DOI: 10.5061/dryad.ngf1vhhzk
title: Microglial cytokines mediate plasticity induced by 10 Hz repetitive magnetic stimulation

description: 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. 
description: <strong>Ethics
 statement. </strong>Mice were maintained in a 12 h light/dark cycle
 with food and water available <em>ad libitum</em>. 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). <strong>Preparation of tissue
 cultures. </strong>Entorhino-hippocampal tissue cultures were
 prepared at postnatal day 3–5 from
 <em>C57BL/6J</em>, <em>HexB-tdTom </em>(Masuda et
 al., 2020), and <em>C57BL/6-Tg(TNFa-eGFP) </em>(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% CO<sub>2</sub> at 35°C.
 Cultivation medium was replaced 3 times per week. <strong>Microglia
 depletion <em>in vitro</em> and <em>in
 vivo</em>. </strong>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 <em>in
 vivo</em> 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. <strong>Repetitive magnetic
 stimulation <em>in vitro</em>. </strong>To learn more
 about the cellular and molecular mechanisms of rTMS-induced plasticity we
 established an <em>in vitro</em> 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 <em>in vitro</em>) were transferred to a 35 mm Petri Dish
 filled with pre-warmed standard extracellular solution containing (in mM):
 129 NaCl, 4 KCl, 1 MgCl<sub>2</sub>, 2
 CaCl<sub>2</sub>, 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 Rapid<sup>2</sup> stimulator (Magstim Company, UK)
 connected to a Double AirFilm<sup>®</sup> 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. <strong>Repetitive magnetic
 stimulation <em>in vivo</em>. </strong>rTMS was carried
 out in adult (~8 weeks old) urethane-anesthetized
 (1.25 g kg<sup>−1</sup>, intraperitoneal;
 0.125 g kg<sup>−1</sup>,
 subcutaneous) <em>C57BL/6J</em> 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<sup>-1</sup> / 20 mg kg<sup>-1</sup>; 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
 NaH<sub>2</sub>PO<sub>4</sub>, 30
 NaHCO<sub>3</sub>, 20 HEPES, 25 glucose, 2 thiourea, 5
 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl<sub>2</sub>, and 10
 MgSO<sub>4</sub>, (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
 NaH<sub>2</sub>PO<sub>4</sub>, 30
 NaHCO<sub>3</sub>, 20 HEPES, 25 glucose, 2 thiourea, 5
 Na-ascorbate, 3 Na-pyruvate, 2 CaCl<sub>2</sub>, and 2
 MgSO<sub>4,</sub> (pH = 7.3–7.4)] at room temperature in which
 slices were maintained at least half an hour until electrophysiological
 assessment. <strong>Propidium iodide
 staining. </strong>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. <strong>Immunostaining and
 imaging. </strong>Tissue<strong> </strong>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<sup>®</sup> 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 <em>HexB-tdTom </em>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. <strong>Live-cell
 imaging. </strong>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
 (<em>HexB<sup>tdT/tdT</sup></em>) and heterozygous
 (<em>HexB<sup>tdT/+</sup></em>) cultures prepared
 from <em>HexB-tdTom </em>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%
 CO<sub>2</sub> / 95% O<sub>2</sub>). 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 <em>C57BL/6-Tg(TNFa-eGFP)</em> 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. <strong>Transcriptome
 Microarray. </strong>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)
 &gt; 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). <strong>Cytokine detection
 assay. </strong>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).
 <strong>RNA Isolation and
 Quantitative reverse transcription PCR (RT-qPCR). </strong>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<sup>®</sup> 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; <strong>#</strong>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<sup> </sup>gene
 expression assays (Applied Biosystems;
 Assay-IDs: <em>Gapdh</em>:
 Mm99999915_g1; <em>Tnf</em>:
 Mm00443258_m1; <em>Il6</em>:
 Mm00446190_m1; <em>Cxcl1</em>: 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. <strong>Whole-cell patch-clamp
 recordings. </strong>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-Na<sub>2</sub>, 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 NaHCO<sub>3</sub>,
 1.25 NaH<sub>2</sub>PO<sub>4</sub>, 2
 CaCl<sub>2</sub>, 2 MgCl<sub>2</sub>, and 10
 glucose (aCSF) and was oxygenated continuously (5%
 CO<sub>2</sub> / 95% O<sub>2</sub>). 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. <strong>Multi-scale
 modeling. </strong>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 × 10<sup>6</sup> nodes and 2.11 ×
 10<sup>7</sup> tetrahedrons. The mean tetrahedron edge length
 was 5.6 µm in the ROI. The physical dimensions of the mesh model were
 adapted from the <em>in vitro</em> 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<sup>-1</sup> and 0.275 S m<sup>-1</sup>,
 respectively. The rate of change of the coil current was set to 1.4 A
 µs<sup>-1</sup> 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). <strong>Experimental Design and Statistical
 Analysis</strong> 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 <em>C57BL/6-Tg(TNFa-eGFP)</em> 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<sup>®</sup> 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 <em>HexB-tdTom </em>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 &lt; 0.05 and fold change &lt; -2 or &gt; 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 <em>Gapdh</em> 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 &lt; 0.05 were considered a significant difference. In the text and figures, values represent mean ± standard error of the mean (s.e.m.). * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 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. <strong>Digital Illustrations. </strong>Figures were prepared using Photoshop graphics software (Adobe, San Jose, CA, USA). Image brightness and contrast were adjusted.
description: Data were analyzed using GraphPad Prism 7 and 9 (GraphPad
 software, USA).