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

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.

Figure 1

Schema illustrating the workflow for sample collection. An upright microscope is used for conducting electrophysiology experiments under video observation. Following the process of establishing the whole-cell configuration as described in the text, negative pressure is applied to the patch pipet and the cytoplasm is withdrawn. The patch pipet is removed and the tip broken off into the bottom of a PCR tube. Extraction solution is then added to the tube and the sample centrifuged and placed on dry ice until analysis by CE–MS.

Figure 2

Images of a patch pipet tip used for collection. (A) Photomicrographs of the intact tip of the patch pipet. (B) An overlay of the pipet tip after being broken off into the PCR tube. (C) Close-up image of the patch pipet tip. (D) Close-up image of the patch pipet tip showing the tip diameter.

Figure 3

Microdissected thalamic nuclei from adjacent regions of a 280-μm-thick section produce dramatically different neurotransmitter profiles when analyzed by CE–MS. The thalamic reticular nucleus (TRN) contains purely GABAergic neurons, but the slice provides sufficient amounts of acetylcholine and glutamate in addition to GABA for MS detection. The dorsal thalamus (DT) contains predominantly glutamatergic neurons (such as those of the dorsal lateral geniculate nucleus, which were patched in this study) but the slice contains abundant amounts of histamine, acetylcholine, GABA, serotonin, norepinephrine, as well as the nitric oxide precursor, argininosuccinate. Additional metabolites from these slices are listed in Table S2 in the Supporting Information.

Figure 4

Data obtained from four distinct cell types: (1) VB thalamocortical neuron; (2) nonburst firing TRN neuron; (3) bursting TRN neuron; (4) astrocyte. (A) Electrophysiological recordings of the individual cells (1–4) shown in the (B) photomicrographs (scale bar = 20 μm). (C) Extracted ion chromatograms corresponding to the cytoplasm sampled from the neurons and glia are shown. Peaks correspond to ornithine (dark red), GABA (light red), glycine (yellow), serine (gold), tryptophan (rainbow), glutamine (light green), glutamate (light blue), tyrosine (dark blue), and proline (indigo).