Investigating the role of cortical microglia in a mouse model of viral infection-induced seizures

ABSTRACT Microglia, resident immune sentinels in the brain, are crucial in responding to tissue damage, infection, damage signals like purines (ATP/ ADP), and clearing cellular debris. It is currently unknown how microglial reactivity progresses and contributes to seizure development following Theiler’s Murine Encephalomyelitis Virus (TMEV) infection. Previously, our group has demonstrated that purinergic signaling in microglia is disrupted in the hippocampus of TMEV-infected mice. However, whether reactive cortical microglia also exhibit changes in purinergic signaling, cytokine levels, and purinergic receptors are unknown. Thus, we seek to evaluate region-based differences in microglial reactivity in the TMEV model. We employed a custom triple transgenic mouse line expressing tdTomato and GCaMP6f under a CX3CR1 Cre promoter and exogenously applied ATP/ADP to acute brain slice preparations from TMEV-infected mice and controls. Interestingly and in contrast to what is observed in hippocampus, we found that despite microglial reactivity in the cortex, microglia can respond to purinergic damage signals and engage calcium signaling pathways, comparable to PBS controls. Using a cytokine panel, we also found that pro-inflammatory cytokine levels (TNF-α, IL-1α and IFN-γ) are brain-region dependent in mice infected with TMEV. Using RNAScope-FISH, we observed increases in expression of purinergic receptors responsible for microglial motility (P2Y12R) and inflammation (P2X7R) in the cortex. Collectively our results suggest that following TMEV infection, microglial response to novel damage signals, as well as the production of proinflammatory cytokines, varies as a function of brain region. Competing Interest Statement The authors have declared no competing interest. Footnotes Revision to submit figures with a white background for better visualization of figures. ABBREVIATIONS - 2-P - Two-photon - 3D - Three dimensional - °C - Degree Celsius - A3 - A3 adenosine receptor - A2A - A2A adenosine receptor - aCSF - artificial cerebrospinal fluid - ADP - Adenosine diphosphate - AMP - Adenosine monophosphate - ATP - Adenosine triphosphate - [Ca2+] - Calcium - CNS - central nervous system - CreERT2 - Cre recombinase – estrogen receptor T2 - CX3CR1 - C-X3-C Motif Chemokine Receptor 1 - ΔF/F0 - Change in fluorescence intensity relative to the baseline fluorescence intensity - dpi - Days post-infection - FISH - fluorescent in situ mRNA hybridization - GPCR - G-Protein coupled receptor - Hz - Hertz - IACUC - Institutional Animal Care and Use Committee - IBA1 - Ionized calcium-binding adapter molecule 1 - IFN-β - Interferon-Beta cytokine - IFN-γ - Interferon-Gamma cytokine - IL-1α - Interleukin 1-alpha cytokine - IL-6 - Interleukin-6 cytokine - IL-10 - Interleukin-10 cytokine - IL-23 - Interleukin-23 cytokine - IL-27 - Interleukin-27 cytokine - i.p. - Intraperitoneal injection - JAX lab - Jackson Laboratory - Lck-GCaMP6f - genetically encoded fast calcium indicator tethered to the membrane - MCP-1 - Monocyte chemoattractant protein-1 - mOsm - milliosmole - Min - Minute(s) - mL - Milliliter - mm - Millimeter - mM - Millimolar - mRNA - Messenger ribonucleic acid - ms - Millisecond - mW - Milliwatt - MΩ - Megaohm - NA - Numerical aperture - NBF - Neutral buffered formalin - NLRP3 - Nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 - nm - Nanometer - PCR - Polymerase Chain Reaction - P2XR - P2X purinergic receptors - P2X7R - Purinergic receptor P2X7 - P2Y12R - Purinergic receptor P2Y12 - P2RY - P2Y purinergic receptors - PBS - Phosphate-buffered saline - PFU - Plaque-forming units - PLC - Phospholipase-C - PSI - Pounds per square inch - RNA - Ribonucleic acid - RO - Reverse Osmosis - ROI - Region of interest - s - Second(s) - RT - Room temperature - STD - Standard deviation - SA-PE - Streptavidin-phycoerythrin - TAM - Tamoxifen - tdTomato/ tdTom - 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/micrometer - µM - Micromolar