Abstract
Our understanding of the physiological and pathological functions of brain lipids is limited by the inability to analyze these molecules at cellular resolution. Here, we present a method that enables the detection of lipids in identified single neurons from live mammalian brains. Neuronal cell bodies are captured from perfused mouse brain slices by patch clamping, and lipids are analyzed using an optimized nanoflow liquid chromatography/mass spectrometry protocol. In a first application of the method, we identified more than 40 lipid species from dentate gyrus granule cells and CA1 pyramidal neurons of the hippocampus. This survey revealed substantial lipid profile differences between neurons and whole brain tissue, as well as between resting and physiologically stimulated neurons. The results suggest that patch clamp-assisted single neuron lipidomics could be broadly applied to investigate neuronal lipid homeostasis in healthy and diseased brains.
Highlights
Our understanding of the physiological and pathological functions of brain lipids is limited by the inability to analyze these molecules at cellular resolution
Present knowledge about neural lipids almost exclusively derives from studies on gross anatomical areas of the brain, in which heterogeneous neuronal populations are intermingled with approximately equal numbers of glial and endothelial cells[1], or on neuronal and glial cells in primary cultures, which are developmentally immature and are maintained in artificial media that can strongly influence their lipid composition
The present report describes an approach in which lipid profiling of individual neurons is achieved by combining patch-clamp with an optimized nanoflow liquid chromatography/high-resolution mass spectrometry protocol
Summary
We used a technique originally devised for single-cell transcriptomics to capture live neurons from acutely dissected mouse hippocampal slices. Considering that relevant signals could be hidden by matrix-derived noise, we extracted ions for a representative set of 236 lipids known to be present in neural tissue (Supplementary Table S1). We focused on lipid-related signals that met the following criteria: (i) signal-to-noise ratio >3; (ii) presence in ≥75% of single-neuron samples; (iii) peak area at least 3x higher than two sets of controls including freshly prepared ACSF (5 μL) and ‘sham-capture’ controls, in which a patch pipette was lowered into the slice but no cell was collected. These included abundant membrane constituents such as cholesterol (Fig. 2a), and less-represented species such as hexosylceramide (d18:1/24:0) and cholesteryl ester (CE) 16:0 (total number of carbons: total number of double bonds) (Fig. 2b,c).
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