Microglial cytokines mediate plasticity induced by 10 Hz repetitive magnetic stimulation
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Mar 24, 2023 version files 598.78 MB
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Abstract
Microglia—the resident immune cells of the central nervous system—sense the activity of neurons and regulate physiological brain functions. They have been implicated in the pathology of brain diseases associated with alterations in neural excitability and plasticity. However, experimental and therapeutic approaches that modulate microglia function in a brain-region-specific manner have not been established. In this study, we tested for the effects of repetitive transcranial magnetic stimulation (rTMS), a clinically employed non-invasive brain stimulation technique, on microglia-mediated synaptic plasticity. 10 Hz electromagnetic stimulation triggered a release of plasticity-promoting cytokines from microglia in organotypic brain tissue cultures, while no changes in microglial morphology or microglia dynamics were observed. Indeed, substitution of tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6) preserved synaptic plasticity induced by 10 Hz stimulation in the absence of microglia. Consistent with these findings, in vivo depletion of microglia abolished rTMS-induced changes in neurotransmission in the medial prefrontal cortex (mPFC) of anesthetized mice. We conclude that rTMS affects neural excitability and plasticity by modulating the release of cytokines from microglia.
Methods
Ethics statement. Mice were maintained in a 12 h light/dark cycle with food and water available ad libitum. Every effort was made to minimize distress and pain of animals. All experimental procedures were performed according to German animal welfare legislation and approved by the competent authority (Regierungspräsidium Freiburg, G-20/154) appropriate animal welfare committee and the animal welfare officer of Albert-Ludwigs-University Freiburg, Faculty of Medicine (X-17/07K, X-17/09C, X-18/02C).
Preparation of tissue cultures. Entorhino-hippocampal tissue cultures were prepared at postnatal day 3–5 from C57BL/6J, HexB-tdTom (Masuda et al., 2020), and C57BL/6-Tg(TNFa-eGFP) (Lenz et al., 2020) mice of either sex as previously described (Del Turco and Deller, 2007). Cultivation medium contained 50% (v/v) MEM, 25% (v/v) basal medium eagle, 25% (v/v) heat-inactivated normal horse serum, 25 mM HEPES buffer solution, 0.15% (w/v) bicarbonate, 0.65% (w/v) glucose, 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamax. The pH was adjusted to 7.3. All tissue cultures were allowed to mature for at least 18 days in a humidified atmosphere with 5% CO2 at 35°C. Cultivation medium was replaced 3 times per week.
Microglia depletion in vitro and in vivo. Tissue cultures were treated immediately after preparation (div 0) with the CSF-1R inhibitor PLX3397 (50 nM; #2501 Axon) for at least 18 days. Vehicle-only treated cultures (DMSO, 0.1 µl) served as age- and time-matched controls. For in vivo microglia depletion, we used the CSF-1R inhibitor BLZ945 (kindly provided by Novartis, Basel, Switzerland) dissolved in 20% (2-hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich, Germany). A dose of 200 mg/kg body weight was applied by oral gavage in adult (8-weeks old) mice for 7 consecutive days as previously described (Hagemeyer et al., 2017; Masuda et al., 2020). No weight loss or any apparent signs of stress could be detected throughout the treatment period. Vehicle-only treated and untreated animals were used as controls.
Repetitive magnetic stimulation in vitro. To learn more about the cellular and molecular mechanisms of rTMS-induced plasticity we established an in vitro model of r(T)MS using mouse entorhino-hippocampal slice cultures, which provide the advantage of a highly laminar fiber- and cyto-architecture ((Frotscher and Heimrich, 1995; Maus et al., 2020; Lenz et al., 2022); further advantages and disadvantages of these preparations were discussed earlier; (Vlachos et al., 2012)). This approach allowed us to assess rMS-induced synaptic plasticity at the level of single identified neurons which are embedded in an organotypic neuronal network (c.f., (Muller-Dahlhaus and Vlachos, 2013)). Moreover, recent work confirmed the maintenance of microglia signatures and functions in organotypic slice cultures akin to what is seen in vivo (Delbridge et al., 2020). Tissue cultures (≥ 18 days in vitro) were transferred to a 35 mm Petri Dish filled with pre-warmed standard extracellular solution containing (in mM): 129 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 4.2 glucose, 10 HEPES, 0.1 mg/ml streptomycin, 100 U/ml penicillin, pH 7.4 adjusted with NaOH, osmolarity adjusted with sucrose to 380–390 mOsm. Cultures were stimulated using the Magstim Rapid2 stimulator (Magstim Company, UK) connected to a Double AirFilm® Coil (coil parameters according to manufacturer’s description: average inductance = 12 μH; pulse rise time approximately 80 μs; pulse duration = 0.5 ms, biphasic; Magstim Company, UK) with a biphasic current waveform. Cultures were positioned approximately 1 cm under the center of the coil and oriented in a way that the induced electric field was parallel to the dendritic tree of CA1 pyramidal neurons. The stimulation protocol consisted of 900 pulses at 10 Hz (50% maximum stimulator output). Cultures were kept in the incubator for at least 2 h after stimulation before experimental assessment. Age- and time-matched control cultures were not stimulated, but otherwise treated identical to stimulated cultures (sham stimulation). For cytokine substitution experiments TNFα (5 ng/ml; #410-MT RD Systems) and IL6 (2.5 ng/ml; #406-ML RD Systems) were added to the stimulation medium.
Repetitive magnetic stimulation in vivo. rTMS was carried out in adult (~8 weeks old) urethane-anesthetized (1.25 g kg−1, intraperitoneal; 0.125 g kg−1, subcutaneous) C57BL/6J mice of either sex. The head was placed under the coil with the medial prefrontal cortex (mPFC) under the center. During the stimulation, the brain-to-coil distance was kept minimal, while brain-to-coil contact was avoided. Repetitive stimulation was performed at fixed intensities of 60% MSO (which corresponds to 90% motor threshold, see (Lenz et al., 2016)) using the same 10-Hz stimulation protocol described above. Control animals placed near the coil during stimulation were not stimulated but otherwise treated identically. All animals were transferred back to their cages with appropriate body temperature control and were held in anesthesia for 2 h under continuous surveillance. After the waiting period, ketamine/xylazin (100 mg kg-1 / 20 mg kg-1; i.p. application) was injected to achieve a suitable analgesia before rapid decapitation.
The brain was prepared as previously described (Ting et al., 2018; Lenz et al., 2021). After dissection, the brain was embedded in low-melting agar (1.8% w/v in PBS; Sigma Aldrich #A9517) and frontal sections (350 µm thickness) containing the mPFC were prepared using a Leica VT1200S vibratome with a cutting angle of 15°. The brain was cut in NMDG-aCSF [containing (in mM) 92 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, and 10 MgSO4, (pH = 7.3–7.4)] at approximately 0°C. After cutting, slices were recovered in NMDG-aCSF at 34°C. Sodium spike-in was performed according to a previously established protocol that is suitable for ~8-week-old animals (Ting et al., 2018; Lenz et al., 2021). After recovery, we transferred the slices to a holding chamber [holding-aCSF; containing (in mM) 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 2 CaCl2, and 2 MgSO4, (pH = 7.3–7.4)] at room temperature in which slices were maintained at least half an hour until electrophysiological assessment.
Propidium iodide staining. Tissue cultures were incubated with propidium iodide (PI, 5 μg/ml; #P3566 Invitrogen) for 2 h, washed in phosphate buffered saline (PBS) and fixed as described below. Cultures treated for 4 h with NMDA (50 μg/ml; #0114 Tocris) served as positive controls in these experiments. Cell nuclei were stained with DAPI, sections were mounted on microscope slides and confocal images were acquired as described below.
Immunostaining and imaging. Tissue cultures were fixed overnight in a solution of 4% (w/v) paraformaldehyde (PFA) in PBS (prepared from 16% PFA stocks in phosphate buffered saline according to manufacturer’s instruction; #28908 Thermo Scientific). After fixation, cultures were washed in PBS (0.1 M, pH 7.4) and consecutively incubated for 1 h with 10% (v/v) normal goat serum (NGS) in 0.5% (v/v) Triton X-100 containing PBS to reduce nonspecific staining and to increase antibody penetration. Subsequently, cultures were incubated overnight at 4°C with rabbit anti-Iba1 (1:1000; #019-19741 Fujifilm Wako) in PBS with 10% NGS and 0.1% Triton X-100. Sections were washed and incubated overnight at 4°C with appropriate Alexa Fluor® dye-conjugated secondary antibodies (1:1000, donkey anti-rabbit Alexa Fluor 488 or 647; #A-21206 or #A-32795 Invitrogen) in PBS with 10% NGS or NHS, 0.1% Triton X-100. For post-hoc visualization of patched pyramidal cells, Streptavidin Alexa Fluor 488 or 633 (Streptavidin A488, 1:1000; #S32354 Invitrogen; Streptavidin A633, 1:1000; #S21375 Invitrogen) was added to the secondary antibody incubation solution. DAPI nuclear stain (1:2000 in PBS for 20 minutes; #62248 Thermo Scientific) was used to visualize cytoarchitecture. Cultures were washed, transferred onto glass slides and mounted for visualization with DAKO anti-fading mounting medium (#S302380-2 Agilent).
A Leica SP8 laser-scanning microscope equipped with a 20x multi-immersion (NA 0.75; Leica), a 40x oil-immersion (NA 1.30; Leica) and a 63x oil-immersion objective (NA 1.40; Leica) was used for confocal image acquisition. Images for analysis of microglia cell density (Figure 1) and images of propidium iodide stainings (Figure 3) were acquired with a 20x objective at 0.75x optical zoom (resolution: 512 x 512 px). Image stacks for spine density and spine volume analysis (Figure 6) were acquired with a 63x oil-immersion objective at 5.0x optical zoom (resolution: 1024 x 1024, Δz = 0.22 µm at ideal Nyquist rate). Image stacks of Iba1 stained HexB-tdTom cultures (Figure 9) were acquired with a 20x objective at 2.0x optical zoom (resolution: 512 x 512 px). Image stacks of Iba1 stained acute cortical slices (Figure 13) were acquired using a 40x oil-immersion objective at 2.0x optical zoom (resolution 1024x1024, Δz = 1 µm). Laser intensity and detector gain were set to achieve comparable overall fluorescence intensity throughout stacks between all groups in each experimental setting.
Live-cell imaging. Live-cell imaging of tissue cultures was performed at a Zeiss LSM800 microscope equipped with a 10x water-immersion (NA 0.3; Carl Zeiss) and a 40x water-immersion objective (NA 1.0; Carl Zeiss). Filter membranes with 2 to 6 cultures were placed in a 35 mm Petri Dish containing pre-oxygenated imaging solution consisting of 50% (v/v) MEM, 25% (v/v) basal medium eagle, 50 mM HEPES buffer solution (25% v/v), 0.65% (w/v) glucose, 0.15% (w/v) bicarbonate, 0.1 mg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamax and 0.1 mM trolox. The cultures were kept at 35°C during the imaging procedure.
Live-cell imaging of homozygous (HexBtdT/tdT) and heterozygous (HexBtdT/+) cultures prepared from HexB-tdTom transgenic animals was performed to assess microglia morphology after rMS. Cultures were stimulated as described above (rMS and sham stimulation) and imaging was started immediately in imaging solution under continuous oxygenation (5% CO2 / 95% O2). For 3 hours, every 2 minutes a z-stack of the same cell was recorded using a 40x water-immersion objective with Δz = 1 μm at ideal Nyquist rate and an optical zoom of 1.0x (resolution 512 x 512 px, 2x line average). Laser intensity and detector gain were initially set and were kept constant over image acquisition time.
Live-cell imaging of C57BL/6-Tg(TNFa-eGFP) cultures was performed to monitor TNFα expression after rMS as an indicator of neuroinflammation. Cultures were stimulated as described above (rMS and sham stimulation) and kept in the incubator after stimulation. After 3 hours a z-stack of each culture was recorded using a 10x water-immersion objective with Δz = 6.3 μm at ideal Nyquist rate and an optical zoom of 0.5x (resolution 1024 x 1024 px). Laser intensity and detector gain were initially set to keep the fluorescent signal in a dynamic range throughout the experiment and were kept constant.
Confocal image stacks were stored as .czi files.
Transcriptome Microarray. Tissue cultures that were cultivated on one filter membrane (3 cultures) were transferred as one sample into RLT buffer (QIAGEN) and RNA was isolated according to the manufacturer’s instructions (RNeasy Plus Micro Kit; #74034 QIAGEN). RNA was eluted in 50 µl water and precipitated in 0.75 M ammonium-acetate and 10 µg glycogen (#R0551 Thermo Scientific) by adding 125 µl ethanol (100%). Samples were incubated at -80°C overnight and consecutively centrifuged for 30 minutes at 4°C. Pellets were washed with 70% ethanol, centrifuged again and dried. Finally, pellets were dissolved in water for further processing. RNA concentration and integrity were consecutively analyzed by capillary electrophoresis using a Fragment Analyser (Advanced Analytical Technologies, Inc., USA). RNA samples with RNA quality numbers (RQN) > 8.0 were further processed with the Affymetrix WT Plus kit and hybridized to Clariom S mouse arrays as described by the manufacturer (Thermo Fisher, Germany). Briefly, labeled fragments were hybridized to arrays for 16 h at 45°C, 60 rpm in a GeneChip™ Hybridization Oven (Thermo Fisher, Germany). After washing and staining, the arrays were scanned with the Affymetrix GeneChip Scanner 3000 7G (Thermo Fisher, Germany). CEL files were produced from the raw data with Affymetrix GeneChip Command Console Software Version 4.1.2 (Thermo Fisher, Germany).
Cytokine detection assay. To analyze protein release upon rMS, cultures were stimulated on incubation medium in interface configuration with three cultures grown on one filter membrane. To test for activity-induced cytokine release during stimulation, we added the voltage-gated sodium channel inhibitor tetrodotoxin (TTX, 2µM; #18660-81-6 Biotrend) to the incubation medium during stimulation in some experiments. Three hours after stimulation, both incubation medium (for detection of protein release) and tissue cultures (for gene expression analysis) were collected and frozen in liquid nitrogen until further processing.
For cytokine detection, a V-Plex Proinflammatory Panel 1 (mouse) Kit Plus (#K15048G Mesoscale Discovery) was used. The collected incubation medium was diluted 1:1 in diluent provided with the kit. Protein detection was performed according to the manufacturer's instructions. A pre-coated plate with capture antibodies on defined spots was incubated with the diluted samples overnight. After washing, samples were incubated overnight with a solution containing electrochemiluminescent MSD SULFO-TAG detection antibodies (Mesoscale Discovery; Antibodies: Anti-ms TNFα Antibody #D22QW, Anti-ms IL6 Antibody #D22QX, Anti-ms CXCL1 Antibody #D22QT). After washing, samples were measured with a MESO QuickPlex SQ 120 instrument (Mesoscale Discovery). The respective protein concentrations were determined using the MSD DISCOVERY WORKBENCH software (Mesoscale Discovery).
RNA Isolation and Quantitative reverse transcription PCR (RT-qPCR). RNA isolation for qPCR analysis was performed as follows. Tissue cultures that were cultivated on one filter membrane (3 cultures) were transferred as one sample into RNA Protection buffer (New England Biolabs) and RNA was isolated according to the manufacturer’s instructions (Monarch® Total RNA Miniprep Kit; #T2010S New England Biolabs). As a quality control, the RIN (RNA integrity number) value of RNA isolated from tissue culture was determined using the Agilent RNA 6000 Pico Kit (#5067-1513 Agilent) with a 2100 Bioanalyzer (#G2939BA Agilent; Mean RIN value: 8.9). Purified RNA was consecutively reverse transcribed (RevertAid RT Kit; #K1691 Thermo Scientific). cDNA was diluted in water to a final concentration of 3 ng/ml. RT-qPCR was performed using a C1000 Touch Thermal Cycler (BIO-RAD) and the CFX 384 Real-Time PCR system (BIO-RAD). 13.5 ng target cDNA diluted in TaqMan Gene Expression Master Mix (#4369016 Applied Biosystems) were amplified using standard TaqMan gene expression assays (Applied Biosystems; Assay-IDs: Gapdh: Mm99999915_g1; Tnf: Mm00443258_m1; Il6: Mm00446190_m1; Cxcl1: Mm04207460_m1). The RT-qPCR protocol was performed as follows: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. Of each sample, three technical replicates were used and no amplification was detected in non-template controls. Amplification curves were excluded from further analysis if efficiency values were less than 90 or exceeded 110 according to automated calculation by the Bio-Rad CFX Maestro software package. Data were exported and stored on a computer as .pcrd-files.
Whole-cell patch-clamp recordings. Whole-cell patch-clamp recordings were carried out at 35°C (3-6 neurons per culture). Patch pipettes contained (in mM) 126 K-gluconate, 10 HEPES, 4 KCl, 4 ATP-Mg, 0.3 GTP-Na2, 10 PO-Creatine, 0.3% (w/v) biocytin (pH 7.25 with KOH, 290 mOsm with sucrose), having a tip resistance of 4-6 MΩ. Pyramidal neurons were visually identified using a LN-Scope (Luigs & Neumann) equipped with an infrared dot-contrast and a 40x water-immersion objective (NA 0.8; Olympus). Electrophysiological signals were amplified using a Multiclamp 700B amplifier, digitized with a Digidata 1550B digitizer, and visualized with the pClamp 11 software package.
For whole-cell patch-clamp recordings of CA1 pyramidal neurons in tissue cultures, the bath solution contained (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, and 10 glucose (aCSF) and was oxygenated continuously (5% CO2 / 95% O2). Spontaneous excitatory postsynaptic currents (sEPSCs) of CA1 pyramidal neurons were recorded in voltage-clamp mode at a holding potential of -60 mV. Series resistance was monitored before and after each recording and recordings were discarded if the series resistance reached ≥ 30 MΩ. For mEPSC recordings of CA1 pyramidal neurons, D-APV (10 μM; #ab120003 Abcam), (-)-Bicuculline-Methiodide (10 µM; #ab120108 Abcam) and TTX (0.5 μM; #18660-81-6 Biotrend) were added to the external solution.
Whole-cell patch-clamp recordings of superficial (layer 2/3) cortical pyramidal neurons in acute mouse brain slices were carried out in a bath solution containing holding-aCSF. For sEPSC recordings, layer 2/3 pyramidal neurons were held at -70 mV in voltage-clamp mode.
For recording of intrinsic cellular properties in current-clamp mode, pipette capacitance of 2.0 pF was corrected and series resistance was compensated using the automated bridge balance tool of the MultiClamp commander. I-V-curves were generated by injecting 1 s square pulse currents starting at -100 pA and increasing in 10 pA steps (sweep duration: 2 s). Series resistance was monitored, and recordings were discarded if the series resistance reached ≥ 30 MΩ.
Multi-scale modeling. A 3-dimensional mesh model was created with two compartments, i.e., bath solution and organotypic tissue culture, using the finite element method and the program Gmsh (4.8.4). Local mesh resolution was increased from 0.01 to 0.004 units in the CA1 region of the culture, i.e., region of interest (ROI), where neurons were placed. The final mesh consisted of 3.55 × 106 nodes and 2.11 × 107 tetrahedrons. The mean tetrahedron edge length was 5.6 µm in the ROI. The physical dimensions of the mesh model were adapted from the in vitro setting.
The coil-to-culture distance was kept at 10 mm and the coil was positioned above the culture. Electrical conductivities for the bath and culture were 1.654 S m-1 and 0.275 S m-1, respectively. The rate of change of the coil current was set to 1.4 A µs-1 at 1% MSO and it was scaled up to 50% MSO. Macroscopic electric field simulations were performed using SimNIBS (3.2.4) and Matlab (2020b). A validated 70 mm MagStim figure-of-eight coil was used in all simulations.
For multi-scale modeling, we used the Neuron Modeling for TMS (NeMo-TMS) framework to study the biological responses of CA1 pyramidal neurons to biphasic single pulse TMS and rTMS (Shirinpour et al., 2021). Axonal morphology was adopted from an example cell (Shirinpour et al., 2021). For all neurons, we implemented the generalized version of the Jarsky model (Shirinpour et al., 2021).
We extracted the membrane potentials and voltage-gated calcium ‘influx’ from the somatic and dendritic compartments (Shirinpour et al., 2021). We analyzed the number of action potentials, calcium spikes and their peak values. Simulations were run on a high-performance computer in the state of Baden-Württemberg, Germany (bwHPC).
Experimental Design and Statistical Analysis
STUDY DESIGN
In this study, we used age-matched organotypic entorhino-hippocampal tissue cultures and ~8 weeks old adult mice of either sex in a prospective study design. Treatment with PLX3397 was used to deplete microglia from organotypic tissue cultures. Vehicle-only treated, age-matched cultures served as controls in these experiments. For microglia depletion in adult mice (~8 weeks old), treatment with BLZ945 was used. Non-treated and vehicle-only treated mice of the same age were used as controls in these experiments. Excitatory inputs onto pyramidal neurons were evaluated in age-matched tissue cultures or ~8 weeks old adult mice after repetitive magnetic stimulation. Sham stimulated age-matched tissue cultures or ~8 weeks old mice served as controls. In experiments that included treatment with TTX, TNFα, or IL6, vehicle-only treatment served as control.
QUANTIFICATION
For the analysis of microglia cell density and spine density, cells or spines were counted manually in maximum intensity projections of the confocal image stacks using the ‘Cell Counter’ plugin of Fiji image processing package [available athttps://fiji.sc/; (Schindelin et al., 2012)].
Spine head volumes were assessed in the confocal image stacks using the Imaris x64 (version 9.5.0) software. The surface tool with the ‘split touching object’ option enabled was used to measure the volume of spine heads. Files were stored as .ims.
Confocal images of PI-stained cultures were processed and analyzed using the Fiji image processing package. After background subtraction (rolling ball radius 50 px), images were binarized and PI positive particles were displayed and counted using the ‘Analyze Particles’ function. Values were normalized to the mean value of the control group.
Confocal image stacks of heterozygous C57BL/6-Tg(TNFa-eGFP) cultures were processed and analyzed as previously described (Lenz et al., 2020) using the Fiji image processing package. Mean fluorescence intensity of the culture area was normalized to the mean value of fluorescence intensity of the sham-stimulated cultures.
Dendritic morphologies were assessed using the Neurolucida® 360 software (version 2020.1.1). Cells were semi-automatically reconstructed with the ‘user guided tree reconstruction’ function. Reconstructions were saved as .DAT files and analysis was performed in the Neurolucida Explorer (version 2019.2.1).
To analyze microglia morphology in HexB-tdTom cultures, confocal image stacks were processed and analyzed using the Fiji image processing package. Of each z-stack a maximum intensity projection was generated and binarized using the ‘Trainable Weka Segmentation’ plugin (Arganda-Carreras et al., 2017). The same classifier was applied to all images of the same microglia over the recorded 3 h. After removing outliers (radius = 2 px, threshold = 50), microglia scanning density and microglia domain of each image were manually assessed as previously described (Pfeiffer et al., 2016). Values were normalized to the mean.
For the analysis of microglial morphology in acute mouse cortical slices, stacks of single cells were also processed and analyzed using the Fiji image processing package. First, each stack was processed using the ‘despeckle’ function, then a maximum intensity projection was generated. After removing outliers (radius = 3 px, threshold = 50), the image was binarized as described before. Again, outliers were removed (radius = 2 px, threshold = 50), microglia scanning density and microglia domain of each image were manually assessed.
Single cell recordings were analyzed off-line using Clampfit 11 of the pClamp11 software package (Molecular Devices). sEPSC and mEPSC properties were analyzed using the automated template search tool for event detection (Lenz et al., 2021). Input resistance was calculated for the injection of -100 pA current at a time frame of 200 ms with maximum distance to the Sag-current. Resting membrane potential was calculated as the mean baseline value. AP detection was performed using the input/output curve threshold search event detection tool, and the AP frequency was assessed upon the number of APs detected during the respective injection step. 4 cells in the microglia-depleted (BLZ945-treated) sham stimulated group were excluded in the analysis of intrinsic properties (Figure 13) due to loss of the integrity of the patch during recording of the I-V-curve. In the control group data of vehicle-only treated and untreated animals were pooled (Figure 13).
Affymetrix GeneChip™ microarray data (CEL files) were analyzed using the Affymetrix Transcriptome Analysis Console (TAC version 4.0.2.15). Gene expression was considered significantly different when FDR p-value < 0.05 and fold change < -2 or > 2. Differentially expressed well-annotated genes were considered ‘microglia-specific’ (Figure 1) if they were part of highly specific microglia markers found by Chiu et al. (Chiu et al., 2013) and ‘microglia-related’ if expression of these genes in microglia was reported in the literature elsewhere. A full list of differentially expressed genes including predicted genes is provided in supplementary table 1-1.
RT-qPCR data were analyzed as previously described (Lenz et al., 2020) with the Bio-Rad CFX Maestro 1.0 software package using the ΔΔCq method with Gapdh as reference gene. Values were normalized to the mean value of the respective vehicle-treated control group.
Mesoscale cytokine detection assay was analyzed using the MSD DISCOVERY WORKBENCH 4.0 platform. mRNA/protein level correlations were visualized by a linear regression fit and analyzed using non-parametric Spearman’s correlation coefficients (r).
STATISTICAL ANALYSIS
Statistical evaluation of the multi-scale modeling was implemented in R (4.0.3; https://www.R-project.org/) and R Studio (1.3.1093; http://www.rstudio.com/) integrated development environment. We ran generalized linear mixed models (GLMM) with predictors TREATMENT (two levels: control, PLX3397) and COMPARTMENT (three levels: soma, apical and basal dendrites). GLMM allows modeling dependent variables from different distributions and model both fixed and random effects (Stroup, 2012). The null model contained the cell as random intercept, and we added each predictor and their interaction terms one-by-one to the subsequent models. The Bayesian information criterion (BIC) was used to compare the current model with the previous one. We selected the winning model if the ΔBIC was at least 10 units for the null or previous model (Kass and Raftery, 1995; Anderson and Burnham, 2004; Fabozzi, 2014).
Data were analyzed using GraphPad Prism 7 and 9 (GraphPad software, USA). Statistical comparisons were made using non-parametric tests, since normal distribution of data could not be assured. For column statistics, Mann-Whitney test (to compare two groups) and Kruskal-Wallis-test followed by Dunn’s multiple comparisons (to compare three groups) were used. For statistical comparison of XY-plots, we used an RM two-way ANOVA test (repeated measurements/analysis) with Sidak’s multiple comparisons. p-values < 0.05 were considered a significant difference. In the text and figures, values represent mean ± standard error of the mean (s.e.m.). * p < 0.05, ** p < 0.01, *** p < 0.001 and not significant differences are indicated by ‘ns’. N-numbers are provided in the figure legends. Statistical differences in XY-plots were indicated in the legend or the title of the figure panels (*) when detected through multiple comparisons.
Digital Illustrations. Figures were prepared using Photoshop graphics software (Adobe, San Jose, CA, USA). Image brightness and contrast were adjusted.
Usage notes
Data were analyzed using GraphPad Prism 7 and 9 (GraphPad software, USA).