz each) separated by 20 sec (at arrow). Both VEH and PLX treated animals exhibited a marked potentiation. Scale bars: x=1 mV, y=10 ms. D) Plot of the mean fEPSP response recorded 55-60 min post-induction showed no difference in the degree of potentiation in slices from VEH and PLX mice (p=0.2768; VEH n=7, PLX n=6; two-tailed unpaired t-test).

Article Snippet: To deplete brain microglia, mice were provided chow containing PLX5622 (1200 ppm) (Cayman Chemical Company, Ann Arbor, MI, USA), an antagonist of the CSF1R which is critical for microglial survival ( ; ).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Microglia Depletion Selectively Eliminates a Singular Form of Hippocampal Long-Term Potentiation

doi: 10.1101/2022.07.29.501926

Figure Lengend Snippet: A) SC input/output curves were comparable between treatment groups. B) Plot shows SC fEPSP responses to a 10 pulse, 40 Hz train were comparable in VEH and PLX mice (F 9,99 = 0.3022, p=0.9725; VEH n=6, PLX5622 n=7; two-way RM ANOVA). C) There was no reliable effect of PLX on the area of the response to individual theta bursts used to induce LTP (F 4,52 =0.0812, p=0.988; VEH n=8, PLX n=7). D) A single train of 5 theta bursts elicited comparable potentiation in slices from VEH- and PLX-mice. Representative fEPSP traces collected before (solid line) and after (dotted line) LTP are shown for the two groups. The bar graph (right) summarizes the normalized slopes at 55-60 post TBS (p=0.2365; VEH n=7, PLX n=9; two-tailed unpaired t-test). Scale bars: x=1 mV, y=10 ms.

Article Snippet: To deplete brain microglia, mice were provided chow containing PLX5622 (1200 ppm) (Cayman Chemical Company, Ann Arbor, MI, USA), an antagonist of the CSF1R which is critical for microglial survival ( ; ).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Microglia Depletion Selectively Eliminates a Singular Form of Hippocampal Long-Term Potentiation

doi: 10.1101/2022.07.29.501926

Figure Lengend Snippet: LPP fEPSP responses were collected from slices from naiive mice and were infused with VEH or 1μM of PLX5622 (solid bar). A) After infusion of VEH or PLX for 1 hour, a HFS (1 sec, 100Hz) was applied to induce LPP-LTP; the two groups exhibited similar levels of potentiation. Scale bars: x=1 mV, y=10 ms. B) Plot of the mean percent potentiation at 55-60 min after HFS shows comparable LPP-LTP in the Veh and PLX groups (p=0.590; VEH n=11, PLX n=8; two-tailed unpaired t-test).

Article Snippet: To deplete brain microglia, mice were provided chow containing PLX5622 (1200 ppm) (Cayman Chemical Company, Ann Arbor, MI, USA), an antagonist of the CSF1R which is critical for microglial survival ( ; ).

Techniques: Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Gestational exposure to PLX5622 significantly depletes CSF1R-expressing cells. (A) Schematic illustrating collection of Csf1r EGFP + cells for flow cytometry. (B) Quantification of EGFP + cells from Csf1r EGFP craniofacial tissues ( n =3-10 embryos per sex/treatment/time-point from two to four dams). (C-W) Immunofluorescence images and quantification of CSF1R + and Csf1r EGFP+ cells in and around the E15.5 nasal septum (C-E), Meckel's cartilage (F-H), ear (I-K), maxillary incisor (L-N), eye (O-Q), trigeminal (R-T) and tongue (U-W). Arrows mark CSF1R and Csf1r EGFP double-positive cells ( n =3 embryos per sex/treatment from two or three dams). c.d., cochlear duct; d.p., dental papilla; e.k., enamel knot; ey, eye; l.s.c., lateral semicircular canal; m.c., Meckel's cartilage; n.s., nasal septum; s.r., stellate reticulum; tg, tongue; ut, utricle. Blue dots represent male and pink dots represent female. Counts represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques: Expressing, Flow Cytometry, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Prenatal exposure to PLX5622 disrupts osteoclast development and function. (A-D′) Csf1r EGFP + osteoclasts (arrows) in the E15.5 premaxilla (A-B′) and maxilla (C-D′). (E-I) Immunofluorescence images (E-H) and quantification (I,I′) of multinucleated CTSK/ Csf1r EGFP double-positive osteoclasts (arrows) (I) and Csf1r EGFP+ single-positive osteoclasts (I′) in the E15.5 mandible. (J-S′) TRAP staining (arrows) in E15.5 premaxilla (J-K′), mandible (L-M′), maxilla (N-O′), frontal (P-Q′) and basioccipital (R-S′) bones. (T-V) Quantification of total TRAP + area in premaxilla (T), mandible (U) and maxilla (V). n =3 embryos per sex/treatment from two or three dams. bo, basioccipital; fr, frontal; m.c, Meckel's cartilage; md, mandible; mx, maxilla pmx, premaxilla. Counts represent mean±s.e.m. and were analyzed by an ART ANOVA (I,I′) or a two-way ANOVA (T-V) with Tukey's post-hoc test. A′-F′ show magnifications of the respective boxed areas in A-F.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques: Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Exposure to PLX5622 during embryogenesis increases bone density in the premaxilla and mandible. (A-J′) E15.5 Sp7 osteoblast staining in the premaxilla (A-B′) mandible (C-D′), maxilla (E-F′), frontal (G-H′) and basioccipital (I-J′) bones of CD1 embryos. (K-T′) E15.5 von Kossa and Alcian Blue staining in the premaxilla (K-L′), mandible (M-N′), maxilla (O-P′), frontal (Q-R′) and basioccipital (S-T′) bones of CD1 embryos. (U-W) Quantification of unmineralized area within the premaxilla (U) and mandible (V), and mineralized area in the maxilla (W). n =3 embryos per sex/treatment from two or three dams. bo, basioccipital; fr, frontal; m.c, Meckel's cartilage; md, mandible; mx, maxilla; pmx, premaxilla. Measurements represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques: Staining

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Prenatal exposure to PLX5622 disrupts craniofacial bone morphogenesis. (A-D′) Lateral (A-B′) and dorsal (C-D′) views of P1 CD1 skulls. Arrows in A-B′ indicate skull doming. Arrowheads in C-D′ mark interfrontal suture. Arrows in C-D′ mark sagittal suture. (E-G) Quantification of skull (E), interparietal (F) and occipital (G) bone lengths. (H-K) Lateral view of P1 CD1 mandibles (H-I′) and quantification of mandible length (J) and height (K). Arrows in H-I′ indicate measured lengths. (L-Q) Lateral view of the P1 CD1 ear (L-M′) and quantification of tympanic ring length (N) and thickness (O), and malleus (P) and stapes (Q) lengths. Arrows mark incus (absent in PLX5622 pups, asterisks), stapes and malleus. Arrowheads in L-M′ mark tympanic ring. Triangular arrowheads in L-M′ mark otic capsule. (R-U′) Ventral views of the P1 CD1 palate. Arrows in R-S″ mark palatine sutures. Arrowheads in R-S″ indicate altered palate morphology. (V) Quantification of premaxilla thickness. Double-headed arrows in R-S″ indicate measured thickness. (W-X′) Ventral view of the P1 CD1 cranial base. n =5-8 pups per sex/treatment from two to four dams. bo, basioccipital; bs, basisphenoid; fr, frontal bone; in, incus; ip, interparietal; ma, malleus; mx, maxilla; na, nasal bone; oc, occipital; pa, parietal bone; p.b., palatine bone; pmx, premaxilla; p.p.mx., palatine process of the maxilla; p.p.p., palatine process of the palatine; pt, pterygoid process; st, stapes; t.r., tympanic ring. Measurements represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques:

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Prenatal exposure to PLX5622 disrupts cranial base and brain development. (A-D) Lateral view of µCT scans of P1 CD1 skulls show presphenoid bone and inter-sphenoid synchondrosis. (E-K) Quantification of posterior cranial base length (E), anterior frontal complex length (F), basioccipital length (G), basisphenoid length (H), presphenoid length (I), spheno-occipital synchondrosis width (J) and inter-sphenoid synchondrosis width (K). (L-S) Coronal view of µCT scans of P1 CD1 skulls across the rostral (L-O) to caudal (P-S) axis. Arrows mark lateral (VL) and third (V3) ventricles. n =3-5 pups per sex/treatment from two or three dams. a.f.c., anterior frontal complex; bo, basioccipital; bs, basisphenoid; et, ethmoid; i.s.s., inter-sphenoid synchondrosis; p.c.b., posterior cranial base; ps, presphenoid bone; s.o.s., spheno-occipital synchondrosis. Measurements represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques:

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Exposure to PLX5622 during embryogenesis disrupts cytokine and chemokine secretion in craniofacial tissue cultures. (A) Schematic illustrating craniofacial tissue culture and Luminex multiplex analysis. (B-O) Cytokine/chemokine analysis of E13.5 craniofacial tissue culture media. n =9 per sex/treatment from three dams. Quantifications represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques: Luminex, Multiplex Assay

Journal: Development (Cambridge, England)

Article Title: CSF1R + macrophage and osteoclast depletion impairs neural crest proliferation and craniofacial morphogenesis

doi: 10.1242/dev.205423

Figure Lengend Snippet: Gestational exposure to PLX5622 decreases neural crest proliferation. (A) Schematic illustrating sphere assay. (B-M) E12.5 Wnt1 Cre ; Rosa26 tdTomato neural crest-derived spheres express PDGFRα (B,C,H,I), SOX2 (D,E,J,K) and SOX10 (F,G,L,M). (N) Primary sphere quantification ( n =6-9 embryos per sex/treatment from two or three dams). (O-Z) Immunofluorescence images of E15.5 Wnt1 Cre -driven tdTomato + signal (O-P′,U-V′) and Ki67 staining (R-S′,X-Y′) in the premaxilla (O-T) and mandible (U-Z). Quantification of tdTomato + (Q,W) and Ki67 + (T,Z) cells. White lines mark the tissue regions used for quantifications ( n =3 embryos per sex/treatment from three dams). DIV, days in vitro ; m.c., Meckel's cartilage; pmx, premaxilla. Counts represent mean±s.e.m. and were analyzed by a two-way ANOVA with Tukey's post-hoc test.

Article Snippet: Depletion of CSF1R + cells was achieved by administering the Plexxikon CSF1R inhibitor PLX5622 (1200 PPM added to chow AIN-76A, Research Diets) to pregnant female mice starting at E3.5.

Techniques: Derivative Assay, Immunofluorescence, Staining, In Vitro

Journal: Nature Communications

Article Title: Ontogeny of arterial macrophages defines their functions in homeostasis and inflammation

doi: 10.1038/s41467-020-18287-x

Figure Lengend Snippet: a – c CSF1R-inhibition in wildtype mice. a Schematic of macrophage depletion by CSF1R-inhibiton for 3 days (PLX5622 chow) followed by 7 days of control chow (recovery) and number of macrophages (Mϕ) in the adventitia of the respective treatment group ( n = 7 for ctrl chow, n = 6 for 3 days of PLX5622; n = 8 for 3 days of PLX5622 followed by 7 days of control chow; two individual experiments for each condition). b Representative FACS analysis of all adventitial macrophages isolated from individual mice fed with either control chow (upper panel), 3 days of PLX5622 (middle panel) or 3 days of PLX followed by 7 days of control chow (lower panel). Adventitial macrophages were gated on CD45+, lineage− (CD11C, Ly6G, TER119, Siglec-F, TCR-ß, Nk1.1), CD11b+, F480 hi . c Representative immunohistology of macrophages in the adventitia ( n = 3, three independent experiments). d – j CSF1R-inhibition in Flt3 Cre Rosa26 mTmG mice. d Percentage of GFP+ macrophages in the adventitia ( n = 7 for ctrl chow, n = 6 for 3 days of PLX5622; n = 4 for 3 days of PLX5622 followed by 7 days of control chow; two individual experiments for each condition). e Flow cytometry of tomato and GFP expression in adventitial macrophages. f Histological quantification of GFP + CD68 + macrophages ( n = 3 for ctrl chow, n = 5 for 3 days of PLX5622; n = 4 for 3 days of PLX5622 followed by 7 days of control chow; 2 individual experiments for each condition). g, h Macrophage proliferation after depletion by PLX5622 in Flt3 Cre Rosa26 eYFP mice and injection of EdU i.p. either g 5, 3, and 1 days before euthanization ( n = 3 for ctrl chow, n = 3 for 3 days of PLX5622 followed by 7 days of control chow; 1 individual experiments for each condition) or i only once 2 h before euthanization (to determine local proliferation; n = 4; 1 individual experiment). h, j Representative immunohistological images from aortas. Scale bar as depicted in images. Two-sided t -tests was performed and mean ± SD is shown.

Article Snippet: Control and PLX5622 (300 ppm formulated in AIN-76A standard chow, Research Diets, Inc.) chows were kindly provided by Plexxikon Inc (Berkeley, CA).

Techniques: Inhibition, Control, Isolation, Hi-C, Flow Cytometry, Expressing, Injection