Name: deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents
Uploaded: 2013-08-07T13:00:00
Description: Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (NS002986). AS wrote the manuscript and implemented the deconvolution analysis. JSD developed the initial version of the deconvolution analysis and commented on the text.

1. Slice Preparation
<ol>
	<li>Prepare 500 ml slicing/storage solution containing (in mM): 119 NaCl, 2.5 KCl, 0.5 CaCl2, 1.3 MgSO4&middot;7H2O, 4 MgCl2, 26.2 NaHCO3, 1 NaH2PO4, and 22 glucose, 320 mOsm, pH 7.4</li>
	<li>Use a 250 ml beaker to prepare a submersion chamber for the slices, fill it with 200 ml slicing/storage solution, warm it in a water bath at 34 &deg;C and bubble it with 95% O2, 5% CO2.</li>
	<li>Keep the ...

<ol>
	<li>Ventura, R., Harris, 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=Three-dimensional+relationships+between+hippocampal+synapses+and+astrocytes.">Three-dimensional relationships between hippocampal synapses and astrocytes.</a> J. Neurosci. 19, 6897-6906 (1999).</li><li>Witcher, M. R., Kirov, S. A., Harris, 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=Plasticity+of+perisynaptic+astroglia+during+synaptogenesis+in+the+mature+rat+hippocampus.">Plasticity of perisynaptic astroglia during synaptogenesis in the mature rat hippocampus.</a> Glia. 55, 13-23 (2007).</li><li>Danbolt, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake.">Glutamate uptake.</a> Prog. Neurobiol. 65, 1-105 (2001).</li><li>Herman, M. A., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glutamate+concentration+in+hippocampal+slice.">Extracellular glutamate concentration in hippocampal slice.</a> J. Neurosci. 27, 9736-9741 (2007).</li><li>Arnth-Jensen, N., Jabaudon, D., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+between+independent+hippocampal+synapses+is+controlled+by+glutamate+uptake.">Cooperation between independent hippocampal synapses is controlled by glutamate uptake.</a> Nat. Neurosci. 5, 325-331 (2002).</li><li>Barbour, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+synapse+independence.">An evaluation of synapse independence.</a> J. Neurosci. 21, 7969-7984 (2001).</li><li>Rusakov, D. A., Kullmann, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrasynaptic+glutamate+diffusion+in+the+hippocampus:+ultrastructural+constraints,+uptake,+and+receptor+activation.">Extrasynaptic glutamate diffusion in the hippocampus: ultrastructural constraints, uptake, and receptor activation.</a> J. Neurosci. 18, 3158-3170 (1998).</li><li>Zerangue, N., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flux+coupling+in+a+neuronal+glutamate+transporter.">Flux coupling in a neuronal glutamate transporter.</a> Nature. 383, 634-637 (1038).</li><li>Eliasof, S., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+cell+glutamate+transporter+is+coupled+to+an+anionic+conductance.">Retinal glial cell glutamate transporter is coupled to an anionic conductance.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 4153-4158 (1996).</li><li>Wadiche, J. I., Amara, S. G., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+fluxes+associated+with+excitatory+amino+acid+transport.">Ion fluxes associated with excitatory amino acid transport.</a> Neuron. 15, 721-728 (1995).</li><li>Wadiche, J. I., Kavanaugh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+and+microscopic+properties+of+a+cloned+glutamate+transporter/chloride+channel.">Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel.</a> J. Neurosci. 18, 7650-7661 (1998).</li><li>Bergles, D. E., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activation+of+glutamate+transporters+in+hippocampal+astrocytes.">Synaptic activation of glutamate transporters in hippocampal astrocytes.</a> Neuron. 19, 1297-1308 (1997).</li><li>Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+the+glutamate+clearance+time+course+from+transporter+currents+in+CA1+hippocampal+astrocytes:+transmitter+uptake+gets+faster+during+development.">Deriving the glutamate clearance time course from transporter currents in CA1 hippocampal astrocytes: transmitter uptake gets faster during development.</a> J. Neurosci. 25, 2906-2916 (2005).</li><li>Scimemi, A., Tian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+transporters+regulate+glutamate+clearance,+NMDA+receptor+activation,+and+synaptic+plasticity+in+the+hippocampus.">Neuronal transporters regulate glutamate clearance, NMDA receptor activation, and synaptic plasticity in the hippocampus.</a> J. Neurosci. 29, 14581-14595 (2009).</li><li>Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+enhances+glutamate+uptake+via+glutamate+transporters+in+rat+glial+cells.">Isoflurane enhances glutamate uptake via glutamate transporters in rat glial cells.</a> Neuroreport. 12, 1077-1080 (2001).</li><li>Barbour, B., Brew, H., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogenic+uptake+of+glutamate+and+aspartate+into+glial+cells+isolated+from+the+salamander+(Ambystoma)+retina.">Electrogenic uptake of glutamate and aspartate into glial cells isolated from the salamander (Ambystoma) retina.</a> J. Physiol. 436, 169-193 (1991).</li><li>Bergles, D. E., Tzingounis, A. V., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+coupled+and+uncoupled+currents+during+glutamate+uptake+by+GLT-1+transporters.">Comparison of coupled and uncoupled currents during glutamate uptake by GLT-1 transporters.</a> J. Neurosci. 22, 10153-10162 (2002).</li><li>Diamond, J. S., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+glutamate+does+not+overwhelm+transporters+on+hippocampal+astrocytes+during+high-frequency+stimulation.">Synaptically released glutamate does not overwhelm transporters on hippocampal astrocytes during high-frequency stimulation.</a> J. Neurophysiol. 83, 2835-2843 (2000).</li><li>Benediktsson, A. M., 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=Neuronal+activity+regulates+glutamate+transporter+dynamics+in+developing+astrocytes.">Neuronal activity regulates glutamate transporter dynamics in developing astrocytes.</a> Glia. 60, 175-188 (2012).</li><li>Hires, S. A., Zhu, Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurement+of+synaptic+glutamate+spillover+and+reuptake+by+linker+optimized+glutamate-sensitive+fluorescent+reporters.">Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4411-4416 (2008).</li><li>Scimemi, A., Meabon, J., Woltjer, R. L., Sullivan, J. M., Diamond, J. S., Cook, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid&#946;1-42+slows+clearance+of+synaptically-released+glutamate+by+mislocalizing+astrocytic+GLT-1.">Amyloid&#946;1-42 slows clearance of synaptically-released glutamate by mislocalizing astrocytic GLT-1.</a> J. Neurosci. 33, 5312-5318 (2013).</li></ol>

Here we describe an experimental approach to obtain electrophysiological recordings from astrocytes, an analytical protocol to isolate glutamate transporter currents in astrocytes and a mathematical method to derive the time course of glutamate clearance from astrocytic transporter currents.
The success of the analysis relies on the ability to obtain high-quality patch clamp recordings from astrocytes and on the accuracy of the fitting algorithms used to describe the transporter currents. The ...

Astrocytter er en av de mest tallrike celletyper i hjernen med stjerne-formet morfologi og fin hinne fremspring som strekker seg gjennom hele neuropil og nå tilgrensende synaptiske kontakter 1,2. De astrocytes &#39;cellemembranen er tettpakket med glutamat transporter molekyler tre. Under fysiologiske betingelser, glutamattransportører hurtig binder den ekstracellulære glutamat ved siden av membranen og overføre den til cellens cytoplasma. Ved å gjøre dette, transportører opprettholde lav basal konsentrasjon av glutamat i det ekstracellulære plass fire. Glutamattransportørene i fin astrocytic prosesser ved siden av eksitatoriske synapser er ideelt posisjonert for å binde glutamat frigjøres under synaptiske hendelser som det sprer bort fra den synaptiske spalten. Ved å gjøre dette, transportører også begrense glutamat ringvirkninger mot peri-og ekstra-synaptiske regioner og på nabokommunene synapser, noe som reduserer den romlige spredningen av eksitatoriske signals i hjernen 5-7. Glutamat transport er en electrogenic prosess stoichiometrically koblet til bevegelsen av tre Na + og en H + langs deres elektrokjemiske gradient og til counter-transport av en K + 8. Glutamat transporten er forbundet med (men ikke støkiometrisk er koplet til) en anionisk konduktans gjennomtrengelig for SCN - (tiocyanat)&gt; NO 3 - (nitrat) ≈ ClO 4 - (perklorat)&gt; I -&gt; Br -&gt; Cl -&gt; F -, ikke til SO CH 3 3 - (metan-sulfonat) og C 6 H 11 O 7 - (glukonat) 9-11. Begge strømmene (støkiometriske og ikke-støkiometriske) kan tas opp ved å skaffe hel-celle patch-clamp opptak fra astrocytes, visuelt identifisert under Dodt belysning eller infrarødt differensial interferens kontrast (IR-DIC) i acute hjernen skiver 12. Den støkiometriske komponenten i den aktuelle forbundet med glutamat transport over membranen kan isoleres ved hjelp av SO CH 3 3 -, eller C 6 H 11 O 7 - intracellulære baserte løsninger, og kan bli fremkalt ved flash-uncaging glutamat på astrocytter 13,14, eller ved å aktivere glutamat frigjøring fra nærliggende synapser, enten elektrisk 12 eller med en målrettet optogenetic kontroll. Tidsforløpet for den støkiometriske komponenten av transportøren strøm er formet av levetiden til glutamat konsentrasjon profil ved astrocytic membraner (dvs. glutamat klaring), kinetikken av glutamattransportører, de passive membran egenskaper av astrocytter, og i løpet av synaptiske stimuleringer, av synkron glutamatfrigiving over de aktiverte synapser 13. Her beskriver vi i detalj: (1) en eksperimentell caoach å isolere den støkiometriske komponent av glutamat-transporter strømmene fra hel-celle patch-clamp opptak fra astrocytter ved hjelp av akutte mus hippokampale skiver som et eksempel eksperimentell fremstilling, (2) en analytisk tilnærming for å utlede tidsforløpet av glutamat klaring fra disse platene 13, 14. Disse metodene kan brukes til å registrere og analysere glutamatreseptor strømmer transporter fra astrocytter på en region av sentralnervesystemet.

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			<td>Company</td>
			<td>Catalogue Number</td>
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			<td>CGP52432</td>
			<td>Tocris</td>
			<td>1246</td>
			<td></td>
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			<td>(R,S)-CPP</td>
			<td>Tocris</td>
			<td>173</td>
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			<td>DPCPX</td>
			<td>Tocris</td>
			<td>439</td>
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			<td>LY341495 disodium salt</td>
			<td>Tocris</td>
			<td>4062</td>
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			<td>Picrotoxin</td>
			<td>Sigma</td>
			<td>P1675</td>
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			<td>Tocris</td>
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			<td>Life Technologies</td>
			<td>A10438</td>
			<td>Optional</td>
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			<td>Matrix electrodes</td>
			<td>Frederick Haer Company</td>
			<td>MX21AES(JD3)</td>
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			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG10165-4</td>
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			<td>Dual-stage glass micro-pipette puller</td>
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			<td>Loctite 404 instant adhesive</td>
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			<td>Igor Pro 6</td>
			<td>Wavemetrics</td>
			<td></td>
			<td></td>
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deriving the time course of glutamate clearance with a deconvolution analysis of astrocytic transporter currents

Den høyeste tetthet av glutamattransportører i hjernen er funnet i astrocytter. Glutamattransportører par bevegelsen av glutamat over membranen ved samtidig transport av 3 Na + og en H +, og telleren-transport av en K +. Den støkiometriske strøm som genereres av transport-prosessen kan overvåkes med hel-celle patch-clamp opptak fra astrocytter. Tidsforløpet for den registrerte strøm er formet av Tidsforløpet for glutamat konsentrasjon profilen til hvilke astrocytter er utsatt for, kinetikken av glutamattransportører, og de passive electrotonic egenskaper astrocytic membraner. Her beskriver vi de eksperimentelle og analytiske metoder som kan brukes til å ta opp glutamat-transporter strømninger i astrocytter og isolere tidsforløpet av glutamat klaring fra alle andre faktorer som former bølgeformen astrocytic transporter strømmer. De fremgangsmåter som er beskrevet her kan brukes til å beregne levetiden of flash-uncaged og postsynaptisk utgitt glutamat på astrocytic membraner i en region i det sentrale nervesystemet under helse og sykdom.

The success of the analytical approach described here critically depends on obtaining high-quality electrophysiological recordings of transporter currents from astrocytes in any region of the central nervous system. In acute mouse hippocampal slices, astrocytes can be readily identified under Dodt illumination or IR-DIC because of their small cell body (&Oslash; = 10 &mu;m) and prominent nucleus (Figure 1). Their distinctive star-shaped morphology can be appreciated with epifluorescence, confocal, or two...

Watch this Scientific Journal Video about Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents at JoVE.com

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Utlede Tid Course of Glutamat Lagersalg med en deconvolution Analyse av astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

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