Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Summary

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission. To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons. Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Abstract

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale. There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation. However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons. Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms. Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation. We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons. This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Introduction

Endocytic trafficking is a ubiquitous cellular mechanism that fine-tunes the plasma membrane presentation of a variety of integral membrane proteins. Endocytosis delivers vital nutrients to the intracellular milieu¹ and desensitizes receptor signaling in response to receptor activation². Endocytic recycling back to the plasma membrane can additionally enhance cellular signaling by increasing protein expression levels at the cell surface³. Moreover, membrane trafficking perturbations are implicated in numerous disease and pathological conditions^4,5, stressing the need to investigate the molecular mechanisms tha....

Protocol

All animal handling and tissue harvesting was performed in accordance with the guidelines of the University of Massachusetts Medical School Institutional Animal Care Use Committee (IACUC), following the approved protocol #A1506 (Melikian, P.I.).

Required solutions

Artificial cerebrospinal fluid (ACSF) - Make fresh daily

125 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.4 mM CaCl₂, 26 mM NaHCO₃, and 11 mM glucose

Note: Prepare ACSF as a 10x stock s....

Results

The neuronal dopamine transporter is internalized in response to PKC activation in cell lines^16-20. Despite many reports demonstrating PKC-induced DAT surface losses in a variety of cell lines and expression systems, it has been challenging to confirm this finding in cultured dopaminergic neurons^21-23. We used mouse striatal slices to directly test whether DAT internalizes in response to PKC activation in adult dopaminergic neurons. Following slice preparation, slices were hemisected along the midli.......

Discussion

Despite longstanding knowledge that endocytic trafficking critically impacts synaptic signaling in the brain, it has proved challenging to quantitatively measure changes in protein surface expression in adult neurons. In this work, we report a reliable approach to label surface protein ex vivo in acute brain slices. Brain slice preparations have a longstanding history of utility for electrophysiological recordings, as they maintain synaptic connections and cell viability up to hours after their prepara.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
sulfo NHS-SS-biotin	Pierce	21331
Streptavidin agarose	Pierce	20347
IgG-free, Protease-free bovine serum albumin	Sigma	A3059
Vibrating microtome sectioner	various
Shaking water bath	various
Millicell mesh-bottomed inserts (8 µm pore size)	Millipore	PI8P 012 50	These can be washed by hand and reused