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

Dette arbejde blev støttet af UCPH 2016 Program of Excellence (U.G., A.R., K.J.), Lundbeck Foundation (M.R.) Lundbeckfonden Center for Biomembraner i Nanomedicin (U.G.), National Institute for sundhed tilskud P01 DA 12408 (U.G.), danske Rådet for uafhængige forskning - medicinske videnskaber (U.G.).

retningslinjer for det danske dyr eksperimenter inspektorat (tilladelse nummer: 2017-15-0201-01160) blev fulgt og eksperimenter udført i et fuldt AAALAC akkrediteret anlæg under tilsyn af en lokal dyrevelfærd Udvalget.
 1. synaptosomal dopamin optagelse (metode 1) Bemærk: denne protokol er til parallel vurdering af to hjerner, men med held kan anvendes til at udføre synaptosomal DA optagelsen eksperimenter med fire hjerner i parallel.
<ol><li>Præpa...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+increases+in+nucleus+accumbens+dopamine+after+systemic+cocaine+administration+are+caused+by+unique+characteristics+of+dopamine+neurotransmission.">Preferential increases in nucleus accumbens dopamine after systemic cocaine administration are caused by unique characteristics of dopamine neurotransmission.</a> J Neurosci. 21, 6338-6347 (2001).</li><li>Schonfuss, D., Reum, T., Olshausen, P., Fischer, T., Morgenstern, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+constant+potential+amperometry+for+investigations+of+dopaminergic+neurotransmission+kinetics+in+vivo.">Modelling constant potential amperometry for investigations of dopaminergic neurotransmission kinetics in vivo.</a> J Neurosci Methods. 112, 163-172 (2001).</li><li>Hoover, B. R., Everett, C. V., Sorkin, A., Zahniser, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+regulation+of+dopamine+transporters+by+tyrosine+kinases+in+rat+neuronal+preparations.">Rapid regulation of dopamine transporters by tyrosine kinases in rat neuronal preparations.</a> J Neurochem. 101, 1258-1271 (2007).</li><li>Hansen, F. H., 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=Missense+dopamine+transporter+mutations+associate+with+adult+parkinsonism+and+ADHD.">Missense dopamine transporter mutations associate with adult parkinsonism and ADHD.</a> J Clin Invest. 124, 3107-3120 (2014).</li><li>Damier, P., Hirsch, E. C., Agid, Y., Graybiel, A. 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J., Park, J., Nunes, E. J., Addy, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Rapid+Dopamine+Dynamics+with+Fast+Scan+Cyclic+Voltammetry+During+Intra-oral+Tastant+Administration+in+Awake+Rats.">Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats.</a> J Vis Exp. , e52468 (2015).</li><li>Phillips, P. E., Robinson, D. L., Stuber, G. D., Carelli, R. M., Wightman, R. 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Dette manuskript beskriver nyttige eksperimentelle protokoller for at afgrænse DA homøostase i en musemodel af valg. Vi leverer detaljerede protokoller for måling niveauer af DA i hjernevæv fra mus ved hjælp af HPLC og synaptosomal DA optagelsen for at vurdere funktionelle DA transport gennem DAT. Procedurer, protokoller og grænser for HPLC eksperiment og synaptosomal DA optagelse assay vil blive uddybet nedenfor.
Synaptosomal optagelse protokol kan give nyttig indsigt til funktionalitet...

Dopamin (DA) er en modulerende neurotransmitter kritiske motor adfærd, belønning og kognitiv funktion1,7,8,9. Ubalancer i DA homeostase er impliceret i flere neuropsykiatriske sygdomme som Parkinsons sygdom1, depression, opmærksomhed underskud hyperaktivitet lidelse og narkotikamisbrug. DA er frigivet fra den præsynaptiske neuron i den synaptiske kløft, hvor det binder sig til og aktiverer receptorer på den præ- og postsynaptiske membran, dermed yderligere formidle signalet. DA niveauet i synapse efter udgivelsen er rumligt og tidsligt kontrolleret af DAT3,10. Transportvirksomheden sequesters DA fra det ekstracellulære rum, og dermed opretholder fysiologiske DA niveauer3,11. Genetiske fjernelse af DAT i mus forårsager en hyperdopaminergic fænotype karakteriseret ved forhøjede synaptic DA niveauer, udtømning af intracellulære DA puljer og dybtgående ændringer i postsynaptiske DAergic signalering10,12.
Her, to adskilte protokoller er præsenteret, en metode til foranstaltning DA væv indhold og en anden for at vurdere funktionaliteten af DAT. kombineret med den overflade biotinylation assay beskrevet af Gabriel et al.13 disse to metoder give oplysninger om DA indhold og funktionelle niveauer af DAT for en grundig vurdering af DA homøostase. Med disse metoder kan DA homeostase af forskellige Transgene mus eller sygdomsmodeller karakteriseret og beskrevet. Disse værktøjer er blevet implementeret og optimeret og er standard brug i vores laboratorier. Nuværende assays har tjent til at undersøge konsekvenser på DA homeostase af at ændre C-terminalen DAT14 eller udtrykke Cre recombinase under tyrosin 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

Dopamin (DA) er en modulerende neurotransmitter kontrollere motorisk aktivitet, belønning processer og kognitiv funktion. Værdiforringelse af dopaminerge (DAergic) neurotransmission er stærkt forbundet med flere centralnervesystemet-associerede sygdomme som Parkinsons sygdom, attention deficit hyperactivity disorder og narkotika afhængighed1,2 ,3,4. Skildrer sygdomsmekanismer involverer DA ubalance er kritisk afhængige af dyremodeller for at efterligne aspekter af sygdomme, og dermed protokoller, der vurderer bestemte dele af DA homeostase er vigtigt at give nye indsigter og mulige terapeutiske mål for disse sygdomme.
Her præsenterer vi to nyttige eksperimentelle protokoller, når de kombineres giver en funktionel læse-out af DAergic systemet i mus. Biokemiske og funktionelle parametre på DA homeostase er opnået gennem vurdering af DA niveauer og dopamin transporter (DAT) funktionalitet5. Når undersøger DA systemet, er evnen til pålideligt måle endogene niveauer af DA fra voksne hjerne afgørende. Derfor præsenterer vi sådan udføre high-performance væskekromatografi (HPLC) på hjernevæv fra mus til at bestemme niveauet af DA. Vi udføre forsøget på væv fra dorsale striatum (dStr) og nucleus accumbens (NAc), men metoden er også egnet til andre områder, DA-innerveres hjernen.
DAT er afgørende for reuptake af DA i den præsynaptiske terminal, derved kontrollere den tidsmæssige og rumlige aktivitet af frigivne DA. Kendskab til niveauer og funktionalitet af DAT i striatum er af stor betydning ved vurderingen af DA homøostase. Her, leverer vi en protokol, der giver mulighed for samtidig udlede oplysninger om overflade niveauer og funktion ved hjælp af en synaptosomal6 DA optagelse assay.
Nuværende metoder kombineret med standard immunoblotting protokoller giver forsker med relevante værktøjer for at karakterisere DAergic system.

Nuværende DA optagelsen protokol (figur 1) omfatter alle trin er nødvendige for at vurdere funktionaliteten af DAT i synaptosomes fra mus. Vores repræsentative data af DA optagelsen metode (figur 2) skildrer en mætning kurve med ujusterede data (figur 2B) og justeret data (figur 2A). Mætning kurve viser optagelsen fra vildtype mus. Normalt ville man gøre DA optagel...

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

Vurdering af dopaminerge homøostase i mus ved hjælp af High-performance væskekromatografi analyse og Synaptosomal dopamin optagelse

Synaptosomal dopamin optagelse og højtydende væskekromatografi analyse repræsenterer eksperimentelle værktøjer til at undersøge dopamin homøostase i mus ved at vurdere funktionen af dopamin-transporter og niveauer af dopamin i striatal væv, henholdsvis. Her præsenterer vi protokoller for at måle dopamin væv indhold og vurdere funktionaliteten af dopamin-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

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-labyrint Tvungen Alternation og Venstre-højre forskelsbehandling Opgaver for vurdering af arbejds-og referencedokument Hukommelse i mus

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

Reproducerbar Mouse iskiasnerven Crush og efterfølgende vurdering af Regeneration af Whole Mount Muskel 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

Vurdere Neurodegenerative fænotyper i<em&gt; Drosophila</em&gt; Dopaminerge neuroner ved at klatre assays og Whole Brain-immunfarvning

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

Et højt indhold Imaging Assay til identifikation af botulinumneurotoksin inhibitorer

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

Miljømæssige Modulationer i antallet af Midbrain dopaminneuroner i voksne mus

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

Vurdering af dendritiske arborization i gyrus dentatus i hippocampus-regionen i mus

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

Identifikation af dopamin D1-Alpha Receptor inden for gnavere kernen Accumbens af en innovativ RNA In Situ opdagelse teknologi

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

Enkelt synapse indikatorer for glutamat frigivelse og optagelse i akut hjerne skiver fra normal og Huntington Mus

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-kvantitativ bestemmelse af dopaminergisk neurontæthed i Substantia Nigra af gnavermodeller ved hjælp af automatiseret billedanalyse

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