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

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...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

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

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.

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 ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.9K Views.  University of Massachusetts Medical School. 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.

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

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

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

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

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

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Biochemistry

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

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

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.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

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.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Biology

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

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 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.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

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

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

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.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

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

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

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.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

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

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

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.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

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

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Chapters in this video

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

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

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

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

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

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

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

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

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons