In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia

Abstract

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

Mitochondrial network adaptations of microglia reveal sex-specific stress response after injury and UCP2 knockout

“… 20 , 21 , 23 , 24 , 25 Microglia isolated from their environment quickly transition to a responsive phenotype, preventing us from deciphering whether novel insights result from the microglia within the tissue environment or from artifacts arising due to the additional stress of tissue dissociation, processing, or the primary culture environment. 26 , 27 , 28 Furthermore, spatial information and environmental factors such as sex are lost in cell isolation studies, which are important contributing factors known to influence microglia. 29 A major limitation in addressing mitochondria in microglia in vivo is the lack of tools to effectively visualize mitochondrial networks specifically in microglia.…”