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Updated: Jul 20, 2025
Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Investigating microglia-neuron crosstalk by characterizing microglial contamination in human and mouse patch-seq
Keon Arbabi1,2, Yiyue Jiang1,3, Derek Howard1
1The Krembil Centre for Neuroinformatics, Centre for Addiction and Mental Health, Toronto, ON, Canada.
Abstract:
Microglia are cells with diverse roles, including the regulation of neuronal excitability. We leveraged Patch-seq to assess the presence and effects of microglia in the local microenvironment of recorded neurons. We first quantified the amounts of microglial transcripts in three Patch-seq datasets of human and mouse neocortical neurons, observing extensive contamination. Variation in microglial contamination was explained foremost by donor identity, particularly in human samples, and additionally by neuronal cell type identity in mice. Gene set enrichment analysis suggests that microglial contamination is reflective of activated microglia, and that these transcriptional signatures are distinct from those captured via single-nucleus RNA-seq. Finally, neurons with greater microglial contamination differed markedly in their electrophysiological characteristics, including lowered input resistances and more depolarized action potential thresholds. Our results generalize beyond Patch-seq to suggest that activated microglia may be widely present across brain slice preparations and contribute to neuron- and donor-related electrophysiological variability in vitro.
Insights
Microglial (immune cell) contamination in Patch-seq data is common and linked to activated microglia. This contamination affects neuronal recordings, influencing excitability and potentially explaining variability in brain slice experiments.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Microglia, the brain's resident immune cells, play crucial roles in regulating neuronal function.
- Patch-seq is a technique combining patch-clamp electrophysiology with single-cell RNA sequencing.
- Understanding microglial influence on neuronal recordings is vital for accurate interpretation of brain slice data.
Purpose of the Study:
- To investigate the presence and impact of microglial contamination in Patch-seq datasets of human and mouse neocortical neurons.
- To determine factors influencing microglial contamination levels and their transcriptional signatures.
- To assess the relationship between microglial contamination and neuronal electrophysiological properties.
Main Methods:
- Quantification of microglial transcripts in three human and mouse neocortical Patch-seq datasets.
- Analysis of variation in microglial contamination based on donor and neuronal cell type identity.
- Gene set enrichment analysis to characterize microglial transcriptional signatures.
- Correlation analysis between microglial contamination levels and neuronal electrophysiological measurements.
Main Results:
- Extensive microglial transcript contamination was observed in human and mouse neocortical Patch-seq datasets.
- Microglial contamination levels varied significantly with donor identity (especially in humans) and neuronal cell type (in mice).
- Transcriptional signatures indicated activated microglia, distinct from those found in single-nucleus RNA-seq.
- Increased microglial contamination correlated with altered neuronal electrophysiology, including lower input resistance and more depolarized action potential thresholds.
Conclusions:
- Microglial contamination is a prevalent issue in Patch-seq, reflecting activated microglia in brain slice preparations.
- This contamination can significantly impact neuronal electrophysiological characteristics, contributing to observed variability.
- Findings highlight the need to account for microglial presence and activation when interpreting Patch-seq data and other brain slice studies.
