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Anal Chem. 2014 Mar 18;86(6):3203-8. doi: 10.1021/ac500168d. Epub 2014 Mar 6.

Patch clamp electrophysiology and capillary electrophoresis-mass spectrometry metabolomics for single cell characterization.

Author information

1
Beckman Institute for Advanced Science and Technology, ‡Department of Pharmacology, §Department of Molecular and Integrative Physiology, ∥Department of Chemistry, and ⊥Neuroscience Program, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

Abstract

The visual selection of specific cells within an ex vivo brain slice, combined with whole-cell patch clamp recording and capillary electrophoresis (CE)-mass spectrometry (MS)-based metabolomics, yields high chemical information on the selected cells. By providing access to a cell's intracellular environment, the whole-cell patch clamp technique allows one to record the cell's physiological activity. A patch clamp pipet is used to withdraw ∼3 pL of cytoplasm for metabolomic analysis using CE-MS. Sampling the cytoplasm, rather than an intact isolated neuron, ensures that the sample arises from the cell of interest and that structures such as presynaptic terminals from surrounding, nontargeted neurons are not sampled. We sampled the rat thalamus, a well-defined system containing gamma-aminobutyric acid (GABA)-ergic and glutamatergic neurons. The approach was validated by recording and sampling from glutamatergic thalamocortical neurons, which receive major synaptic input from GABAergic thalamic reticular nucleus neurons, as well as neurons and astrocytes from the ventral basal nucleus and the dorsal lateral geniculate nucleus. From the analysis of the cytoplasm of glutamatergic cells, approximately 60 metabolites were detected, none of which corresponded to the compound GABA. However, GABA was successfully detected when sampling the cytoplasm of GABAergic neurons, demonstrating the exclusive nature of our cytoplasmic sampling approach. The combination of whole-cell patch clamp with single cell cytoplasm metabolomics provides the ability to link the physiological activity of neurons and astrocytes with their neurochemical state. The observed differences in the metabolome of these neurons underscore the striking cell to cell heterogeneity in the brain.

PMID:
24559180
PMCID:
PMC3964733
DOI:
10.1021/ac500168d
[Indexed for MEDLINE]
Free PMC Article

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