Reactive microglia fail to respond to environmental damage signals in a viral-induced mouse model of temporal lobe epilepsy

Microglia are highly adaptable innate immune cells that rapidly respond to damage signals in the brain through adoption of a reactive phenotype and production of defensive inflammatory cytokines. Microglia express a distinct transcriptome, encoding receptors that allow them to dynamically respond to pathogens, damage signals, and cellular debris. Expression of one such receptor, the microglia-specific purinergic receptor P2ry12 , is known to be downregulated in reactive microglia. Here, we explore the microglial response to purinergic damage signals in reactive microglia in the TMEV mouse model of viral brain infection and temporal lobe epilepsy. Using two-photon calcium imaging in acute hippocampal brain slices, we found that the ability of microglia to detect damage signals, engage calcium signaling pathways, and chemoattract towards laser-induced tissue damage was dramatically reduced during the peak period of seizures, cytokine production, and infection. Using combined RNAscope in situ hybridization and immunohistochemistry, we found that during this same stage of heightened infection and seizures, microglial P2ry12 expression was reduced, while the pro-inflammatory cytokine TNF-a expression was upregulated in microglia, suggesting that the depressed ability of microglia to respond to new damage signals via P2ry12 occurs during the time when local elevated cytokine production contributes to seizure generation following infection. Therefore, changes in microglial purinergic receptors during infection likely limit the ability of reactive microglia to respond to new threats in the CNS and locally contain the scale of the innate immune response in the brain. ### Competing Interest Statement The authors have declared no competing interest. * 2-P : two-photon 3D : three dimensional A1 : A1 adenosine receptor A2A : A2A adenosine receptor aCSF : artificial cerebrospinal fluid ADP : adenosine diphosphate AMP : adenosine monophosphate ATP : adenosine triphosphate CA1 & CA3 : subfields 1 and 3, respectively, of the cornu ammonis region of the hippocampus CNS : central nervous system CreERT2 : Cre recombinase – estrogen receptor T2 Cx3cr1 : C-X3-C Motif Chemokine Receptor 1 DAMP : damage-associated molecular pattern dF/F or ΔF/F : the change in fluorescence intensity relative to the baseline fluorescence intensity DPI : days post-infection EYFP : enhanced yellow fluorescent protein FISH : fluorescent in situ mRNA hybridization FOV : field of view G5 : genetically encoded green calcium indicator variant 5G GCaMP : genetically encoded calcium indicator Hrs : hours Hz : hertz IBA1 : ionized calcium-binding adapter molecule 1 IL-6 : Interleukin 6 cytokine i.p. : intraperitoneal injection mOsm : milliosmole Min : minute(s) mL : milliliter mm : millimeter mM : millimolar mRNA : messenger ribonucleic acid ms : millisecond mW : milliwatt MΩ : megaohm NA : numerical aperture NADH : nicotinamide adenine dinucleotide + hydrogen NBF : neutral buffered formalin NG2 : Nerve/glial antigen 2, also known as chondroitin sulfate proteoglycan 4 (CSPG4) nm : nanometer OD : outer diameter P2RY12 : purinergic receptor P2Y12 P2RY : P2Y purinergic receptors PBS : phosphate-buffered saline PC : Polr2a gene Pfu : plaque-forming units PSI : pounds per square inch RNA : ribonucleic acid ROI : region of interest ROS : reactive oxygen species s second(s) SEM : standard error of the mean SNR : signal-to-noise ratio STD : standard deviation TAM : tamoxifen TdT : tdTomato fluorescent protein TLE : temporal lobe epilepsy TMEV : Theiler’s murine encephalomyelitis virus TNF-α : tumor necrosis factor alpha µg : microgram µL : microliter µm : micron (also known as micrometer) µM : micromolar µs : microsecond.