DOI: 10.5061/dryad.612jm648h
title: Microglia modulate TNFα‐mediated synaptic plasticity

description: The pro-inflammatory cytokine tumor necrosis factor α (TNFα) tunes the
 capacity of neurons to express synaptic plasticity. It remains, however,
 unclear how TNFα mediates synaptic positive (=change) and negative
 (=stability) feedback mechanisms. We assessed effects of TNFα on microglia
 activation and synaptic transmission onto CA1 pyramidal neurons of mouse
 organotypic entorhino–hippocampal tissue cultures. TNFα mediated changes
 in excitatory and inhibitory neurotransmission in a
 concentration-dependent manner, where low concentration strengthened
 glutamatergic neurotransmission via synaptic accumulation of
 GluA1-only-containing AMPA receptors and higher concentration increased
 inhibition. The latter induced the synaptic accumulation of
 GluA1-only-containing AMPA receptors as well. However, activated,
 pro-inflammatory microglia mediated a homeostatic adjustment of excitatory
 synapses, that is, an initial increase in excitatory synaptic strength at
 3 h returned to baseline within 24 h, while inhibitory neurotransmission
 increased. In microglia-depleted tissue cultures, synaptic strengthening
 triggered by high levels of TNFα persisted and the impact of TNFα on
 inhibitory neurotransmission was still observed and dependent on its
 concentration. These findings underscore the essential role of microglia
 in TNFα-mediated synaptic plasticity. They suggest that pro-inflammatory
 microglia mediate synaptic homeostasis, that is, negative feedback
 mechanisms, which may affect the ability of neurons to express further
 plasticity, thereby emphasizing the importance of microglia as gatekeepers
 of synaptic change and stability.
description: Transcriptome
 analysis Tissue cultures (<em>n</em> = 3)
 were transferred as one sample into 250 μL RNAlater (Thermo Fisher
 Scientific, Cat# AM7020) and stored at −20°C. RNA was isolated after
 homogenization with TRIzol (Thermo Fisher Scientific, Cat# 15596018) using
 the Direct-zol RNA Microprep-Kit (Zymo Research, Freiburg im Breisgau,
 Germany, Cat# R2061) according to the manufacturer's instructions.
 RNA was eluted in 50 μL water and precipitated with 0.75 M
 ammonium-acetate and 10 μg glycogen (Thermo Fisher Scientific, Cat# R0551)
 by adding 125 μL ethanol (100%). Samples were incubated at −80°C overnight
 and consecutively centrifuged for 30 min 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
 Analyzer (Advanced Analytical Technologies, Inc., Ankeny, IA, USA) and the
 Agilent RNA 6000 Pico Kit (Agilent, Cat# 5067-1513). RNA samples with RNA
 integrity numbers (RIN) &gt; 8.0 were further processed with the
 Affymetrix WT Plus kit and hybridized to Clariom S mouse arrays (Thermo
 Fisher Scientific, Cat# 902931) as described by the manufacturer. Briefly,
 labeled fragments were hybridized to arrays for 16 h at 45°C, 60 rpm in a
 GeneChip™ Hybridization Oven (Thermo Fisher Scientific). After washing and
 staining, the arrays were scanned with the Affymetrix GeneChip Scanner
 3000 7G (Thermo Fisher Scientific). CEL files were produced from the raw
 data with Affymetrix GeneChip Command Console Software Version 4.1.2
 (Thermo Fisher Scientific). CEL files were processed with the Oligo R
 package and RNA and intensity were normalized using Robust Multichip
 Average method. A linear-model-based analysis, <em>limma</em>
 R package (Ritchie et al., 2015), was used to identify differentially regulated genes. An adjusted <em>p-</em>value (Benjamini & Hochberg) below 0.05 was considered as significant. Gene-set enrichment analysis was done using GSEA 4.1.0 (Mootha et al., 2003; Subramanian et al., 2005), Cytoscape (Shannon et al., 2003) and custom R-scripts to produce heatmaps.
description: Transcriptome Analysis Console 4.0 (Applied
 Biosystems). R-4.3.1, <em>oligo</em> R
 package, <em>limma</em> R package, GSEA 4.1.0.