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Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

ABSTRACT: In this project, we enabled mass spectrometry based proteomics characterization of single neurons in sections of the mouse brain. We accessed cells in a whole-cell patch clamp configuration and aspirated a portion of the neuronal soma into the patch clamp probe. The collected aspirate was expelled into a microvial, where proteins were extracted and processed for shot-gun analysis. The resulting trace amounts of protein digests were analyzed on a custom-built microanalytical capillary electrophoresis platform that was connected to an electrospray ionization high-resolution mass spectrometer. This “patch proteomics” technology identified ~150 proteins from triplicate measurement of single dopaminergic neurons in the mouse substantia nigra.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Primary Neuron

SUBMITTER: Peter Nemes

LAB HEAD: Peter Nemes

PROVIDER: PXD028040 | Pride | 2022-02-18

REPOSITORIES: pride

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			Action	DRS
	2018-08-08_SC02.zip	Other
	2018-08-08_SC02_50_nL_4_neurons_wit_bsa.RAW	Raw
	2018-08-08_SC03.zip	Other
	2018-08-08_SC03_50_nL_4_neurons_wit_bsa.RAW	Raw
	2018-08-08_SC04.zip	Other

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Project description:Aims: This study aimed to identify and characterize the intrinsic properties of locus coeruleus (LC) noradrenergic neurons in male and female mice using a genetic approach. We also sought to investigate sex-specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. Methods: Utilizing a genetic mouse model by crossing Dbhcre knock-in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal properties, including capacitance, action potential frequency, and rheobase, were assessed using patch-clamp recordings. Spontaneous and evoked activity between sexes was compared. Proteomic analyses of individual LC neuron soma was conducted using mass-spectrometry to discern protein expression profiles. Results: Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed inherent differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. Conclusions: Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex-based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells.

Dataset Information

Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry

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