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...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical,+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat. Neurosci. 8, 881-888 (2005).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J. Biol. Chem. 278, 22168-22174 (2003).</li><li>Melikian, H. E., Buckley, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+trafficking+regulates+the+activity+of+the+human+dopamine+transporter.">Membrane trafficking regulates the activity of the human dopamine transporter.</a> J. Neurosci. 19, 7699-7710 (1999).</li><li>Daniels, G. M., Amara, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+trafficking+of+the+human+dopamine+transporter.+Clathrin-mediated+internalization+and+lysosomal+degradation+in+response+to+phorbol+esters.">Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters.</a> J. Biol. 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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 border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</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

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Neuroscience

12.8K vues.  University of Massachusetts Medical School.  Trafic membranaire neuronal contrôle dynamiquement la membrane plasmique disponibilité des protéines et un impact significatif sur la neurotransmission. À ce jour, il a été difficile de mesurer le trafic endocytose neuronale dans les neurones adultes. Ici, nous décrivons une méthode très efficace, quantitative pour mesurer les changements rapides dans l'expression des protéines de surface ex vivo dans des tranches de cerveau de courte durée. 