Name: brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons
Uploaded: 2014-04-03T15:00:00
Description: 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

Dette arbeidet ble finansiert av NIH tilskudd DA15169 og DA035224 til HEM

Alle dyr håndtering og vev høsting ble utført i samsvar med retningslinjene fra University of Massachusetts Medical School Institutional Animal Care bruk Committee (IACUC), etter den godkjente protokollen # A1506 (Melikian, PI). Nødvendige løsninger Kunstig cerebrospinalvæsken (ACSF) - Make fersk daglig 125 mM NaCl, 2,5 mM KCl, 1,2 mM NaH 2 PO 4, 1,2 mM MgCl 2, 2...

<ol>
	<li>Conner, S. D., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+portals+of+entry+into+the+cell.">Regulated portals of entry into the cell.</a> Nature. 422, 37-44 (2003).</li><li>Zastrow, M., Williams, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+neuromodulation+by+receptor+membrane+traffic+in+the+endocytic+pathway.">Modulating neuromodulation by receptor membrane traffic in the endocytic pathway.</a> Neuron. 76, 22-32 (2012).</li><li>Leto, D., Saltiel, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+transport+by+insulin:+traffic+control+of+GLUT4.">Regulation of glucose transport by insulin: traffic control of GLUT4.</a> Nat. Rev. Mol. Cell Biol. 13, 383-396 (2012).</li><li>Liu, Y. W., Lukiyanchuk, V., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+membrane+trafficking+defects+of+disease-associated+dynamin+2+mutations.">Common membrane trafficking defects of disease-associated dynamin 2 mutations.</a> Traffic. 12, 1620-1633 (2011).</li><li>Li, X., DiFiglia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recycling+endosome+and+its+role+in+neurological+disorders.">The recycling endosome and its role in neurological disorders.</a> Prog. Neurobiol. , 127-141 (2012).</li><li>Sandvig, K., Pust, S., Skotland, T., van Deurs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-independent+endocytosis:+mechanisms+and+function.">Clathrin-independent endocytosis: mechanisms and function.</a> Curr. Opin. Cell Biol. 23, 413-420 (2011).</li><li>Kumari, S., Mg, S., Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+unplugged:+multiple+ways+to+enter+the+cell.">Endocytosis unplugged: multiple ways to enter the cell.</a> Cell Res. 20, 256-275 (2010).</li><li>Barry, M. F., Ziff, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+trafficking+and+the+plasticity+of+excitatory+synapses.">Receptor trafficking and the plasticity of excitatory synapses.</a> Curr. Opin. Neurobiol. 12, 279-286 (2002).</li><li>Bredt, D. S., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+at+excitatory+synapses.">AMPA receptor trafficking at excitatory synapses.</a> Neuron. 40, 361-379 (2003).</li><li>Kerchner, G. A., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+synapses+and+the+emergence+of+a+postsynaptic+mechanism+for+LTP.">Silent synapses and the emergence of a postsynaptic mechanism for LTP.</a> Nat. Rev. Neurosci. 9, 813-825 (2008).</li><li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+and+synaptic+plasticity.">AMPA receptor trafficking and synaptic plasticity.</a> Annu. Rev. Neurosci. 25, 103-126 (2002).</li><li>Borgland, S. L., Malenka, R. C., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+cocaine-induced+potentiation+of+synaptic+strength+in+the+ventral+tegmental+area:+electrophysiological+and+behavioral+correlates+in+individual+rats.">Acute and chronic cocaine-induced potentiation of synaptic strength in the ventral tegmental area: electrophysiological and behavioral correlates in individual rats.</a> J. Neurosci. 24, 7482-7490 (2004).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine-induced+potentiation+of+synaptic+strength+in+dopamine+neurons:+Behavioral+correlates+in+GluRA(-/-)+mice.">Cocaine-induced potentiation of synaptic strength in dopamine neurons: Behavioral correlates in GluRA(-/-) mice.</a> PNAS. 101, 14282-14287 (2004).</li><li>Hyman, S. E., Malenka, R. C., Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+addiction:+the+role+of+reward-related+learning+and+memory.">Neural mechanisms of addiction: the role of reward-related learning and memory.</a> Annu. Rev. Neurosci. 29, 565-598 (2006).</li><li>Thomas, M. J., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+mesolimbic+dopamine+system.">Synaptic plasticity in the mesolimbic dopamine system.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358, 815-819 (2003).</li><li>Sorkina, T., Hoover, B. R., Zahniser, N. R., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+and+protein+kinase+C-induced+internalization+of+the+dopamine+transporter+is+mediated+by+a+clathrin-dependent+mechanism.">Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.</a> Traffic. 6, 157-170 (2005).</li><li>Holton, K. L., 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=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. Chem. 274, 35794-35801 (1999).</li><li>Sorkina, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screen+reveals+an+essential+role+of+Nedd4-2+in+dopamine+transporter+ubiquitination+and+endocytosis.">RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.</a> J. Neurosci. 26, 8195-8205 (2006).</li><li>Eriksen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+dopamine+transporter+trafficking+in+live+neurons+by+use+of+fluorescent+cocaine+analogs.">Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs.</a> J. Neurosci. 29, 6794-6808 (2009).</li><li>Rao, A., Simmons, D., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+subcellular+distribution+of+endosomal+compartments+and+the+dopamine+transporter+in+dopaminergic+neurons.">Differential subcellular distribution of endosomal compartments and the dopamine transporter in dopaminergic neurons.</a> Mol. Cell Neurosci. 46, 148-158 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-752 (2011).</li></ol>

Til tross for lang erfaring som endocytic menneskehandel kritisk påvirker synaptiske signalering i hjernen, har det vist seg utfordrende å kvantitativt måle endringer i protein overflate uttrykk hos voksne nevroner. I dette arbeidet, rapporterer vi en pålitelig tilnærming til å merke overflateprotein ex vivo i akutte hjernen skiver. Brain slice forberedelser har en lang historie av verktøy for elektrofysiologiske opptak, som de opprettholder synaptiske forbindelser og celleviabilitet opptil timer etter t...

Endocytic menneskehandel er en allestedsnærværende cellulær mekanisme som fintuner plasma membran presentasjon av en rekke integrerte membran proteiner. Endocytose gir viktige næringsstoffer til det intracellulære miljøet 1 og desensitizes reseptor signal som respons på reseptoraktivering 2.. Endocytic resirkulering tilbake til plasmamembranen kan i tillegg øke cellulær signalisering ved å øke protein uttrykk nivåer på celleoverflaten 3.. Videre er membran trafficking forstyrrelsene innblandet i en rekke sykdommer og patologiske tilstander 4,5, understreker behovet for å undersøke de molekylære mekanismer som styrer protein endocytic menneskehandel. Mens mange proteiner utnytte klassiske clathrin avhengige internalise mekanismer, monterings bevis i løpet av de siste årene viser at flere clathrin uavhengig endocytiske mekanismer styrer endocytic potensialet i et økende utvalg avproteiner 6,7. Dermed har behovet for å undersøke endocytiske mekanismer tilrettelegge menneskehandel i fysiologiske relevante systemer vokst betraktelig. I hjernen har endocytic smugling av reseptorer, ionekanaler og nevrotransmitter transportører en primær rolle i etableringen av synaptisk plastisitet 8-11 og respons til narkotika misbruk 12-15, til syvende og sist påvirker nevronal eksitabilitet og synaptiske responser. Hittil flertallet av nevrale trafficking studier er avhengige av enten heterologe ekspresjonssystemer eller dyrkede primære nevroner, kan verken som pålitelig reflektere mekanismer som spiller inn i voksen nevroner. Her rapporterer vi en tilnærming som bruker overflaten biotinylering å kvantitativt måle overflateproteinnivåer i akutte hjernen skiver avledet fra voksne gnagere. Ved hjelp av denne tilnærmingen, presenterer vi data som viser at musen striatal dopamin transporter internalizes raskt i resom reaksjon på phorbol ester-mediert protein kinase C (PKC) aktivering.

<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

Regulert endocytic trafficking er den sentrale mekanismen tilrettelegge en rekke neuromodulatory hendelser, ved dynamisk styring reseptor, ionekanal, og transporter celleoverflaten presentasjon på en minutters tidsskala. Det er et bredt mangfold av mekanismer som styrer endocytic smugling av enkelte proteiner. Studier som undersøker de molekylære grunnlaget for menneskehandel har først og fremst stole på overflaten biotinylering å kvantitativt måle endringer i membran protein overflate uttrykk som svar på eksogene stimuli og genmanipulering. Imidlertid har denne tilnærmingen i hovedsak vært begrenset til dyrkede celler, noe som kanskje ikke trofast gjenspeiler de fysiologisk relevante mekanismer som spiller inn i voksen nevroner. Videre kan dyrkede celle tilnærminger undervurdere regionsspesifikke forskjeller i trafficking mekanismer. Her beskriver vi en tilnærming som strekker celleoverflaten biotinylering til den akutte hjernen skive forberedelse. Viviser at denne metoden gir et high-fidelity tilnærming for å måle raske endringer i membranproteinoverflatenivået hos voksne nevroner. Denne tilnærmingen er sannsynlig å ha bred nytte innen nevronale endocytic menneskehandel.

Den neuronal dopamin transportøren er internalisert i respons til aktivering av PKC i cellelinjer 16-20. Til tross for mange rapporter som viser PKC-indusert DAT overflate tap i en rekke cellelinjer og ekspresjonssystemer, har det vært vanskelig å få bekreftet dette funnet i dyrkede dopaminerge nevroner 21-23. Vi brukte mus striatum-skiver til direkte å teste om DAT internalizes som reaksjon på PKC aktivering hos voksne dopaminerge nevroner. Etter skive fremstilling, ble skiver hemisected lan...

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

Nevronale membran menneskehandel dynamisk styrer plasma membran protein tilgjengelighet og betydelig påvirker nevrotransmisjon. Til dags dato har det vært utfordrende å måle nevronale endocytic menneskehandel i voksen nevroner. Her beskriver vi en svært effektiv, kvantitativ metode for å måle hurtige forandringer i overflateprotein ekspresjon ex vivo i akutte hjerneskiver.

Brain Slice Biotinvlerina: An<em&gt; Ex Vivo</em&gt; Approach to Measure Region spesifikke Plasma Membrane Protein Trafficking in Adult Neuroner

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

Research

JoVE Journal

Neuroscience

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

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Funksjonell Bildediagnostiske Bruke Ultrasonic blod-hjerne barrieren avbrudd og mangan forbedret MR

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Using an ÃƒÅ½Ã‚Â±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Ved hjelp av en α-bungarotoxin Binding Side Tag til Study GABA A-reseptoren Membran Lokalisering og menneskehandel

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Slice It Hot: Akutt Adult Brain Slicing i Fysiologisk Temperatur

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

En<em&gt; Ex Vivo</em&gt; Laser-indusert ryggmargsskade Model å vurdere Mekanismer av aksonal degenerasjon i sanntid

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo optogenetic Disseksjon av Fear kretser i hjernen skiver

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

I Vivo Functional Brain Imaging tilnærming basert på Bioluminescent Kalsium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Live bilder av GLUT4 Protein menneskehandel musen primære hypothalamus nevroner bruker Deconvolution mikroskopi

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

En alternativ tilnærming til studere primære hendelser i Neurodegeneration bruker Ex Vivo rotte hjernen skiver

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Bruke nær-infrarød fluorescens og høyoppløselig skanning å måle protein uttrykk i gnagere Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...