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

Bu eser UCPH 2016 Program of Excellence (U.G., A.R., KJ) tarafından desteklenen, Lundbeck Vakfı (MR) Lundbeck Vakfı Merkezi Biomembranes içinde Nanomedicine (U.G.), Ulusal Enstitüsü, sağlık hibe P01 DA 12408 (U.G.), Danimarka için Bağımsız araştırma - tıbbi Bilimler (U.G.) Konseyi.

Danimarkalı hayvan deney Müfettişliği yönergeleri (izni numarası: 2017-15-0201-01160) takip etti ve bir tam akredite AAALAC tesis yerel hayvan refahı gözetiminde yapılan deneyler Komitesi.
 1. synaptosomal dopamin alımı (yöntem 1) Not: Bu protokol iki beyin paralel değerlendirilmesi için ama başarılı bir şekilde synaptosomal DA alımı deneyler ile dört beyinlerinde gerçekleştirmek için kullanılabilir paralel.
<ol><li>Başına Tabl...

<ol>
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S., Dempsey, C., Fields, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+profiling+of+dopamine+regulation+in+substantia+nigra+and+ventral+tegmental+area.">Comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area.</a> J Vis Exp. , (2012).</li><li>Van Dam, D., 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=Regional+distribution+of+biogenic+amines,+amino+acids+and+cholinergic+markers+in+the+CNS+of+the+C57BL/6+strain.">Regional distribution of biogenic amines, amino acids and cholinergic markers in the CNS of the C57BL/6 strain.</a> Amino Acids. 28, 377-387 (2005).</li><li>Barth, C., Villringer, A., Sacher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+hormones+affect+neurotransmitters+and+shape+the+adult+female+brain+during+hormonal+transition+periods.">Sex hormones affect neurotransmitters and shape the adult female brain during hormonal transition periods.</a> Front Neurosci. 9 (37), (2015).</li><li>Corthell, J. 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Bu el yazması DA seçim herhangi bir fare modeli homeostazı betimlemek için yararlı deneysel iletişim kurallarını açıklar. Biz ayrıntılı iletişim kuralları DA ölçüm düzeyde beyin dokusu içinde HPLC ve synaptosomal DA alımı yoluyla DAT. fonksiyonel DA ulaşım değerlendirmek için kullanarak fare üzerinden sağlar Yordamlar, iletişim kuralları ve HPLC deney ve synaptosomal DA alımı tahlil aşağıda gelişmiş.
Synaptosomal alımını Protokolü DAT. kombine işlevsel...

Dopamin (DA) düzenleyici nörotransmitter motor davranış, ödül ve bilişsel işlev1,7,8,9için kritik olduğunu. Dengesizlikler DA Homeostazı, dikkat eksikliği hiperaktivite bozukluğu, uyuşturucu bağımlılığı, depresyon ve Parkinson hastalığı1gibi birkaç nöropsikiyatrik hastalıkların karıştığı. DA presynaptic nöron böylece daha fazla sinyal taşıma nerede bağlar ve tarih öncesi ve postsinaptik membran reseptörleri aktive, sinaptik yarık serbest bırakılır. Synapse yayımlanmasından sonra DA kademede dağınık şekilde ve geçici DAT3,10tarafından kontrol edilir. Taşıyıcı DA ekstrasellüler uzaydan tüketiyor ve böylece fizyolojik DA düzeyleri3,11ayakta tutan. DAT genetik kaldırılması farelerde yükseltilmiş sinaptik DA düzeyleri tarafından karakterize bir hyperdopaminergic fenotip hücre içi DA havuzları ve postsinaptik DAergic10,12sinyal derin değişiklikler tükenmesi neden olur.
Burada, iki ayrı protokol sundu, ölçü birimi DA doku içerik ve Gabriel vd.13 tarafından açıklanan yüzey biotinylation tahlil ile buluşman kombine işlevselliğini değerlendirmek için başka bir yöntem bu iki yöntem DA bilgileri DAT içerik ve işlevsel düzeyleri DA homeostazı ayrıntılı bir değerlendirme için. Bu yöntemler ile DA homeostazı çeşitli transgenik fareler veya hastalık modelleri ile karakterize ve açıklanan. Bu araçlar uygulanan ve en iyi duruma getirilmiş ve laboratuarlarımızda standart kullanılmaktadır. Geçerli deneyleri sonuçları C-terminal DAT14 değiştirme veya Cre recombinase tirozin hidroksilaz (TH) organizatörü 5altında ifade DA homeostazı üzerinde araştırmak için hizmet etmişlerdir.

<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) bir düzenleyici nörotransmitter kontrol motor aktivite, ödül süreçleri ve bilişsel işlev var. Dopaminerjik (DAergic) neurotransmission bozulma şiddetle Parkinson hastalığı, dikkat eksikliği hiperaktivite bozukluğu ve uyuşturucu bağımlılığı1,2 gibi merkezi sinir sistemi ile ilişkili çeşitli hastalıklarla ilişkilidir ,3,4. Hastalık mekanizmaları DA dengesizlik içeren operasyona eleştirel yönleri hastalıkları taklit etmek için hayvan modellerinde bağımlı, ve böylece DA homeostazı belirli bölümlerini değerlendirmek protokolleri roman yorumlara ve tedavi mümkün sağlamak önemlidir Bu hastalıklar için hedefler.
Burada, o zaman birlikte iki yararlı deneysel protokol mevcut farelerde DAergic sisteminin fonksiyonel bir okuma sağlar. Biyokimyasal ve fonksiyonel parametrelere DA homeostazı DA düzeyleri ve dopamin taşıyıcı (DAT) işlevselliği5değerlendirilmesi ile elde edilir. Ne zaman DA sistemi araştıran, güvenilir bir şekilde endojen DA--dan yetişkin beyin düzeylerini ölçmek için temel yeteneğidir. Bu nedenle, biz mevcut yüksek performanslı sıvı Kromatografi (HPLC) gerçekleştirmek nasıl beyin dokusunda da düzeylerini belirlemek için fare Dorsal striatum (dStr) ve nucleus accumbens (NAc) doku üzerinde deney gerçekleştirmek, ama yöntemi Ayrıca diğer DA innervated beyin bölgesi için uygun mu.
DAT DA geri presynaptic terminal içine Alım için gerekli böylece serbest bırakmak da zamansal ve mekansal etkinliği denetleme Düzeyleri ve DAT işlevselliğini striatum içinde bilerek büyük DA homeostazı değerlendirirken önemlidir. Burada, aynı anda yüzey düzeyleri ve işlev bir synaptosomal6 DA alımı tahlil kullanarak bilgi anlamak için izin veren bir protokol sağlar.
Geçerli yöntemleri standart immunoblotting iletişim kuralları ile kombine DAergic sistemi karakterize etmek için araştırmacı ile ilgili araçlar sağlar.

Geçerli DA Alım Protokolü (şekil 1) DAT işlevindeki fare üzerinden synaptosomes değerlendirmek için gereken tüm adımları içerir. Temsilcisi verilerimiz DA Alım yöntemi (Şekil 2) bir doygunluk eğrisi ayarsız veri (şekil 2B) ile gösteriyor ve veri (şekil 2A) ayarlanabilir. Doygunluk eğrisi alımını vahşi türü Fareler gösteriyor. Genellikle bir DA...

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

Dopaminerjik Homeostasis farelerde yüksek performanslı sıvı Kromatografi analizi ve Synaptosomal dopamin alımı kullanımı tarafından değerlendirilmesi

Synaptosomal dopamin alımı ve yüksek performanslı sıvı Kromatografi analiz temsil deneysel araçları tarafından değerlendirilmesi dopamin taşıyıcı işlevi ve striatal dokusunda dopamin düzeyleri dopamin homeostazı farelerde araştırmak için anılan sıraya göre. Burada protokolleri dopamin doku içeriği ölçmek ve dopamin taşıyıcı işlevselliğini değerlendirmek için mevcut.

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

Denge kullanarak Fare Motor Denge ve Koordinasyon Değerlendirme

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-labirent Zorla Değişim ve Çalışma ve Fareler Referans Bellek değerlendirilmesi için sağ-sol Ayrımcılık Görevler

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

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

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

Içinde Nörodejeneratif fenotipleri değerlendirilmesi<em&gt; Drosophila</em&gt; Tırmanma Tahliller tarafından Dopaminerjik nöronlar ve Tüm Beyin immün

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

Botulinum nörotoksininin önleyicilerinin tanımlanmasına daha yüksek bir miktarda Görüntüleme Deneyi

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

Yetişkin Farelerde Orta beyin dopamin Nöronlar Sayısının Çevre Modülleri

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

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Farelerde Hipokampal Bölgesi Dentate Gyrus dendritik arborization Değerlendirilmesi

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

Dopamin D1-Alfa reseptör kemirgen Nucleus Accumbens bir yenilikçi RNA Situ içinde hafiye teknoloji tarafından içinde tanımlaması

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

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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

Otomatik Görüntü Analizi Kullanılarak Kemirgen Modellerinin Substantia Nigra'sında Dopaminerjik Nöron Yoğunluğunun Yarı Nicel Belirlenmesi

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