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.
<ol>
	<li><strong...

<ol>
	<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. 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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. 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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) ...

We are introducing two techniques in this protocol. The goal of the first, is to assess dopamine transporter function by measuring dopamine uptake from synaptosomal preparations. The second, is for assessing levels of dopamine in tissue by HPLC analysis.These methods provide important parameters of dopamine homeostasis by demonstrating how to measure dopamine tissue content and assess functionality of the dopamine transporter in mice. The main advantage of these techniques is that they can be performed post in-vivo manipulations such as, chemogenetic manipulations, in-vivo drug treatments or behavioral training of the animals. Demonstrating the HPLC procedure will be Pia Weikop, Associate Professor at the Laboratory of Neuropsychiatry.To begin this procedure, reveal the skull by cutting the skin and removing any excess tissue. Next, cut the skull along the sutura sagittalis, all the way anteriorly. Place the scissor tips in the eyes and remove the remaining skull by cutting through it.Rapidly remove the brain and place it in ice cold PBS. After that, using a brain matrix, dissect a three millimeter coronal slice of the striatum. Followed by finer dissection with a puncture.Subsequently, transfer the tissues to a 1.5 millimeter micro centrifuge tube for weighing. Then, repeat the procedures for the second brain. Once wait has been obtained for both brains, transfer the tissues to a homogenization glass containing one milliliter of ice cold homogenization buffer.After that, homogenize the tissue using a motor driven pestle. Next, transfer the homogeny to a 1.5 milliliter micro centrifuge tube and collect the remaining homogeny from the homogenization glass by rinsing it with 0.5 milliliters of additional homogenization buffer. Following that, pellet the nuclei and cell debris by centrifugation.Then, transfer the supernatant containing the cell membranes and cytoplasm to the new 1.5 milliliter micro centrifuge tubes and centrifuge for 20 minutes at four degree celsius. Afterward, discard the supernatant containing the cytoplasm and re-suspend the pellet containing crude synaptosomes in 40 microliters per milligram tissue in homogenization buffer. In this step, add 440 microliters of uptake buffer, ligand and cocaine to 12 designated 1.5 milliliter micro centrifuge tubes and 440 microliters of uptake buffer and ligand to the 36 remaining 1.5 milliliter micro centrifuge tubes.Label six two milliliter micro centrifuge tubes as 10, 5, 2.5, 1.25, 0.62 and 0.31. Then, add 750 microliters of uptake buffer and ligand to the five micro centrifuge tubes with the lowest number. Next, add 1, 455.4 microliters of uptake buffer and ligand, 14.6 microliters of unlabeled dopamine and 30 microliters of tritiated dopamine to the tube labeled, 10.Next, transfer 750 microliters of the mixture from the tube labeled 10, to the tube labeled 5. Mix well and transfer 750 microliters of the mixture from this tube to the tube labeled 2.5. Repeat this dilution with the remaining tubes.After that, pre-rinse the glass microfiber filters with four milliliters of uptake buffer after placing on a 12 well filter bucket. Following that, add 10 microliters of the synaptosomal membrane suspension to the first 24 1.5 milliliter micro centrifuge tubes containing 440 microliters buffer. Vortex carefully and spin down shortly to ensure that the synaptosomes are submerged in the buffer.Then, leave them at 37 degree celsius for 10 minutes. After 10 minutes, add 50 microliters of dopamine of 10 micromolar and five micromolar to the first two columns and leave them at 37 degrees celsius for five minutes with shaking. After five minutes, stop the reaction by adding one milliliter of ice cold uptake buffer, repeat it with the 2.5 micromolar and 1.25 micromolar and then, with the 0.62 micromolar and 0.31 micromolar.Add the samples to the pre-rinsed micro fiber filters and wash them with five, four milliliters of ice cold uptake buffer. After the first 12 samples have been added to the filters, move the filters to scintillation tubes. Repeat this with the next 12 samples.Prepare three max counting tubes by placing a microfiber filter in the bottom of scintillation tubes, 49 to 51 and adding 25 microliters of the maximum dopamine of 10 micromolar on top. Following this, leave all 51 scintillation tubes in a fume hood for one hour. Then, add three milliliters of scintillation fluid to each scintillation tube and shake vigorous on a shaker for one hour.To count tritiated dopamine in a beta counter for one minute, open the program and choose the suitable settings. For protein determination, use a standard BCA protein assay kit, to determine protein concentrations of the synaptosomes for adjustments and for proper comparison of uptake between samples. For tissue preparation, add 500 microliters of homogenization solution to each sample and homogenize all the samples using an ultra homogenizer on full speed for approximately 30 seconds.Keep the samples in iced water during homogenization. Next, centrifuge the samples for 30 minutes at four degree celsius, 14, 000 x G.After that, transfer approximately 200 microliters of each sample to a glass 0.22 micron filter before analyzing the samples by EC-HPLC methodology. To set up the detector, set the output to 700 millivolts and temperature at 32 degree celsius on the detector oven.Place the vials in the autosampler. Make a range program using the online manual and add the time program according to table two. Set up the system by opening the program.Next, type user ID and password and click okay. Then, wait for the program to connect to the instruments. Then, go to batch table and fill in data information.Save file by going to file, then click, save batch file as, followed by save. Subsequently, inject the first sample by pressing start batch. Afterward, go to data acquisition to follow the chromatogram when the samples are finished.Then, go to LC post run analysis and choose the file name. Then, click on view. On the manual integration bar, add all the peaks to be integrated, then, go to data report and print the readout.After the program has been added to the detector, make a three point calibration curve by injecting the prepared standard in five, 10 and 15 microliters. In this figure, the representative data depicts the saturation curve of dopamine uptake through the dopamine transporter from synaptosomal preparations in wild type mice. Black dots are the values of the four mice shown separately.The green curve shows how data would normally be depicted by combining data from four to six mice per group. Here is the saturation graph of the raw data before adjusting for actual protein concentration of the samples. This graph shows the counts from the cocaine containing samples which are used to subtract background from the uptake data.This control is essential to determine the reliability of the uptake data. This histogram shows the uptake capacity of the dopamine transporter and this histogram shows that Km for the dopamine transporter to be 0.1 plus or minus 0.03 micromolar which corresponds well to the rotating disk voltammetry method that depicts Km values of the dopamine transporter to be 0.6 to 1.2 micromolar. While attempting the synaptosomal optic procedure, it's important to keep tissue on ice at all times by preparing the synaptosomes.Following the synaptosome of dopamine uptake assay, additional procedures such as the surface biotinylation assay can be performed in order to determine whether a perturbed dopamine uptake can be explained by an altered surface expression of the dopamine transporter. After watching this video, you should have a good understanding of how to investigate dopamine homeostasis in mice by accessing the functionality of the dopamine transporter and dopamine levels in striatal tissue preparations.

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Research

JoVE Journal

Neuroscience

12.3K 次观看.  University of Copenhagen. We are introducing two techniques in this protocol. The goal of the first, is to assess dopamine transporter function by measuring dopamine uptake from synaptosomal preparations. The second, is for assessing levels of dopamine in tissue by HPLC analysis.These methods provide important parameters of dopamine homeostasis by demonstrating how to measure dopamine tissue content and assess functionality of the dopamine transporter in mice. The main advantage of these techniques is that they...