Name: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons (Video) | JoVE Journal
Uploaded: 2014-04-03T15:00:00.000+00:00
Duration: 6 min 18 s
Description: Learn brain slice biotinylation to quantify acute changes in neuronal surface proteins ex vivo. This method enables drug treatment studies and protein trafficking analysis in adult neurons using streptavidin affinity chromatography.

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5 minutes

This work was funded by NIH grants DA15169 and DA035224 to H.E.M.

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 solution, excluding NaHCO3 and glucose. Make 1x working solutions daily from the 10x stock, supplementing with fresh NaHCO3 and glucose.
Sucrose-supplemented ACSF (SACSF) &#8211; Make fresh daily
250 mM sucrose, 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 SACSF as a 10x stock solution, excluding NaHCO3 and glucose. Make 1x working solutions daily from the 10x stock, supplementing with fresh NaHCO3 and glucose.
Sulfo-N-hydroxysuccinyl-SS-Biotin (sulfo-NHS-SS-biotin, Pierce Chemical Company)
Stock solutions should be 200 mg/ml in DMSO and are resistant to multiple freeze/thaw cycles. Aliquots are stored at -20 &#176;C. The succinyl ester is rapidly hydrolyzed in aqueous solution, so working solutions should be prepared immediately prior to applying to slices.
Slice Quench Solution
ACSF supplemented with 100 mM glycine
RIPA Lysis Buffer
10 mM Tris, pH 7.4, 150 mM NaCl, 1.0 mM EDTA, 1% Triton-X-100, 0.1% SDS, 1% Na deoxycholate
RIPA with Protease Inhibitors (RIPA/PI) &#8211; Make fresh daily
RIPA supplemented with 1 &#956;M leupeptin, 1 &#956;M pepstatin, 1 &#956;M aprotinin, and 1 mM phenylmethyl sulfonyl fluoride.
1. Prepare Brain Slices
<ol>
	<li>Make fresh 1x SACSF and 1x ACSF</li>
	<li>Chill SACSF on ice in a small beaker. This will be used to hold the freshly harvested mouse brain.</li>
	<li>Saturate the ACSF and SACSF with oxygen by bubbling with 95%/5% O2/CO2, 20 min on ice.</li>
	<li>P30-38 mice should be used for optimal tissue viability. Sacrifice animals by cervical dislocation and decapitation, and rapidly remove brains into prechilled, oxygenated SACSF.</li>
	<li>Using a vibrating microtome, make 300 &#956;m brain sections in region of interest.</li>
	<li>If desired, slices can be further dissected prior to recovery to enrich for particular brain regions or separate right and left hemispheres to use as control and experimental slices, respectively.</li>
	<li>Using a fire-polished Pasteur pipette, transfer slices to mesh-bottomed chambers set into 24-well plates.</li>
	<li>Allow slices to recover for 40 min, 31 &#176;C in oxygenated ACSF, with continual, gentle bubbling.</li>
</ol>
2. Drug Treatment (if appropriate) and Slice Biotinylation
<ol>
	<li>Following recovery, wash slices 3x in prewarmed (37 &#176;C), oxygenated ACSF bubbling constantly with 95%/5% O2/CO2.</li>
	<li>Add test compounds and incubate with continuous oxygenation.
	<ol>
		<li>For convenience, add a 1/10th volume of 10x concentrated drug, and mix by gently inverting.</li>
		<li>Shake plates gently in a water bath at the desired temperature.</li>
	</ol>
	</li>
	<li>Following drug treatment, rapidly chill slices by washing 3x in ice cold ACSF.</li>
</ol>
3. Biotinylate Surface Proteins
<ol>
	<li>Prepare 1.0 mg/ml sulfo-NHS-SS-biotin in ice cold ACSF immediately prior to labeling.</li>
	<li>Add 0.75 ml sulfo-NHS-SS-biotin to slices and incubate slices on ice, 45 min.</li>
	<li>Wash slices three times quickly with ice cold ACSF, then incubate for 10 min in ice cold ACSF on ice.</li>
	<li>Wash slices three times with ice cold slice quench buffer and incubate with 0.75 ml slice quench buffer two times, 25 min, on ice to quench free sulfo-NHS-SS-biotin.</li>
</ol>
4. Prepare Tissue Lysates
<ol>
	<li>Wash slices three times in ice cold ACSF and transfer each slice to a microcentrifuge tube using a fire-polished Pasteur pipette.</li>
	<li>Gently pellet slice by centrifuging 200 x g, 1 min and carefully aspirate remaining ACSF.</li>
	<li>Add 400 &#181;l ice cold RIPA/PI and break up tissue by pipetting up and down once through a P200 pipette.</li>
	<li>Transfer dissociated slice/RIPA to a fresh tube and incubate 30 min, 4 &#176;C, rotating, to complete lysis.</li>
	<li>Pellet cellular debris by centrifuging, 18,000 x g, 15 min, 4 &#176;C.</li>
	<li>Determine lysate protein concentrations using the BCA protein assay, with bovine serum albumin (BSA) as a standard.</li>
</ol>
5. Isolate Biotinylated Proteins
<ol>
	<li>Optimize Bead/total protein ratio. 
	Note: Individual protein expression levels can vary widely across different brain regions. Unless all of the biotinylated protein in a given amount of tissue lysate is captured, it is not possible to accurately detect any potential changes in surface expression. Therefore, it is imperative to empirically determine the optimal bead/total protein ratio for a particular protein/brain region prior to embarking upon a new slice biotinylation study.
	<ol>
		<li>Incubate 25 &#181;l streptavidin agarose beads with increasing amounts of tissue lysate (approximate range 25-200 &#181;g), and then proceed as described below for binding, washing and elution steps.</li>
		<li>Quantify the resulting immunoreactive bands and choose a bead/lysate ratio in the linear range of binding that will permit accurate quantification of either increased or decreased protein surface expression.</li>
	</ol>
	</li>
	<li>Prepare Streptavidin-Agarose Beads
	<ol>
		<li>Determine total bead volume needed for all samples. Prepare a sufficient bead volume for that amount plus one extra (i.e. 4 samples at&#160;25 &#181;l/sample = 100 &#181;l beads + one extra = 125 &#181;l beads.</li>
		<li>Vortex Streptavidin bead stock and pipette out desired volume. If using a P200 pipette, cut off the end of the tip to prevent blockage or bead damage.</li>
		<li>Wash beads 3 times in 0.5-1.0 ml RIPA/PI to remove preservatives, vortexing after each RIPA addition and collecting beads between washes by centrifuging 18,000 x g, 1 min, at room temperature.</li>
		<li>Aspirate off buffer between washes using a glass Pasteur pipette attached to a vacuum flask. A plastic P200 tip attached to the end of the glass Pasteur pipette provides finer control when aspirating. It is not necessary to remove absolutely all the buffer for the first two&#160;washes, as it risks aspirating beads into the pipette. After the final wash, remove as much excess buffer as possible without aspirating the beads.</li>
		<li>Add RIPA/PI to bring beads back to their original volume, pipetting up and down to resuspend. Avoid vortexing at this point, as beads will stick to the tube wall.</li>
		<li>Aliquot beads into microcentrifuge tubes using a P200 with the tip cut off. Pipette up and down several times between sampling to assure beads remain evenly suspended and dispersed among the tubes.</li>
	</ol>
	</li>
	<li>Bind biotinylated proteins to streptavidin beads
	<ol>
		<li>Distribute cell lysates to the tubes containing the agarose beads. For experiments where multiple samples are being compared, make sure to use the same amount of protein for each sample for accurate comparisons.</li>
		<li>Add additional RIPA/PI to bring to samples to a 200 &#181;l minimal volume. This assures that samples will mix adequately during the incubation and also unifies the protein concentrations across samples.</li>
		<li>Place tubes in a tube rotator and mix overnight at 4 &#176;C.</li>
		<li>In separate tubes, dispense an equivalent of the total lysate volume used for each sample. Alternatively, if the sample volumes are high, a fraction of total lysate volume can be reserved instead, to accommodate maximal load volumes on SDS-PAGE gels. These will be used to normalize the surface expression to the total protein amount.</li>
		<li>Add either an equal volume of 2x or 1/5 volume of 6x SDS-PAGE sample buffer and either incubate at 4 &#176;C, in parallel with bead samples, or store at -20 &#176;C until samples are analyzed.</li>
	</ol>
	</li>
</ol>
6. Elute and Analyze Samples
<ol>
	<li>Pellet beads by centrifuging 18,000 x g, 2 min, at room temperature.</li>
	<li>Aspirate supernatant and wash beads three times with 0.75 ml RIPA. To minimize bead loss, leave a small head volume of buffer above beads between washes. After the final wash, remove as much RIPA as possible, without disrupting the bead pellet.</li>
	<li>Elute biotinylated proteins from streptavidin beads by reducing the disulfide linkage. Add 25 &#181;l 2x SDS-PAGE reducing sample buffer, vortex well and rotate samples 30 min, room temperature. 
	Note: Many membrane proteins have a high tendency to aggregate when boiled in SDS-PAGE sample buffer, severely impairing their electrophoretic mobility. If this is the case for the protein being investigated, avoid heating samples and, instead, elute slowly at room temperature. If boiling is absolutely necessary (e.g. if another protein assessed in parallel requires boiling), sample buffer can be supplemented with 2 M urea (final concentration) to minimize aggregation. While effective in reducing aggregation in some cases, urea often compromises band appearances.</li>
	<li>Analyze samples by immunoblot.</li>
	<li>Thaw total lysate samples and rotate in parallel with bead samples, 30 min, room temperature.</li>
	<li>Separate proteins on SDS-PAGE gels.</li>
	<li>Identify protein(s) of interest by immunoblotting.
	<ol>
		<li>Be certain that bands are detected in the linear range of detection for proper quantification.</li>
		<li>To assure that the biotinylation reagent has not gained access to intracellular proteins via damaged/compromised cells. This is best accomplished by immunoblotting in parallel for an intracellular protein specific to the cell type being investigated.</li>
		<li>Quantify band densities and calculate relative protein surface density as a percent&#160;of the total protein expression level.</li>
	</ol>
	</li>
</ol>

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The authors have no financial interests that conflict with this work.

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.&#160;In this work, we report a reliable approach to label surface protein ex vivo in acute brain slices.&#160;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...

Endocytic trafficking is a ubiquitous cellular mechanism that fine-tunes the plasma membrane presentation of a variety of integral membrane proteins.&#160;Endocytosis delivers vital nutrients to the intracellular milieu1 and desensitizes receptor signaling in response to receptor activation2.&#160;Endocytic recycling back to the plasma membrane can additionally enhance cellular signaling by increasing protein expression levels at the cell surface3.&#160;Moreover, membrane trafficking perturbations are implicated in numerous disease and pathological conditions4,5, stressing the need to investigate the molecular mechanisms that govern protein endocytic trafficking.&#160;While many proteins utilize classic clathrin-dependent internalization mechanisms, mounting evidence over the past several years demonstrates that multiple clathrin-independent endocytic mechanisms govern the endocytic potential of an increasing array of proteins6,7.&#160;Thus, the need to investigate endocytic mechanisms facilitating trafficking in physiological relevant systems has grown considerably.
In the brain, endocytic trafficking of receptors, ion channels and neurotransmitter transporters has a primary role in establishing synaptic plasticity8-11 and response to drugs of abuse12-15, ultimately impacting neuronal excitability and synaptic responses.&#160;To date the majority of neuronal trafficking studies rely on either heterologous expression systems or cultured primary neurons, neither of which may reliably reflect mechanisms at play in adult neurons.&#160;Here, we report an approach that uses surface biotinylation to quantitatively measure surface protein levels in acute brain slices derived from adult rodents. Using this approach, we present data that demonstrate that the mouse striatal dopamine transporter rapidly internalizes in response to phorbol ester-mediated protein kinase C (PKC) activation.

<table><tbody><tr><td>sulfo NHS-SS-biotin</td><td>Pierce</td><td>21331</td><td></td></tr><tr><td>Streptavidin agarose</td><td>Pierce</td><td>20347</td><td></td></tr><tr><td>IgG-free, Protease-free bovine serum albumin</td><td>Sigma</td><td>A3059</td><td></td></tr><tr><td>Vibrating microtome sectioner</td><td>various</td><td></td><td></td></tr><tr><td>Shaking water bath</td><td>various</td><td></td><td></td></tr><tr><td>Millicell mesh-bottomed inserts (8 &amp;micro;m pore size)</td><td>Millipore</td><td>PI8P 012 50</td><td>These can be washed by hand and reused</td></tr></tbody></table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

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

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.&#160;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.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

The neuronal dopamine transporter is internalized in response to PKC activation in cell lines16-20. 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 neurons21-23. 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...

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

The overall goal of this procedure is to quantify acute changes in neuronal surface proteins. In ex vivo brain slice preparations, this is accomplished by first preparing viable acute brain slices. Next, the slices are treated with drugs of interest.Then the cell surface proteins are labeled using membrane imperian tonation reagents. Finally, surface biotinylated proteins are isolated using strep avid and affinity chromatography. Ultimately, immuno plotting is used to quantify changes in protein surface expression following drug treatments.The main advantage of this technique over existing methods, such as cell-based ation, is that neuronal protein trafficking is examined in situ rather than in heterologous expression systems or primary neuronal cultures. Generally, individual new to this method will struggle to prepare viable brand slices.Okay. To prepare brain slices begin by making fresh one x artificial cerebral spinal fluid or A CSF and sucrose supplemented A CSF or S-A-C-S-F according to the text protocol, chill.The S-A-C-S-F on ICE use 95%oxygen with 5%carbon dioxide to saturate the buffers with oxygen by bubbling for 20 minutes on ice. After sacrificing P 30 to 38 mice by cervical dislocation and decapitation and rapidly removing the brains, place them in pre chilled oxygenated S-A-C-S-F using a vibrating microtome. Make 300 micron brain sections in the region of interest and if desired, separate right and left hemispheres to use as control and experimental slices respectively.Using a fire polished pastier pipette transfer slices to mesh bottom holding chambers containing oxygenated A CSF. Allow the slices to recover for 40 minutes at 31 degrees Celsius with continuous gentle bubbling. After the incubation, transfer the slices to individual mesh chambers.In 24 well plates and wash the slices three times in prewarm oxygenated A CSF with constant bubbling. If testing compounds, add one 10th the volume of a 10 x concentrated drug and mix by gently inverting after gently shaking the plates in a water bath at the desired temperature. Use ice cold A CSF to rapidly chill the slices by washing them three times to buy aate surface proteins.Prepare fresh 1.0 milligram per milliliter sulfa N-H-S-S-S biotin in ice cold A CSF Add 0.75 milliliters of the solution to the slices and incubate on ice for 45 minutes. After using ice cold A CSF to quickly wash the slices three times, incubate them for 10 minutes in ice cold A CSF on ice, then use ice cold slice quench buffer to wash the slices three times and incubate them with 0.75 milliliters of slice, quench buffer two times for 25 minutes each on ice to quench free suo N-H-S-S-S biotin to prepare tissue lysates. Wash the slices three times in ice cold A CSF before using a fire polished pasti pipette to transfer each slice to a micro centrifuge tube.Gently pellet the slices by centrifusion at 200 GS for one minute and carefully aspirate the remaining A CSF. Then add 400 microliters of ice cold, rip a pi and use a P 200 pipette to break up the tissue by pipetting up and down once to complete lysis. Transfer the dissociated tissue to a fresh tube and incubate for 30 minutes at four degrees Celsius with rotating centrifuge at 18, 000 Gs for 15 minutes at four degrees Celsius.To pellet the cellular debris isolate the biotinylated proteins and analyze by immuno blot. According to the text protocol, the neuronal dopamine transporter is internalized in response to PKC activation in cell lines. Despite many reports demonstrating PKC induced dopamine, active transporter, or debt surface losses in a variety of cell lines and expression systems, it has been challenging to confirm this finding in cultured dopaminergic neurons, we used mouse stri AAL slices to directly test whether dat internalizes in response to PKC activation in adult dopaminergic neurons as seen.Here we detected robust DA surface expression in mouse striatum under basil or vehicle treated conditions with 81.4 plus or minus 5.8%of total debt at the cell surface PKC activation with four ball 12. Myra State 13 acetate significantly decreased dat surface expression to 60.8 plus or minus 5.2%of total debt, which corresponds to an about 30%loss of debt from the plasma membrane. In contrast, only 1.6 plus or minus 0.4%of total tyrosine hydroxylase was biotinylated consistent with its intracellular localization and confirming that the tonation reagent was excluded from the cell interior of dopaminergic neurons.After watching this video, you should have a good understanding of how to measure changes in plus membrane protein expression in intact neuronal terminals.

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Research

JoVE Journal

Neuroscience

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins.  Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein.  The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function.  We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

Learn brain slice biotinylation to quantify acute changes in neuronal surface proteins ex vivo. This method enables drug treatment studies and protein trafficking analysis in adult neurons using streptavidin affinity chromatography.

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

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