Name: assessment of dopaminergic homeostasis in mice by use of high-performance liquid chromatography analysis and synaptosomal dopamine uptake
Uploaded: 2017-09-21T12:30:00
Description: Watch this Scientific Journal Video about Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake at JoVE.com

Dit werk werd ondersteund door de UCPH 2016 programma van Excellence (U.G., A.R., K.J.), de Stichting van Lundbeck (M.R.) de Lundbeck Stichting Centrum voor biomembranen in nanogeneeskunde (U.G.), het Nationaal Instituut van gezondheid subsidies P01 DA 12408 (U.G.), de Deense Raad voor onafhankelijk onderzoek - medische wetenschappen (U.G.).

de richtsnoeren van het inspectoraat van de Deense dier experimenten (toestemming nummer: 2017-15-0201-01160) werd gevolgd en experimenten uitgevoerd in een volledig AAALAC geaccrediteerde faciliteit onder toezicht van een lokale dierenwelzijn Comité.
 1. synaptosomal Dopamine opname (methode 1) Opmerking: dit protocol is voor parallelle beoordeling van twee hersenen, maar kan met succes worden gebruikt voor het uitvoeren van synaptosomal DA opname experimenten met...

<ol>
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Dit manuscript beschrijft nuttige experimentele protocollen te bakenen DA homeostase in een muismodel van keuze. We bieden gedetailleerde protocollen voor meten niveaus van DA in hersenweefsel van muizen met behulp van HPLC en synaptosomal DA opname te beoordelen van functionele DA vervoer door middel van DAT. De procedures, de protocollen en de grenzen voor de HPLC-experiment en synaptosomal DA opname bepaling zal hieronder worden uitgewerkt.
Het protocol synaptosomal opname kan nuttig inzich...

Dopamine (DA) is een cruciaal voor motor gedrag, de beloning en de cognitieve functie1,,7,,8,9f neurotransmitter. Onevenwichtigheden in de homeostase van de DA zijn betrokken bij verschillende neuropsychiatrische ziekten zoals de wanorde van de Hyperactiviteit van de aandacht-tekort, drugsverslaving, depressie en de ziekte van Parkinson1. DA is vrijgelaten uit het presynaptische neuron in de synaptische spleet, waar het aan bindt en activeert de receptoren op de pre- en postsynaptisch membraan, waardoor verdere overbrengen van het signaal. Het niveau van DA in de synaps na release is ruimtelijk en stoffelijk gecontroleerd door DAT3,10. De vervoerder sequesters DA van de extracellulaire ruimte, en dus voedt fysiologische DA niveaus-3,11. Genetische verwijdering van DAT bij muizen veroorzaakt een hyperdopaminergic fenotype gekenmerkt door verhoogde synaptic DA niveaus, uitputting van intracellulaire DA zwembaden en ingrijpende veranderingen in postsynaptisch DAergic signalering van10,12.
Hier twee afzonderlijke protocollen worden gepresenteerd, een methode maatregel DA weefsel inhoud en een andere om te beoordelen van de functionaliteit van DAT. gecombineerd met de bepaling van de oppervlakte biotinylation beschreven door Gabriel et al.13 deze twee methoden bevatten informatie over DA inhoud en functionele niveaus van DAT voor een grondige beoordeling van DA homeostase. Met deze methoden kunt DA homeostase van verschillende transgene muizen of modellen van de ziekte worden gekenmerkt en beschreven. Deze hulpprogramma's zijn uitgevoerd en geoptimaliseerd en zijn standaard gebruik in onze laboratoria. Huidige testen hebben gediend om te onderzoeken van de gevolgen voor de DA-homeostase van het veranderen van de C-terminal van DAT14 of uiting van Cre recombinase onder de tyrosine hydroxylase (TH) promotor 5.

<table>
	<tbody>
		<tr>
			<td>COMT inhibitor</td>
			<td>Sigma Aldrich, Germany</td>
			<td>RO-41-0960</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>[3H]-Dopamine</td>
			<td>Perkin-Elmer Life Sciences, Boston, MA, USA</td>
			<td>NET67-3001MC</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Glass microfiber filters</td>
			<td>GF/C Whatman, GE Healthcare Life Sciences, Buckinghamshire</td>
			<td>1822-024</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>HiSafe Scintillation fluid</td>
			<td>Perkin Elmer</td>
			<td>1200-437</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>MicroBeta2</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>BCA Protein Assay kit</td>
			<td>Thermo Scientific Pierce</td>
			<td>23225</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Life Science</td>
			<td>H3375</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Life Science</td>
			<td>S7903</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Life Science</td>
			<td>S3014</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Life Science</td>
			<td>P9541</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck KGaA</td>
			<td>10043-52-4</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Life Science</td>
			<td>63065</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Life Science</td>
			<td>A0278</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Life Science</td>
			<td>G7021</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Pargyline</td>
			<td>Sigma Aldrich</td>
			<td>P-8013</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Desipramine</td>
			<td>Sigma Aldrich</td>
			<td>D3900</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Dopamine</td>
			<td>Sigma Life Science</td>
			<td>H8502</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Cocaine</td>
			<td>Sigma Life Science</td>
			<td>C5776</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Brain matrix</td>
			<td>ASI instruments</td>
			<td>RBM2000C</td>
			<td>For synaptosomal DA uptake protocol</td>
		</tr>
		<tr>
			<td>Cafano mechanical teflon disrupter</td>
			<td>Buch &#38; Holm</td>
			<td>Discontinued</td>
			<td>For synaptosomal DA uptake protocol (homogenization)</td>
		</tr>
		<tr>
			<td>Antec Decade (Amperometric detector)</td>
			<td>Antec, Leiden, The Netherlands</td>
			<td>Discontinued: new model DECADE Elite / Lite&#8482; Electrochemical Detector type 175 and 176</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Avantec 0.22 &#956;m glass filter</td>
			<td>Frisenette ApS, Denmark</td>
			<td>13CP020AS</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Column: Prodigy 3 &#956; ODS-3 C18</td>
			<td>Phenomenex, YMC Europe, Chermbeck, Germany</td>
			<td>Part Number:00A-3300-E0</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>LC solution software</td>
			<td>Shimadzu</td>
			<td>LabSolutions Series Workstation</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Perchlor acid 0.1M</td>
			<td>Fluka Analytical</td>
			<td>35418-500ml</td>
			<td>For HPLC protocol (Tissue preparation)</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134-50g</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Natriumdihydrogenphosphar</td>
			<td>Bie&#38;Berntsen</td>
			<td>1.06346 1000g</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Sodium 1-octanesulfonate monohydrate</td>
			<td>Aldrich</td>
			<td>74885 -10g</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Acetonitrile, isocratic HPLC grade</td>
			<td>Scharlau</td>
			<td>AC03402500</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Filtre 0.22um</td>
			<td>Frisenette ApS, Denmark</td>
			<td>Avantec 13CP020AS</td>
			<td>For HPLC protocol (Tissue preparation)</td>
		</tr>
		<tr>
			<td>ortho-Phosphoric acid 85%</td>
			<td>Merck</td>
			<td>1.00563. 1000ml</td>
			<td>For HPLC protocol</td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td>Antec, Leiden, The Netherlands</td>
			<td>AN1161300</td>
			<td>For HPLC protocol (see manual online)</td>
		</tr>
		<tr>
			<td>Detector program on DECADE II electrochemical detector</td>
			<td>Antec, Leiden, The Netherlands</td>
			<td>Lite&#8482; Electrochemical Detector type 175 and 176</td>
			<td>For HPLC protocol</td>
		</tr>
	</tbody>
</table>

assessment of dopaminergic homeostasis in mice by use of high-performance liquid chromatography analysis and synaptosomal dopamine uptake

Dopamine (DA) is een f neurotransmitter beheersing van motorische activiteit, beloning processen en cognitieve functie. Bijzondere waardevermindering van de Dopaminerge (DAergic) transmissie is sterk geassocieerd met diverse centrale zenuwstelsel-geassocieerde ziekten zoals de ziekte van Parkinson, aandacht-deficit-hyperactivity disorder en drug verslaving1,2 ,3,4. Uitgezet ziekte mechanismen waarbij DA onbalans is kritisch afhankelijk van diermodellen om na te bootsen van aspecten van de ziekten, en dus protocollen die specifieke delen van de DA-homeostase te beoordelen zijn belangrijk om te bieden van nieuwe inzichten en mogelijke therapeutische doelstellingen voor deze ziekten.
Hier presenteren we twee nuttige experimentele protocollen die combinatie bieden een functionele uitlezing van het systeem van de DAergic in muizen. Biochemische en functionele parameters op DA homeostase zijn verkregen door middel van de beoordeling van de niveaus van de DA en dopamine transporter (DAT) functionaliteit5. Bij het onderzoeken van de DA-systeem, is de mogelijkheid voor het betrouwbaar meten endogene voor DA van volwassen hersenen essentieel. Daarom presenteren we het uitvoeren van krachtige vloeibare chromatografie (HPLC) op hersenweefsel van muizen om niveaus van DA. Wij voeren het experiment op weefsel van de dorsale striatum (dStr) en nucleus accumbens (NAc), maar de methode is ook geschikt voor andere DA-geïnnerveerd hersengebieden.
DAT is essentieel voor reuptake voor DA in de presynaptische terminal, waardoor beheersing van de temporele en ruimtelijke activiteit van vrijgegeven DA. Het kennen van de niveaus en de functionaliteit van DAT in het striatum is van groot belang bij de beoordeling van DA homeostase. Hier, bieden wij een protocol waarmee gelijktijdig om informatie te deduceren op oppervlakte niveaus en functie met behulp van een synaptosomal6 DA opname assay.
Huidige methoden gecombineerd met standaard immunoblotting protocollen bieden de onderzoeker met relevante tools karakteriseren van het DAergic-systeem.

Huidige DA opname protocol (Figuur 1) omvat alle noodzakelijke stappen voor het beoordelen van de functionaliteit van DAT in synaptosomes van muizen. Onze representatieve gegevens van de DA opname methode (Figuur 2) toont een verzadiging kromme met niet-gecorrigeerde gegevens (figuur 2B) en gezuiverde gegevens (figuur 2A). De verzadiging curve toont opname van wild type ...

Watch this Scientific Journal Video about Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake at JoVE.com

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Beoordeling van Dopaminerge homeostase in muizen door gebruik van High-performance Liquid Chromatography analyse en Synaptosomal Dopamine opname

Synaptosomal dopamine opname en krachtige vloeibare chromatografie vertegenwoordigen experimentele analysetools te onderzoeken van dopamine homeostase in muizen door beoordeling van de functie van de dopamine transporter en niveaus van dopamine in striatale weefsel, respectievelijk. Hier presenteren we protocollen voor dopamine weefsel inhoud meten en beoordelen van de functionaliteit van de dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Research

JoVE Journal

Neuroscience

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Beoordeling van de Motor Balans en coördinatie in Muizen met behulp van de Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

T-doolhof Gedwongen Afwisseling en links-rechts Discriminatie Taken voor de beoordeling van Werken en Reference geheugen in Muizen

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Reproduceerbare Mouse heupzenuw Crush en de daaropvolgende beoordeling van Regeneratie door Whole Mount Muscle Analyse

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Het beoordelen Neurodegenerative Fenotypes in<em&gt; Drosophila</em&gt; Dopaminerge neuronen reduceert door Klimmen Testen en Whole Brain Immunokleuring

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Een hoog gehalte Imaging Assay voor Identificatie van botulineneurotoxine Remmers

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Milieu Modulaties van het aantal dopaminesysteem Neuronen in volwassen muizen

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Beoordeling van Dendritische arborization in de gyrus dentatus van de hippocampus regio in Muizen

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identificatie van Dopamine D1-Alpha Receptor binnen knaagdier Nucleus Accumbens door een innovatieve RNA In Situ de technologie van de opsporing

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Enkele Synapse indicatoren van glutamaat release en opname in acute hersenen plakjes van normale en Huntington muizen

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Semi-kwantitatieve bepaling van dopaminerge neurondichtheid in de substantia Nigra van knaagdiermodellen met behulp van geautomatiseerde beeldanalyse

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...