Embryonic neocortical microglia express Toll-like receptor 9 and respond to plasmid DNA injected into the ventricle: technical considerations regarding microglial distribution in electroporated brain walls

Abstract

Microglia, the resident immune cells in the central nervous system, play multiple roles during development. In the embryonic cerebral wall, microglia modulate the functions of neural stem/progenitor cells through their distribution in regions undergoing cell proliferation and/or differentiation. Previous studies using CX3CR1-GFP transgenic mice demonstrated that microglia extensively survey these regions. To simultaneously visualize microglia and neural-lineage cells that interact with each other, we applied the in utero electroporation (IUE) technique, which has been widely used for gene-transfer in neurodevelopmental studies, to CX3CR1-GFP mice (males and females). However, we unexpectedly faced a technical problem: although microglia are normally distributed homogeneously throughout the mid-embryonic cortical wall with only limited luminal entry, the intraventricular presence of exogenously derived plasmid DNAs induced microglia to accumulate along the apical surface of the cortex and aggregate in the choroid plexus. This effect was independent of capillary needle puncture of the brain wall or application of electrical pulses. The microglial response occurred at plasmid DNA concentrations lower than those routinely used for IUE, and was mediated by activation of Toll-like receptor 9 (TLR9), an innate immune sensor that recognizes unmethylated cytosine-phosphate guanosine (CpG) motifs abundant in microbial DNA. Administration of plasmid DNA together with oligonucleotide (ODN) 2088, the antagonist of TLR9, restored the normal (dispersed) intramural localization of microglia and decreased luminal accumulation of these cells. Thus, via TLR9, intraventricular plasmid DNA administration causes aberrant distribution of embryonic microglia, suggesting that the behavior of microglia in brain primordia subjected to IUE should be carefully interpreted.

Significance statement Microglia have been recently shown to play multiple roles in the embryonic brain. In the trials for labeling neural-lineage cells using IUE technique in CX3CR1-GFP mice, in which microglia express GFP, to achieve dual live-imaging of these cell types, we unexpectedly found that intra-ventricular administration of plasmid DNA caused microglial aberrant accumulation along the luminal surface of the cerebral wall and in the choroid plexus. Notably, Co-administration of TLR9 antagonist into the ventricle together with plasmid DNA restored the normal dispersed localization of microglia in the mid-embryonic (E14) cortex, suggesting that massive microglial accumulation induced by plasmid DNA is primarily mediated by TLR9 activation. Our findings have implications for the application of IUE to investigate embryonic microglia.