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

This work was supported by the UCPH 2016 Program of Excellence (U.G., A.R., K.J.), the Lundbeck Foundation (M.R.) the Lundbeck Foundation Center for Biomembranes in Nanomedicine (U.G.), the National Institute of Health Grants P01 DA 12408 (U.G.), the Danish Council for independent Research - Medical Sciences (U.G.).

The guidelines of the Danish Animal Experimentation Inspectorate (permission number: 2017-15-0201-01160) was followed and experiments performed in a fully AAALAC accredited facility under the supervision of a local animal welfare committee.
1. Synaptosomal Dopamine Uptake (Method 1)
NOTE: This protocol is for parallel assessment of two brains, but can be successfully used to perform synaptosomal DA uptake experiments with four brains in parallel.
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	<li>Tritsch, N. X., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+modulation+of+synaptic+transmission+in+cortex+and+striatum.">Dopaminergic modulation of synaptic transmission in cortex and striatum.</a> Neuron. 76, 33-50 (2012).</li><li>Cartier, E. A., 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=A+biochemical+and+functional+protein+complex+involving+dopamine+synthesis+and+transport+into+synaptic+vesicles.">A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.</a> J Biol Chem. 285, 1957-1966 (2010).</li><li>Kristensen, A. 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=SLC6+neurotransmitter+transporters:+structure,+function,+and+regulation.">SLC6 neurotransmitter transporters: structure, function, and regulation.</a> Pharmacol Rev. 63, 585-640 (2011).</li><li>Gainetdinov, R. R., Caron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoamine+transporters:+from+genes+to+behavior.">Monoamine transporters: from genes to behavior.</a> Annu Rev Pharmacol Toxicol. 43, 261-284 (2003).</li><li>Runegaard, A. 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=Preserved+dopaminergic+homeostasis+and+dopamine-related+behaviour+in+hemizygous+TH-Cre+mice.">Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice.</a> Eur J Neurosci. 45, 121-128 (2017).</li><li>Whittaker, V. P., Michaelson, I. A., Kirkland, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+separation+of+synaptic+vesicles+from+nerve-ending+particles+('synaptosomes').">The separation of synaptic vesicles from nerve-ending particles ('synaptosomes').</a> Biochem J. 90, 293-303 (1964).</li><li>Hornykiewicz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+(3-hydroxytyramine)+and+brain+function.">Dopamine (3-hydroxytyramine) and brain function.</a> Pharmacol Rev. 18, 925-964 (1966).</li><li>Schultz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+dopamine+signals.">Behavioral dopamine signals.</a> Trends Neurosci. 30, 203-210 (2007).</li><li>Beaulieu, J. M., Gainetdinov, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology,+signaling,+and+pharmacology+of+dopamine+receptors.">The physiology, signaling, and pharmacology of dopamine receptors.</a> Pharmacol Rev. 63, 182-217 (2011).</li><li>Giros, B., Jaber, M., Jones, S. R., Wightman, R. M., Caron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperlocomotion+and+indifference+to+cocaine+and+amphetamine+in+mice+lacking+the+dopamine+transporter.">Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter.</a> Nature. 379, 606-612 (1996).</li><li>Torres, G. E., 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=Glutamate+and+monoamine+transporters:+new+visions+of+form+and+function.">Glutamate and monoamine transporters: new visions of form and function.</a> Curr Opin Neurobiol. 17, 304-312 (2007).</li><li>Jones, S. R., 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=Profound+neuronal+plasticity+in+response+to+inactivation+of+the+dopamine+transporter.">Profound neuronal plasticity in response to inactivation of the dopamine transporter.</a> Proc Natl Acad Sci U S A. 95, 4029-4034 (1998).</li><li>Gabriel, L. R., Wu, S., 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=Brain+slice+biotinylation:+an+ex+vivo+approach+to+measure+region-specific+plasma+membrane+protein+trafficking+in+adult+neurons.">Brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons.</a> J Vis Exp. , (2014).</li><li>Rickhag, 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=A+C-terminal+PDZ+domain-binding+sequence+is+required+for+striatal+distribution+of+the+dopamine+transporter.">A C-terminal PDZ domain-binding sequence is required for striatal distribution of the dopamine transporter.</a> Nat Commun. 4, 1580 (2013).</li><li>Dunkley, P. R., Jarvie, P. E., Robinson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+Percoll+gradient+procedure+for+preparation+of+synaptosomes.">A rapid Percoll gradient procedure for preparation of synaptosomes.</a> Nat Protoc. 3, 1718-1728 (2008).</li><li>Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty+years+of+synaptosome+research.">Thirty years of synaptosome research.</a> J Neurocytol. 22, 735-742 (1993).</li><li>Schmitz, Y., Benoit-Marand, M., Gonon, F., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+regulation+of+dopaminergic+neurotransmission.">Presynaptic regulation of dopaminergic neurotransmission.</a> J Neurochem. 87, 273-289 (2003).</li><li>Yang, L., Beal, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+neurotransmitter+levels+in+models+of+Parkinson's+disease+by+HPLC-ECD.">Determination of neurotransmitter levels in models of Parkinson's disease by HPLC-ECD.</a> Methods Mol Biol. 793, 401-415 (2011).</li><li>Earles, C., Schenk, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotating+disk+electrode+voltammetric+measurements+of+dopamine+transporter+activity:+an+analytical+evaluation.">Rotating disk electrode voltammetric measurements of dopamine transporter activity: an analytical evaluation.</a> Anal Biochem. 264, 191-198 (1998).</li><li>Wu, Q., Reith, M. E., Kuhar, M. J., Carroll, F. I., Garris, P. 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. 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+substantia+nigra+of+the+human+brain.+II.+Patterns+of+loss+of+dopamine-containing+neurons+in+Parkinson's+disease.">The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinson's disease.</a> Brain. 122 (Pt 8), 1437-1448 (1999).</li><li>Atack, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+dopamine+by+a+modification+of+the+dihydroxyindole+fluorimetric+assay.">The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay.</a> Br J Pharmacol. 48, 699-714 (1973).</li><li>Yoshitake, 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=High-sensitive+liquid+chromatographic+method+for+determination+of+neuronal+release+of+serotonin,+noradrenaline+and+dopamine+monitored+by+microdialysis+in+the+rat+prefrontal+cortex.">High-sensitive liquid chromatographic method for determination of neuronal release of serotonin, noradrenaline and dopamine monitored by microdialysis in the rat prefrontal cortex.</a> J Neurosci Methods. 140, 163-168 (2004).</li><li>Decressac, M., Mattsson, B., Lundblad, M., Weikop, P., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+neurodegenerative+and+behavioural+changes+induced+by+AAV-mediated+overexpression+of+alpha-synuclein+in+midbrain+dopamine+neurons.">Progressive neurodegenerative and behavioural changes induced by AAV-mediated overexpression of alpha-synuclein in midbrain dopamine neurons.</a> Neurobiol Dis. 45, 939-953 (2012).</li><li>Huot, P., Johnston, T. H., Koprich, J. B., Fox, S. H., Brotchie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-DOPA+pharmacokinetics+in+the+MPTP-lesioned+macaque+model+of+Parkinson's+disease.">L-DOPA pharmacokinetics in the MPTP-lesioned macaque model of Parkinson's disease.</a> Neuropharmacology. 63, 829-836 (2012).</li><li>Mikkelsen, 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=MPTP-induced+Parkinsonism+in+minipigs:+A+behavioral,+biochemical,+and+histological+study.">MPTP-induced Parkinsonism in minipigs: A behavioral, biochemical, and histological study.</a> Neurotoxicol Teratol. 21, 169-175 (1999).</li><li>Salvatore, M. F., Pruett, B. 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. T., Stathopoulos, A. M., Watson, C. C., Bertram, R., Trombley, P. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+bulb+monoamine+concentrations+vary+with+time+of+day.">Olfactory bulb monoamine concentrations vary with time of day.</a> Neuroscience. 247, 234-241 (2013).</li><li>Zhuang, X., 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=Hyperactivity+and+impaired+response+habituation+in+hyperdopaminergic+mice.">Hyperactivity and impaired response habituation in hyperdopaminergic mice.</a> Proc Natl Acad Sci U S A. 98, 1982-1987 (2001).</li><li>Ungerstedt, U., Pycock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+correlates+of+dopamine+neurotransmission.">Functional correlates of dopamine neurotransmission.</a> Bull Schweiz Akad Med Wiss. 30, 44-55 (1974).</li><li>Wickham, R. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurements+of+phasic+changes+in+extracellular+dopamine+concentration+in+freely+moving+rats+by+fast-scan+cyclic+voltammetry.">Real-time measurements of phasic changes in extracellular dopamine concentration in freely moving rats by fast-scan cyclic voltammetry.</a> Methods Mol Med. 79, 443-464 (2003).</li><li>Callaghan, P. D., Irvine, R. J., Daws, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+in+vivo+dynamics+of+neurotransmitter+release+and+serotonin+uptake+after+acute+para-methoxyamphetamine+and+3,4-methylenedioxymethamphetamine+revealed+by+chronoamperometry.">Differences in the in vivo dynamics of neurotransmitter release and serotonin uptake after acute para-methoxyamphetamine and 3,4-methylenedioxymethamphetamine revealed by chronoamperometry.</a> Neurochem Int. 47, 350-361 (2005).</li></ol>

This manuscript describes useful experimental protocols to delineate DA homeostasis in any mouse model of choice. We provide detailed protocols for measuring levels of DA in brain tissue from mice using HPLC and synaptosomal DA uptake to assess functional DA transport through DAT. The procedures, protocols and limits for the HPLC experiment and synaptosomal DA uptake assay will be elaborated below.
The synaptosomal uptake protocol can provide useful insight to the functionality of DAT. Combine...

Dopamine (DA) is a modulatory neurotransmitter critical for motor behaviour, reward and cognitive function1,7,8,9. Imbalances in DA homeostasis are implicated in several neuropsychiatric diseases such as attention-deficit hyperactivity disorder, drug addiction, depression and Parkinson&#39;s disease1. DA is released from the presynaptic neuron into the synaptic cleft, where it binds to and activates receptors on the pre- and postsynaptic membrane, thereby further conveying the signal. The level of DA in the synapse after release is spatially and temporally controlled by DAT3,10. The transporter sequesters DA from the extracellular space, and thus sustains physiological DA levels3,11. Genetic removal of DAT in mice causes a hyperdopaminergic phenotype characterized by elevated synaptic DA levels, depletion of intracellular DA pools and profound changes in postsynaptic DAergic signalling10,12.
Here, two separate protocols are presented, one method to measure DA tissue content and another to assess the functionality of DAT. Combined with the surface biotinylation assay described by Gabriel et al.13 these two methods provide information on DA content and functional levels of DAT for a thorough assessment of DA homeostasis. With these methods DA homeostasis of various transgenic mice or disease models can be characterized and described. These tools have been implemented and optimized and are standard use in our laboratories. Current assays have served to investigate the consequences on the DA homeostasis of altering the C-terminal of DAT14 or expressing Cre recombinase under the tyrosine hydroxylase (TH) promoter 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 a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Current DA uptake protocol (Figure 1) includes all steps necessary to assess the functionality of DAT in synaptosomes from mice. Our representative data of the DA uptake method (Figure 2) depicts a saturation curve with unadjusted data (Figure 2B) and adjusted data (Figure 2A). The saturation curve shows uptake from wild type mice. Usually one would make DA uptake for co...

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

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

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

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JoVE Journal

Neuroscience

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