1. Volume 155, Issue 7, Pages 1596-1609 (December 2013)
Microglia Promote Learning-Dependent Synapse Formation through Brain-Derived Neurotrophic Factor 
Christopher N. Parkhurst, Guang Yang, Ipe Ninan, Jeffrey N. Savas, John R. Yates, Juan J. Lafaille, Barbara L. Hempstead, Dan R. Littman, Wen-Biao Gan 
Cell 
Volume 155, Issue 7, Pages (December 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Generation of Mice Carrying the CX3CR1CreER Allele
(A) Schematic of the targeting strategy used for knockin of CreER-IRES-YFP at the CX3CR1 locus.
(B) Southern blot analysis of AflII digested genomic DNA from untargeted (WT), CX3CR1CreER-targeted, or CX3CR1CreER mice after deletion of the neomycin resistance sequence.
(C) Coronal sections of motor cortex from P45 CX3CR1CreER mice stained for EYFP, Iba1, and NeuN.
(D) Coronal sections of motor cortex from mice of the indicated genotypes and treatments (scale bar, 100 μm).
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4. Figure 2
A Strategy to Restrict Cre-Mediated Manipulation of Gene Function, Including Deletion of Microglia
(A) CX3CR1-EYFP+ CD11b+ populations in various tissues from mice of the indicated genotypes 5 or 30 days after tamoxifen treatment.
(B) Coronal sections of motor cortex from CX3CR1CreER/+:R26DsRed/+ mice stained for EYFP and DsRed 30 days after tamoxifen treatment.
(C) Quantification of flow-cytometry fluorescence-activated cell sorting (FACS) analysis showing the percentage of CX3CR1-EYFP+ cells coexpressing DsRed in multiple tissues at 5 or 30 days after tamoxifen treatment.
(D) Time course of tamoxifen/DT administration and analysis.
(E) FACS analysis of microglia in the brain of control and microglia-depleted mice at the indicated time points after DT administration. Dot plots show the total number of CX3CR1-EYFP+ CD11b+ cells gated on DAPI− CD3− CD19− CD45int.
(F) Coronal sections of motor cortex from control or microglia-depleted mice stained for Iba1 1 day after DT administration.
(G) Number of CX3CR1-EYFP+ CD11b+ microglia in the brain after DT administration at various time points.
(H) FACS analysis showing the percentage of CX3CR1-EYFP+ CD11b+ cells in the spleen and blood of mice after DT administration.
(I) Quantification of data shown in (H). n = 4 animals for each experimental condition. Data are represented as mean ± SEM. ∗∗∗∗p < , ∗p < Scale bar, 100 μm.
See also Figures S1, S2, and S3.
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5. Figure 3
Microglia Are Important for Learning-Dependent Spine Remodeling and Performance Improvement
(A) Timeline of tamoxifen/DT administration and in vivo imaging in CX3CR1-iDTR mice.
(B) Transcranial two-photon imaging of dendritic spines in control and microglia-depleted mice. Filled and empty arrowheads indicate spines formed or eliminated between two views. Asterisk indicates filopodia.
(C and D) The percentage of spines that were formed or eliminated within 4 days in the motor cortex was significantly reduced after microglia depletion in both P19 (C) and P30 animals (∗p < 0.05, ∗∗p < 0.01, n = 4–6).
(E) Timeline of tamoxifen/DT administration, rotarod training, and in vivo imaging.
(F) Motor-learning-related spine remodeling was significantly reduced in P30 mice with microglia depletion (∗p < 0.05, ∗∗p < 0.01, n = 4–5).
(G) Motor-learning-related spine formation was significantly reduced in P60 mice with microglia depletion (∗∗p < 0.01, n = 4–5).
(H) Average speed reached during the first rotarod training session in P30 mice (n = 6–7).
(I) Average speed reached during the first rotarod training session in P60 mice (n = 8).
(J) Microglia-depleted mice showed impaired performance improvement compared with nondepleted control mice over 1 or 2 days of training (∗p < 0.05, n = 6–7).
(K) P60 microglia-depleted mice showed impaired performance improvement compared with nondepleted control mice over 1 or 2 days of training (∗p < 0.05, n = 8).
(L) Percentage of freezing in control or microglia-depleted mice before (pre-CS) and during (CS) presentation of the conditioned stimulus in the recall test (∗p < 0.05, n = 8).
(M) The discrimination ratio of time spent interacting with a novel object versus a familiar object in a NOR assay was significantly altered in microglia-depleted mice (∗p < 0.05, n = 8). Data are represented as mean ± SEM.
See also Figure S4.
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6. Figure 4
Biochemical and Electrophysiological Properties of Synapses Are Altered in Microglia-Depleted Brains
(A) Quantitative proteomic scheme to identify CNS proteins altered after microglial depletion. Control (n = 3) or microglia-depleted (n = 3) brain homogenates were mixed 1:1 with 15N internal standard and prepared together. Samples were then analyzed by LCLC-MS/MS shotgun proteomics. Green dots represent microglia.
(B) Proteomic summary volcano plot (x axis = log2 CX3CR1CreER/+/CX3CR1CreER/+:R26iDTR/+; y axis = −log10 ANOVA p value). Black open circles: quantified proteins; red open circles: significantly altered proteins; green filled circles: significantly altered proteins with known synaptic functions.
(C) Synaptosome fractions from control or microglia-depleted brains probed with indicated antibodies by western blot.
(D) Densitometric quantification of western blots in (C) (∗p < 0.05, n = 6).
(E) Examples of NMDA mEPSCs in layer V pyramidal neurons from control and microglia-depleted mice.
(F) Average NMDA mEPSC frequency, amplitude, and decay time in control (n = 17 cells) and microglia-depleted mice (n = 17 cells). mEPSC frequency and decay time were significantly reduced in microglia-depleted mice (p < 0.001).
(G) Examples of AMPA mEPSCs in layer V pyramidal neurons from control and microglia-depleted mice.
(H) Average mEPSC frequency, amplitude, and decay time in control (n = 9 cells) and microglia-depleted mice (n = 8 cells). mEPSC frequency was significantly reduced in microglia-depleted mice (p < 0.05). Data are represented as mean ± SEM.
See also Table S1.
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7. Figure 5
Loss of Microglial BDNF Results in Altered Synaptic Protein Levels, Synaptic Structural Plasticity, and Performance Improvement after Learning
(A) PCR-based analysis of WT (BDNFWT), conditional undeleted (BDNFflox), and conditional deleted (BDNFΔ) BDNF alleles from CX3CR1-EYFP− and CX3CR1-EYFP+ cells sorted from the CNS of CX3CR1CreER/+:BDNFflox/+ or CX3CR1CreER/+:BDNFflox/flox after tamoxifen treatment.
(B) Quantitative real-time PCR analysis of BDNF mRNA isolated from CX3CR1-EYFP+ microglia purified from BDNFflox/+ or BDNFflox/flox mice (∗∗p < 0.01, n = 3).
(C) Average protein levels of total BDNF in the cortex or hippocampus of CX3CR1CreER/+:BDNFflox/+ or CX3CR1CreER/+:BDNFflox/flox mice as measured by ELISA (n = 4).
(D) Synaptosome fractions from the brains of CX3CR1CreER/+:BDNFflox/+ or CX3CR1CreER/+:BDNFflox/flox mice probed with indicated antibodies.
(E) Densitometric quantification of western blots in (D) (∗p < 0.05, n = 6).
(F) Transcranial two-photon imaging of dendritic spines in Thy1 YFP mice crossed with CX3CR1CreER/+:BDNFflox/+ or CX3CR1CreER/+:BDNFflox/flox mice before or after rotarod training. Filled and empty arrowheads indicate spines that were formed or eliminated, respectively, between two views. Asterisk indicates filopodia. Scale bar, 2 μm.
(G) Percentage of existing spines that were eliminated or new spines that formed over 2 days of training in the motor cortex of BDNFflox/+ or BDNFflox/flox mice (∗∗∗p < 0.001, n = 4).
(H) Average speed reached during the first rotarod training session (n = 5–7).
(I) Performance increase in motor-learning task over 1 or 2 days of rotarod training (∗p < 0.05; error bars, SEM; n = 5–7).
(J) Percentage of freezing in control CX3CR1CreER/+:BDNFflox/+ and CX3CR1CreER/+:BDNFflox/flox mice before (pre-CS) and during (CS) presentation of the conditioned stimulus in the recall test (∗p < 0.05, n = 6–7).
(K) The discrimination ratio of time spent interacting with a novel object versus a familiar object in a NOR assay was significantly altered in mice depleted of microglial BDNF (∗p < 0.05, n = 8). Data are represented as mean ± SEM.
See also Figure S5.
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8. Figure 6
Microglia Produce Both Pro-BDNF and mBDNF to Phosphorylate Neuronal TrkB
(A) Neurons or microglia were cultured from P1 mice from BDNF-HA or WT animals. Cell lysates or culture media were immunoprecipitated with a rabbit antibody to HA. Pro-BDNF and mBDNF were detected by immunoblotting with a second antibody to HA (mouse HA1.1).
(B) Synaptosome westerns for p-TrkB from CX3CR1CreER/+:BDNFflox/+ or CX3CR1CreER/+:BDNFflox/flox mice (∗p < 0.05, n = 6).
(C) Representative immunoblots of E18 rat neurons at 8 days in vitro, treated as indicated.
(D) Densitometric quantification of p-TrkB western blots in (C) (∗p < 0.05, ∗∗p < 0.005, n = 9). Data are represented as mean ± SEM.
See also Figure S6.
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9. Figure S1
Characterization of Multiple Myeloid Populations in CX3CR1CreER Mice and Tamoxifen Pulse Labeling Strategy for Microglia, Related to Figure 2
(A) Representative FACS analysis of CNS, blood, or spleen tissues from WT or CX3CR1CreER/+ mice at P14 or P30. Dot plots show percentage of cells in boxed regions gated on DAPI− CD3− CD19−.
(B) Percentage of indicated cell populations in (A) in various tissues in WT or CX3CR1CreER/+ mice at P14 or P30 (error bars = SEM, n = 4).
(C) Diagram of pulse labeling strategy for the restriction of tamoxifen induced recombination to microglia. (i) Prior to tamoxifen, CX3CR1CreER/+:R26DsRed/+ mice express only EYFP in both CNS and peripheral CX3CR1+ populations. Peripheral CX3CR1+ cells are continuously replaced from a CX3CR1− progenitor population within the bone marrow (white cells). (ii) 5 days after tamoxifen treatment, both microglia and peripheral CX3CR1+ cells express both EYFP and DsRed. (iii) Over a period of 25 days, peripheral CX3CR1+ cells turnover and are replenished from a pool of bone marrow progenitors by new CX3CR1+ cells that have not undergone recombination. After 30 days, most peripheral CX3CR1+ cells have turned over and therefore express only EYFP, while microglia remain recombined and coexpress EYFP and DsRed.
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10. Figure S2
Depletion of Microglia Does Not Result in Inflammatory and Astrocytic Changes to Surrounding Tissue, Related to Figure 2
(A) Percent of starting weight measured over 1 week after administration of DT in control or microglia-depleted mice (n = 7 mice per group).
(B) Real-time PCR measurement of mRNA expression for cytokines in CNS tissue from CX3CR1CreER or CX3CR1CreER:R26iDTR/+ mice (error bars = SEM, n = 4).
(C and D) ELISA measurements of protein levels of cytokines from cortex (C) or hippocampus (D) of control or microglia-depleted mice (error bars = SEM, n = 4).
(E) Coronal sections of motor cortex or hippocampal CA1 region stained for the astrocytic marker GFAP in CX3CR1CreER/+ or CX3CR1CreER/+:R26iDTR/+ mice at indicated time-points after DT administration (scale bar = 50 μm).
(F) Coronal sections of motor cortex or hippocampal CA1 region stained for the glutamate transporter GLT-1 in CX3CR1CreER or CX3CR1CreER/+:R26iDTR/+ mice at indicated time points after DT administration (scale bar = 50 μm).
(G–J) Percentage of GFAP (G and H) or GLT-1 (I and J) immunoreactivity within imaged cortical or CA1 region in control or microglia-depleted mice (error bars = SEM, n = 16 fields in 4 mice per group).
(K) BBB permeability as measured by the amount of Evans Blue dye per mg of CNS tissue in control or microglia-depleted mice (error bars = SEM, n = 4).
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11. Figure S3
Depletion of Microglia Does Not Cause Changes in the Number of Neurons, Synaptic Density, or Levels of Apoptosis in Cortex or Hippocampus, Related to Figure 2
(A) Coronal sections from motor cortex and hippocampal CA1 region from control or microglia-depleted mice stained for the neuronal marker NeuN, presynaptic marker SV2, or marker of apoptosis cleaved caspase 3 (scale bar = 50 μm). No staining of cleaved caspase 3 was detected.
(B and C) Quantification of number of NeuN+ cells (B) or percentage of SV2 immunoreactivity (C) in motor cortex and hippocampal CA1 region (error bars = SEM, n = 16 fields in 4 mice per group).
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12. Figure S4
Loss of One Allele of CX3CR1 Does Not Alter Baseline Spine Plasticity or Rotarod Performance Improvement, Related to Figure 3
(A) Spine formation and elimination over 4 days in P30 Thy1 YFP-H line mice or CX3CR1CreER/+:Thy1 YFP-H mice injected with DT (n = 4).
(B) Rotarod performance improvement in P30 Thy1 YFP-H line mice or CX3CR1CreER/+:Thy1 YFP-H mice injected with DT (n = 7-8). Error bars represent SEM.
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13. Figure S5
Sorting Microglia from CX3CR1CreER:BDNFflox Mice for the Measurement of Total Brain BDNF Levels and Characterizing the Number of Neurons, Synaptic Density, or Levels of Apoptosis in Cortex or Hippocampus after Microglial BDNF Removal, Related to Figure 5
(A) DAPI− cells were isolated, and CD45− and CD45+ cell populations were selected. Cells were then sorted based on CX3CR1-EYFP and CD11b into EYFP+ (microglia) and EYFP− (undefined CNS cell type) populations for further analysis. Postsorting analysis confirmed a high degree of purity for each sorted population.
(B) Coronal sections from indicated genotypes and CNS regions stained for the neuronal marker NeuN, presynaptic marker SV2, or marker of apoptosis cleaved caspase 3 (scale bar = 50 μm). No staining of cleaved caspase 3 was detected.
(C and D) Quantification of number of NeuN+ cells (C) or percentage of SV2 immunoreactivity (D) in motor cortex and hippocampal CA1 region (error bars = SEM, n = 16 fields in 4 mice per group).
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14. Figure S6
Determining the Potential Effect of Loss of Microglial BDNF on Microglial Proliferation, Cell Number, Cell Distribution, or the Percentage of Dendritic Spines Located in Close Proximity to Microglial Processes, Characterizing the Purity of Microglial Cultures, and Quantifying BDNF mRNA Levels in CX3CR1CreER/+:BDNFflox/flox Primary Microglia Cultures, Related to Figure 6
(A) Representative FACS analysis of CNS tissue from CX3CR1CreER/+:BDNFflox/+ (BDNFflox/+) or CX3CR1CreER/+:BDNFflox/flox mice (BDNFflox/flox) mice after tamoxifen treatment. Dot plots show percentage of microglia (left) among DAPI− Ly6c− cells and percentage of BrdU+ microglia (right).
(B) Percentage of CX3CR1+ CD11b+ microglia among live cells in BDNFflox/+ or BDNFflox/flox mice (error bars = SEM, n = 4).
(C) Percentage of BrdU+ cells among microglia in BDNFflox/+ or BDNFflox/flox mice (error bars = SEM, n = 4).
(D) Coronal sections of motor cortex or hippocampal CA1 region from indicated genotypes stained for the neuronal marker NeuN (blue) or the microglial marker Iba1 (red) (scale bar = 50 μm). The distribution and morphology of microglia appear similar between BDNFflox/+ and BDNFflox/flox mice.
(E) Quantification of number of Iba1+ cells in indicated brain regions and genotypes (error bars = SEM, n = 16 fields in 4 mice per group).
(F) Schematic depicting the method used for quantifying potential association of microglial processes with dendritic spines.
(G) Percent of dendritic spines located within 1 μm to Iba1+ microglial processes was not significant different between CX3CR1CreER/+:BDNFfl/fl and CX3CR1CreER/+:BDNFfl/+ control mice (p > 0.3; error bars = SD, CX3CR1CreER:BDNFflox/+ n = 394 spines in 4 mice; CX3CR1CreER:BDNFflox/flox n = 400 spines in 4 mice).
(H) Representative FACS analysis of primary microglia cultures from BDNF-HA/HA mice. Dot plot is gated on DAPI− cells.
(I) mRNA levels of primary microglial cultures from CX3CR1CreER/+:BDNFflox/flox or CX3CR1CreER/+ control mice after treatment with 4-OHT (error bars = SEM, n = 4).
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