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>Preparations
	<ol>
		<li>Label 48 1.5 mL micro-centrifuge tubes per Table 1 (24 for each brain). Use different colours for the tubes belonging to each brain to prevent confusion between samples</li>
		<li>Label 51 scintillation tubes #1-51.</li>
		<li>Place phosphate buffered saline (PBS) on ice. This will be used to keep brains chilled.</li>
		<li>Turn on the centrifuge to allow to pre-cool to 4 &#176;C.</li>
		<li>Pre-weigh two micro-centrifuge tubes to get the exact tissue weight during the synaptosomal preparation (section 2.6).</li>
		<li>Prepare homogenization buffer by mixing 4 mM HEPES and 0.32 M sucrose. Adjust the pH to 7.4 and keep on ice. 
		NOTE: Homogenization buffer can be kept in aliquots at -20 &#176;C and thawed the day of the experiment.</li>
		<li>Prepare uptake buffer by combining the following: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1 mM ascorbic acid (0.194 g/L), 5 mM D-glucose (0.991 g/L). Adjust pH to 7.4 with NaOH (leads to a sodium concentration of approximately 130 mM) and keep on ice. 
		NOTE: For 2 brains, prepare 1500 mL uptake buffer. Prepare uptake buffer as a 10x stock solution without ascorbic acid and glucose kept at 4 &#176;C. Make 1x working solution freshly on the day, supplementing with ascorbic acid and glucose. Adjust pH with NaOH to 7.4.</li>
		<li>Prepare uptake buffer + ligand by combining the following: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1 mM ascorbic acid (0.194 g/L), 5 mM D-glucose (0.991 g/L), 1 &#956;M pargyline, 100 nM desipramine, 10 nM Catechol-O-methyl-transferase (COMT) inhibitor. Adjust to pH 7.4 with NaOH and keep on ice. 
		NOTE: Use 50 mL uptake buffer and add ligand. Pargyline is added to help prevent degradation of DA by oxidation through monoamine oxidase (MAO). COMT inhibitor (RO-41-0960) is added to prevent degradation of DA (catecholamines in general) through COMT. Desipramine is added to inhibit uptake through the norepinephrine and serotonin transporters, and thereby make the uptake results specific for DAT.</li>
		<li>Prepare uptake buffer + ligand + cocaine by combining the following: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1 mM ascorbic acid (0.194 g/L), 5 mM D-glucose (0.991 g/L), 1 &#956;M pargyline, 100 nM desipramine, 10 nM COMT inhibitor, 500 &#956;M cocaine. Adjust pH to 7.4 with NaOH and keep on ice. 
		NOTE: Use 7 mL uptake buffer + ligand and add cocaine. Cocaine is added to obtain a background measurement of non-specific DA uptake to subtract from the data, leaving only the DAT-specific uptake (since SERT and NET are inhibited by desipramine as described above). Alternatively, a highly potent and specific DAT inhibitor GBR-12935, can be used instead. 
		Important: Keep everything on ice from now on.</li>
	</ol>
	</li>
	<li>Synaptosomal preparation
	<ol>
		<li>Sacrifice one mouse at a time by cervical dislocation and decapitation.</li>
		<li>Cut the skin using scissors to reveal the skull and cut off any excess tissue posterior of skull left from decapitation. Cut the skull with small straight scissors along the sutura sagittalis ending up as anteriorly as possible.</li>
		<li>Place the two scissor tips in each eye of the mouse and cut through skull to remove the remainder of the skull and the intact brain. Rapidly remove the brain and place it in ice-cold PBS. This will keep it cold, while the second brain is dissected.</li>
		<li>Using a brain matrix, dissect a 3 mm coronal slice of the striatum (anterior-posterior: +1.5 mm to -1.5 mm) followed by finer dissection with a puncher. See Figure 1 for punch area.</li>
		<li>Keep the tissue on ice at all times moving forward.</li>
		<li>Transfer the tissue to a 1.5 mL micro-centrifuge tube for weighing. 
		NOTE: This weight will be used in step 1.2.14.</li>
		<li>Repeat step 1.2.1-1.2.6 for the second brain.</li>
		<li>Once weight has been obtained for both brains, transfer the tissue to a homogenization glass containing 1 mL of ice-cold homogenization buffer.</li>
		<li>Homogenize the tissue using a motor driven pestle at 800 rpm, 10 even strokes. 
		NOTE: One stroke equals one up and down action, the first stroke should be about 5 s, the following 3-4 s. Homogenization in isotonic medium is important to prevent bursting of the synaptosomes6. Using appropriate force and isotonic medium will allow the presynaptic terminals to reseal enclosing cytoplasm, synaptic vesicles, mitochondria and cytoskeleton15.</li>
		<li>Transfer the homogenate to a 1.5 mL micro-centrifuge tube and collect the remaining homogenate from the homogenization glass by rinsing with 0.5 mL additional homogenization buffer.</li>
		<li>Keep the sample on ice while tissue from the other brain is homogenized. Run the two brains in parallel in steps 1.2.12-1.2.13.</li>
		<li>Pellet the nuclei and cell debris by centrifugation (1,000 x g, 10 min, 4&#176;C).</li>
		<li>Transfer the supernatant (S1) (containing cell membranes and cytoplasm) to new 1.5 mL microcentrifuge tubes and centrifuge at 16,000 x g for 20 min at 4&#176;C.</li>
		<li>Discard the supernatant (containing cytoplasm) and resuspend the pellet (P2) (containing crude synaptosomes) in 40 &#956;L homogenization buffer/ mg tissue (based on the weight obtained in step 1.2.6). Gently resuspend the crude synaptosomal fraction with a p1000 pipette as too much force will break the synaptosomes. 
		NOTE: It is important to end up with at least 280 &#956;L. If not, diluting with more than 40 &#956;L per mg will be necessary. Steps 1.2.1-1.2.13 must be performed as fast as possible and everything has to be kept on ice. As soon as the crude synaptosomes have been resuspended in homogenization buffer they are fairly stable as long as they are kept on ice6,15,16.</li>
	</ol>
	</li>
</ol>
2. Uptake Experiment
<ol>
	<li>Add 440 &#956;L uptake buffer + ligand + cocaine to the designated 12 1.5 mL microcentrifuge tubes and 440 &#956;L uptake buffer + ligand to the 36 remaining 1.5 mL microcentrifuge tubes (see Table 1 for the layout). 
	NOTE: Remove them from ice 15 min before the start of the experiment to bring them to room temperature, but keep on ice until then to conserve inhibitors.</li>
	<li>Prepare saturation curve (dopamine (2, 5, 6-[3H]-DA) (3-H dopamine) (91.1 Ci/mmol) at various final concentrations (0.031, 0.0625, 0.125, 0.25, 0.5, and 1.0 &#956;M)).
	<ol>
		<li>Label six 2 mL microcentrifuge tubes as 10, 5, 2.5, 1.25, 0.62, and 0.31.</li>
		<li>Add 750 &#956;L uptake buffer + ligand to the 5 microcentrifuge tubes with lowest number.</li>
		<li>Add 1,455.4 &#956;L uptake buffer + ligand, 14.6 &#956;L dopamine (1 mM), 30 &#956;l 3-H dopamine (11 &#956;M) to the tube labelled 10.</li>
		<li>Transfer 750 &#956;L from the tube labelled 10 to the tube labelled 5. Mix well and transfer 750 &#956;L from this tube to the 2.5 tube. Repeat this dilution with the remaining tubes.</li>
	</ol>
	</li>
	<li>Pre-rinse the glass microfiber filters with 4 mL uptake buffer after placing them on a 12 well filter bucket.</li>
	<li>Add 10 &#956;L of the synaptosomal membrane suspension to the first 24 1.5 mL micro-centrifuge tubes (see Table 1 for the layout) containing 440 &#956;L buffer. Vortex carefully and spin down shortly to ensure that the synaptosomes are submerged in the buffer. Leave at 37 oC for 10 min. 
	NOTE: It is important to be exact on the times during the uptake experiment for fair comparison between brains. Use a stopwatch for precision.</li>
	<li>Add 50 &#956;L dopamine of concentration 10 and 5 &#956;M (section 2.2) to the first two columns and leave at 37 oC for 5 min with shaking. After exactly 5 min, stop the reaction by adding 1 mL ice-cold uptake buffer. Do the same with concentration 2.5 and 1.25 &#956;M (section 2.2) and then with concentration 0.62 and 0.31 &#956;M.</li>
	<li>Add the samples to the pre-rinsed microfiber filters and wash with 5 x 4 mL ice-cold uptake buffer. When the first 12 samples have been added to the filters, move the filters to scintillation tubes. Repeat this with the next 12 samples.</li>
	<li>Repeat step 2.4-2.6 for the next brain.</li>
	<li>Prepare 3 max-counting tubes by placing a microfiber filter in the bottom of scintillation tubes 49-51 and adding 25 &#181;L of the maximum dopamine concentration on top (10 &#956;M).</li>
	<li>Leave all 51 scintillation tubes in a fume hood for 1 h.</li>
	<li>Add 3 mL scintillation fluid to each scintillation tube and shake vigorous on a shaker for 1h.</li>
	<li>Count [3H]-DA in a beta-counter for 1 min.
	<ol>
		<li>Open the program and choose: Label: H-3, Plate/Filter: 4 mL vial, 4 by 6, Assay type: Normal and Counting time: 1 min. 
		NOTE: This step can be performed the following day if more convenient. Leave samples covered with tin foil over night.</li>
	</ol>
	</li>
	<li>Protein determination
	<ol>
		<li>Use a standard BCA Protein Assay kit to determine protein concentrations of the synaptosomes for adjustments from counts per min (cpm) to fmol/min/&#956;g and for proper comparison of uptake between samples.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>Use the data from the uptake experiment to make a saturation curve for each group and calculate the rate of the reaction (Vmax) and the Michaelis Menten constant (KM). 
	NOTE: The KM value reflects the substrate concentration required to reach half of Vmax. Accordingly, Vmax directly reflects the function of DAT (maximum uptake capacity), which depends on the number of DAT on the surface and on how its activity might be modulated by posttranslational modifications and/or interacting proteins as well as on alterations in ion gradients. The KM value is an indirect measure of substrate affinity for the transporter that, importantly also can be modulated by posttranslational modifications and/or interacting proteins. To fully assess functional changes it is therefore important to directly determine surface expression levels by for instance a surface biotinylation assay. A description of dopamine reuptake and an elaboration on meaning of the KM and Vmax values have been reviewed by Schmitz et al.17.</li>
</ol>
4. High-performance Liquid Chromatography (Method 2)
<ol>
	<li>Brain dissection
	<ol>
		<li>Label and weigh microcentrifuge tubes; one per region per mouse.</li>
		<li>Sacrifice one mouse at a time by cervical dislocation and decapitation. Rapidly remove the brain as described in section 1.2. Perform further dissection immediately.</li>
		<li>Using a brain matrix, dissect a 3 mm coronal slice of the striatum followed by finer bilateral dissection of NAc and dStr with a puncher. See Figure 3 for punch area.</li>
		<li>Transfer the tissue to 1.5 mL microcentrifuge tubes and quickly place on dry ice. Weigh the tissue and place it back on the dry ice immediately. 
		NOTE: The tissue weight is important for the concentration calculation later on.</li>
		<li>Repeat steps 4.1.1-4.1.4 with the next mouse. 
		NOTE: Place the tissue at 80oC until further processing or immediately proceed to tissue processing.</li>
	</ol>
	</li>
</ol>
5. Tissue Preparation
<ol>
	<li>Turn on centrifuge to allow it to pre-cool to 4 &#176;C</li>
	<li>Prepare the homogenization solution (0.1 N perchloric acid (HClO4)) and keep on ice.</li>
	<li>Using homogenization solution, prepare a fresh 0.1 pmol/&#956;L dopamine standard from a 1 mM stock solution (a 10,000 x dilution). 
	NOTE: The stock solution is prepared by dissolving dopamine in homogenization buffer to a stock concentration of 1 mM, which can be kept at 20 &#176;C for about one month. If other areas of the brain are of interest, concentration of the standard will have to be modified, since other brain areas including prefrontral cortex, hippocampus, substantia nigra and ventral tegmental area possess up to 100 times less DA compared to striatum.</li>
	<li>Add 500 &#956;L homogenization solution to each sample (i.e. NAc and dStr tissue; section 4.1) and homogenize samples using an ultra-homogenizer on full speed for approximately 30 s. Keep the sample in iced water during homogenization. Between each sample, clean the ultra homogenizer with water (full speed for 30 s).</li>
	<li>Centrifuge the samples for 30 min at 4 &#176;C, 14,000 x g.</li>
	<li>Transfer approximately 200 &#956;L sample to a glass 0.22 &#956;m filter (1 cm diameter) using a 1 mL plastic syringe. 
	NOTE: The use of glass filters is not of important, as long as the filters chosen are 0.22 &#956;m to remove the high molecular weight substances.</li>
	<li>Analyze samples by electro-chemical detection (EC)-HPLC methodology18 as described in section 6.</li>
</ol>
6. High-performance Liquid Chromatography Analysis
<ol>
	<li>Prepare the following solution for mobile phase: Sodium acetate 55 mM, octanesulfonic acid 1 mM, Na2EDTA 0.1 mM, acetonitrile 8 %, acetic acid 0.1 M, pH = 3.2. 
	NOTE: Adjust pH with 0.1 M acetic acid. It is important to be precise with the pH. The solution should be degassed by the on-line degasser (integrated part of the HPLC system). Make 2 L of mobile phase and change it about once a month (change it when the noise gets noticable). Due to fluctuations it takes about 24 h to adjust after changing the mobile phase.</li>
	<li>Set-up of detector:
	<ol>
		<li>Set volt output to 700 mV and temperature for 32 &#176;C on the detector oven; this is very important. Make a range program using the online manual. See the manual for further detail). Add the time program as per Table 2. 
		NOTE: In this study, the program is set to automatically change the current at set retention times, to keep the HPLC at the optimal current and to keep the peaks of the chromatogram as enlarged as possible, but still within view. This has been optimized for striatal brain lysates from mice.</li>
	</ol>
	</li>
	<li>When the program has been added to the detector, make a 3 point calibration curve, by injecting the standard three times (5, 10 and 15 &#956;L). 
	NOTE: Standard (10 &#956;L (10 &#956;L x 0.1 pmol/&#956;L = 1 pmol DA)) should be injected every tenth sample, and samples calibrated to the nearest standard. Fine tune the system the day prior by adjusting the 3-point standard, and checking if the chromatogram comes out within the expected range. If substantial noise is observed, change the mobile phase. If a peak is higher than 990 mV then re-inject the sample in a lower volume, or make a new range program and a new standard curve. The detection limit is measured as 3 times the noise value in mV to the lowest peak value injected for each standard (NA, DOPAC, DA, 5-HIAA, HVA, 3-MT and 5-HT).
	<ol>
		<li>Set-up system by opening the LC solution and activating analysis 1; this starts in the online mode. Type user ID and password and click ok. Wait for LC solution to connect to instruments. Go to batch table and fill in data information (e.g., sample type: unknown for samples and std for standard, analysis type: IT QT, inj volume: 10, ISTD amt: Level 1 con). Save file (Go to File -&#62; save Batch file as -&#62; Save).</li>
		<li>Make the system inject the first sample by pressing &#39;Start Batch&#39;. This will result in the injection of 10 &#956;L sample by the autosampler with a solvent delivery module. 
		NOTE: Use a pump flow rate of 0.15 mL/min and a C18 column with reversal phase. Here an amperometric detector for electrochemical detection was used. The glassy carbon electrode should be set to 0.8 V with an Ag/AgCl reference electrode. In order to assay dopamine, use Chromatography 3 &#181; ODS (3) C18 (DA 2 mm x 100 mm, particle size 3 &#181;m) to achieve chromatographic separation.</li>
		<li>Extract the recorded and calculated peak areas.
		<ol>
			<li>Go to data acquisition to follow the chromatogram when the samples have finished. Go to Lc post run analysis -&#62; chose the file name (the chromatogram will open) -&#62; click on view. On the manual integration bar, add all the peaks to be integrated -&#62; go to data report and print the read out. 
			NOTE: Here the LC solution software was used for data analysis and system control.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>From the peak areas, calculate the concentration of dopamine in a sample, as follows by using the known concentration of the standard:
	<ol>
		<li>Use the peak area of the standard (equals 1 pmol) to acquire factor A. 
		<img alt="Equation" src="/files/ftp_upload/56093/56093eq1.jpg" /></li>
		<li>Use factor A to get the concentration of the samples. 
		Concentration of sample (in pmol per 10 &#956;L) = Factor A &#215; peak area of the sample</li>
		<li>Use this formula to get the sample concentration in ng. 
		Sample concentration pmol / 10 &#181;L x 500 &#181;L x MW of dopamine = sample concentration in ng 
		Sample concentration (in ng) = Sample concentration (in pmol per 10 &#956;L)&#160; x 500 &#956;L x MW of dopamine</li>
		<li>Use the sample concentration in ng to get the sample concentration in pg/g tissue (sample concentration in pg / tissue g = pg/g tissue).</li>
	</ol>
	</li>
</ol>

<ol><li>Tritsch, N. X., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopaminergic+modulation+of+synaptic+transmission+in+cortex+and+striatum%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((A+biochemical+and+functional+protein+complex+involving+dopamine+synthesis+and+transport+into+synaptic+vesicles%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((SLC6+neurotransmitter+transporters%3A+structure%2C+function%2C+and+regulation%5BTitle%5D)+AND+%22Pharmacol+Rev%22%5BJournal%5D)">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/pubmed?term=((Monoamine+transporters%3A+from+genes+to+behavior%5BTitle%5D)+AND+%22Annu+Rev+Pharmacol+Toxicol%22%5BJournal%5D)">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/pubmed?term=((Preserved+dopaminergic+homeostasis+and+dopamine-related+behaviour+in+hemizygous+TH-Cre+mice%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((The+separation+of+synaptic+vesicles+from+nerve-ending+particles+%28%27synaptosomes%27%29%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">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/pubmed?term=((Dopamine+%283-hydroxytyramine%29+and+brain+function%5BTitle%5D)+AND+%22Pharmacol+Rev%22%5BJournal%5D)">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/pubmed?term=((Behavioral+dopamine+signals%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">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/pubmed?term=((The+physiology%2C+signaling%2C+and+pharmacology+of+dopamine+receptors%5BTitle%5D)+AND+%22Pharmacol+Rev%22%5BJournal%5D)">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/pubmed?term=((Hyperlocomotion+and+indifference+to+cocaine+and+amphetamine+in+mice+lacking+the+dopamine+transporter%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Glutamate+and+monoamine+transporters%3A+new+visions+of+form+and+function%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">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/pubmed?term=((Profound+neuronal+plasticity+in+response+to+inactivation+of+the+dopamine+transporter%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Brain+slice+biotinylation%3A+an+ex+vivo+approach+to+measure+region-specific+plasma+membrane+protein+trafficking+in+adult+neurons%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((A+C-terminal+PDZ+domain-binding+sequence+is+required+for+striatal+distribution+of+the+dopamine+transporter%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">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/pubmed?term=((A+rapid+Percoll+gradient+procedure+for+preparation+of+synaptosomes%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((Thirty+years+of+synaptosome+research%5BTitle%5D)+AND+%22J+Neurocytol%22%5BJournal%5D)">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/pubmed?term=((Presynaptic+regulation+of+dopaminergic+neurotransmission%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">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/pubmed?term=((Determination+of+neurotransmitter+levels+in+models+of+Parkinson%27s+disease+by+HPLC-ECD%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Rotating+disk+electrode+voltammetric+measurements+of+dopamine+transporter+activity%3A+an+analytical+evaluation%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Preferential+increases+in+nucleus+accumbens+dopamine+after+systemic+cocaine+administration+are+caused+by+unique+characteristics+of+dopamine+neurotransmission%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Modelling+constant+potential+amperometry+for+investigations+of+dopaminergic+neurotransmission+kinetics+in+vivo%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Rapid+regulation+of+dopamine+transporters+by+tyrosine+kinases+in+rat+neuronal+preparations%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">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/pubmed?term=((Missense+dopamine+transporter+mutations+associate+with+adult+parkinsonism+and+ADHD%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((The+substantia+nigra+of+the+human+brain.+II.+Patterns+of+loss+of+dopamine-containing+neurons+in+Parkinson%27s+disease%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">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/pubmed?term=((The+determination+of+dopamine+by+a+modification+of+the+dihydroxyindole+fluorimetric+assay%5BTitle%5D)+AND+%22Br+J+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((High-sensitive+liquid+chromatographic+method+for+determination+of+neuronal+release+of+serotonin%2C+noradrenaline+and+dopamine+monitored+by+microdialysis+in+the+rat+prefrontal+cortex%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">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/pubmed?term=((Progressive+neurodegenerative+and+behavioural+changes+induced+by+AAV-mediated+overexpression+of+alpha-synuclein+in+midbrain+dopamine+neurons%5BTitle%5D)+AND+%22Neurobiol+Dis%22%5BJournal%5D)">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/pubmed?term=((L-DOPA+pharmacokinetics+in+the+MPTP-lesioned+macaque+model+of+Parkinson%27s+disease%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">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/pubmed?term=((MPTP-induced+Parkinsonism+in+minipigs%3A+A+behavioral%2C+biochemical%2C+and+histological+study%5BTitle%5D)+AND+%22Neurotoxicol+Teratol%22%5BJournal%5D)">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/pubmed?term=((Comprehensive+profiling+of+dopamine+regulation+in+substantia+nigra+and+ventral+tegmental+area%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Regional+distribution+of+biogenic+amines%2C+amino+acids+and+cholinergic+markers+in+the+CNS+of+the+C57BL%2F6+strain%5BTitle%5D)+AND+%22Amino+Acids%22%5BJournal%5D)">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/pubmed?term=((Sex+hormones+affect+neurotransmitters+and+shape+the+adult+female+brain+during+hormonal+transition+periods%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Olfactory+bulb+monoamine+concentrations+vary+with+time+of+day%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Hyperactivity+and+impaired+response+habituation+in+hyperdopaminergic+mice%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/4371656">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/pubmed?term=((Examination+of+Rapid+Dopamine+Dynamics+with+Fast+Scan+Cyclic+Voltammetry+During+Intra-oral+Tastant+Administration+in+Awake+Rats%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Real-time+measurements+of+phasic+changes+in+extracellular+dopamine+concentration+in+freely+moving+rats+by+fast-scan+cyclic+voltammetry%5BTitle%5D)+AND+%22Methods+Mol+Med%22%5BJournal%5D)">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/pubmed?term=((Differences+in+the+in+vivo+dynamics+of+neurotransmitter+release+and+serotonin+uptake+after+acute+para-methoxyamphetamine+and+3%2C4-methylenedioxymethamphetamine+revealed+by+chronoamperometry%5BTitle%5D)+AND+%22Neurochem+Int%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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. Combined with a surface biotinylation experiment13, knowledge on the total amount, surface level, and functionality of DAT can be obtained. Given the major role of DAT and its influence on DA transmission and its participation in various diseases, it has been a major goal to establish assays that can model DAT function. One of the advantages of the synaptosomal DA uptake experiment is that it can be performed post in vivo manipulations such as upon chemogenetic manipulation as well as different in vivo drug treatments or behavioral training in addition to investigations on genetically modified mice. Drug-treatment can be performed after synaptosomal preparation, instead of in vivo if preferred, making it possible to test the effect of drugs on DAT directly22.
Alternatives to performing synaptosomal DA uptake, is to perform uptake experiments either on DAT transfected cell cultures or in neuronal cultures naturally expressing the transporter. Cell culture assays might be preferred for initial investigations into different modifications of DAT23, whereas neuronal primary cultures with the endogenous transporter may provide a more physiologically trustworthy picture of the transporter function in vivo. Even though neuronal cultures are made directly from animals, there are advantages of using synaptosomes instead. Neuronal cultures are usually made from prenatal or immature neurons, which might influence the function and expression of DAT, whereas synaptosomal preparations represent physiological preparations that can be obtained from adult and even old animals without difficulties6,14.
There are several advantages of using the synaptosomal uptake experiment to investigate function of DAT, but important limitations have to be considered. The synaptosomes have limited viability6. Keeping them on ice is essential to obtain reliable results with low variations. If kept on ice and provided necessary nutrients, purified synaptosomes are viable for hours and take up and release neurotransmitters efficiently15. It is possible to freeze synaptosomes, but the method of freezing is of great importance15. Small variations in the experimental procedure can lead to extensive variations in outcome. Therefore, experimental protocols should be optimized on wild type conditions (e.g. wild type mice) until reproducible results are obtained and then comparisons can be made following various genetic or pharmacological manipulations. Synaptosomal DA uptake assay is an easy, reliable and valid experimental tool to acquire reproducible data with a very low variation in a key parameter in DA homeostasis, DAT functionality (Figure 2A). The limitations are heavily outweighed by the advantages of being able to perform the experiment on preserved nerve endings from adult mice6.
The presently used methods to analyze DA levels in tissue are supported by histochemical methods developed in the 1950&#39;s. The significance of developing methods like HPLC to measure DA levels, has been obvious since discovering the substantial decrease of DA in the basal ganglia of Parkinson&#39;s patients, thereby founding the principle of treating patients suffering from Parkinson&#39;s with L-DOPA24. Since this discovery, more advanced techniques for tissue analysis of DA levels have been developed, but as with any other techniques there are pitfalls. One of the major pitfalls of these techniques is the unstable nature of monoamines (dopamine, noradrenaline and serotonin). How to correctly prepare the tissue preparation to avoid loss of monoamines has been discussed in great detail by Atack et al.25 and will not be discussed further in this article, except to stress the importance of placing the tissue on dry ice directly after dissection and not adding homogenization solution until immediately before the HPLC analysis. From our experience, tissue can be kept at -80 oC for up to one month without any degradation of DA if no solution has been added. Atack et al discuss tissue preparation for methods ranging from the fluoro spectrometric method to advanced HPLC methods allowing a detection limit down to 3 ng/mL tissue25. The method we describe in this paper is based on the same principles. Current advanced technologies enable more refined analyses and detection of DA levels at fmol concentrations. By using a fluorescent HPLC technique, even more robust monoamine analysis can be obtained26. Due to the robustness of the method, HPLC is widely used to obtain information about changes in the levels of monoamines and precursors and metabolites in various brain regions, such as DA in striatum, to validate disease models of Parkinson&#39;s in mice, monkeys and minipigs27,28,29. Here, we perform the experiment on tissue from dStr and NAc, but the method is also suitable for other DA-innervated brain areas, such as the prefrontral cortex, hippocampus, substantia nigra and ventral tegmental area 30. In these areas, a more diluted standard sample will be necessary for proper determination of DA levels. Our analysis of DA content show higher levels in striatal subcompartments compared to previous investigations, but this can be explained experimentally. First, we have dissected two parts of the striatum (NAc and dStr) as opposed to investigating the whole striatum, which might account for the difference compared to previous reports12,31. Striatal measurements will have lower DA levels compared to measurements in pure dStr, since levels in NAc are substantially lower compared to dStr. We also have previous studies confirming our DA levels5.
Every assay has its limitations. Assays are developed in an attempt to model and provide information on specific aspects of a cellular process, and they might leave out possible important details or provide a too generalized picture of the real world process. An important limitation to consider when choosing HPLC, is that it only provides a snapshot of the neurotransmitter levels. However, neurotransmitter levels are prone to fluctuate over a day, week or month32,33, which emphasizes the need to obtain samples at a narrow time window, instead of comparing samples taken hours, days or months apart as though they were taken within the same hour. However, HPLC data can provide useful information on DA content and reveal aberrant altered levels such as those demonstrated by the DAT-KO and DAT-KD transgenic mouse lines, where genetic deletion or knock-down of DAT significantly influences DA homeostasis by perturbing DA reuptake. These data furthermore demonstrate that striatal DA pools primarily consist of sequestered DA rather than de-novo synthesized DA and that replenishment of intracellular striatal DA pools is critically dependent on the reuptake process12,34. One important pitfall to consider is that tissue dissection may limit a more specific and accurate description of the DA content in a brain area at any given time. The variation in DA concentration in different brain areas varies greatly. Therefore, the accuracy of dissection is of great importance, which can be improved by dissecting smaller areas to ensure only tissue from the area of interest is included.
A more functional measure of the endogenous DA pools can be analyzed using microdialysis. This has been developed and pioneered by Ungerstedt et al. using the vertical microdialysis probe35. The microdialysis technique makes it possible to measure monoamine concentrations in the brain of freely moving animals and in different brain structures. Another advantage of the microdialysis technique over the tissue sampling through HPLC is the option to measure and follow monoamine changes over a large window of time. This is a considerable advantage compared to sampling of brain tissue where only one time point is possible per animal as in the HPLC protocol. While, microdialysis can provide insight into the release of DA, tissue sampling followed by HPLC will instead reveal changes in the endogenous pools and vesicular DA. To obtain real-time information on DA release kinetics in various brain areas, methods like fast-scan cyclic voltammetry36,37 or high-speed chronoamperometry38 can be implemented.

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 &amp;amp; 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&amp;trade; Electrochemical Detector type 175 and 176</td><td>For HPLC protocol</td></tr><tr><td>Avantec 0.22 &amp;mu;m glass filter</td><td>Frisenette ApS, Denmark</td><td>13CP020AS</td><td>For HPLC protocol</td></tr><tr><td>Column: Prodigy 3 &amp;mu; 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&amp;amp;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&amp;trade; 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 comparison with a mutant mouse, which would lead to a saturation curve for each genotype5. In that case, differences between wild type and mutant mice in 2A and 2B can be explained by multiple factors. The more thorough the experimenter is in following every step of the protocol, the less difference there will be between depicting raw (2B) and protein adjusted (2A) data. The most obvious reasons for differences are i) imprecise weighing of the tissue in step 1.2.6, ii) loss of tissue while transferring to and from homogenization glass in step 1.2.8-1.2.10 and iii) imprecision while transferring supernatant in step 1.2.13 and removing supernatant in step 1.2.14. We recommend performing a preliminary experiment in wild type mice for better precision.
The dopamine EC-HPLC method (Figure 3) includes all steps necessary to asses amount of DA in dStr and NAc of mice. Our representative data (Figure 4 and Table 3) depict the outcome of an experiment performed on wild type mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56093/56093fig1.jpg" /> 
Figure 1: Schematic workflow depicting the different steps in the synaptosomal DA uptake experiment protocol. The mouse is sacrificed by cervical dislocation followed by brain dissection and placement in a brain matrix. A 3 mm thick coronal slice is dissected from the brain, and fine dissection is performed by a bilateral punch of a small area immediately underneath the corpus callosum. Dorsal striatum is homogenized in 1 mL homogenization buffer by mechanical disruption followed by 10 min centrifugation at low speed. Supernatant is transferred to a clean tube and centrifuged at high speed for 20 min. The pellet, containing the synaptosomes, is resuspended and ready for the uptake experiment. Uptake is performed by adding triplicates of the different [3H]-DA concentrations, ranging from a final concentration of 0.031 to 1 &#956;M. In addition, the varying concentrations of tritiated DA are added to a control sample containing 500 &#956;M cocaine. Lastly, the samples are counted in a beta-counter and data analysis is performed revealing the saturation curve of DAT. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56093/56093fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56093/56093fig2.jpg" /> 
Figure 2: Representative results from a synaptosomal DA uptake experiment of four C57Bl/6 wild type mice from a Drd1a-cre mouse strain. (A) Representative data depicting the saturation curve of DA uptake through the DAT of synaptosomal preparations from wild type mice (n = 4). 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 4-6 mice per group. This graph combines data from four mice. (B) The saturation graph of the raw data, without adjusting for actual protein concentration of the samples. Essentially, it is the unadjusted version of the data in A. (C) Control data. 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, but is rarely shown in articles. If this data for some reason does not show linearity, it indicates a critical issue with the set-up, which needs to be identified and solved to deduce anything from the data. R squared: n1 = 0.9852, n2 = 0.9584, n3 = 0.9606, n4 = 0.9913. (D) Histogram showing the uptake capacity of DAT (VMAX). VMAX = 43.13 &#177; 3.2 fmol/min/&#956;g protein). Data are shown as mean &#177; SEM. (E) Histogram showing the KM for DAT to be 0.1 &#177; 0.03 &#956;M corresponding well to the rotating disk voltammetry method depicting KM values of DAT to be 0.6 &#956;M19. A KM value of 0.1 &#956;M also corresponds well to the KM values of 0.22 &#956;M that have been obtained by stimulation models of stimulated DA overflow in striatum20,21. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56093/56093fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56093/56093fig3.jpg" /> 
Figure 3: Schematic workflow depicting the different steps in the high-performance liquid chromatography protocol. The mouse is sacrificed by cervical dislocation followed by brain dissection and placement in brain matrix. A 3 mm thick coronal slice is dissected from the brain, and fine dissection is performed by bilaterally punching two smaller areas, dividing the striatum into the dStr and the NAc. Tissue is homogenized, followed by fast centrifugation. A standard with known DA concentrations as well as supernatants from the samples are run in the HPLC, producing chromatograms. The areas of the different peaks are used to calculate the concentration of DA in the samples depicted in a histogram. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56093/56093fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56093/56093fig4.jpg" /> 
Figure 4: Representative results of a high-performance liquid chromatography experiment. (A) HPLC chromatogram for 10 &#956;L sample from dStr injected to a C18 (2 mm x 100 mm) column used for small molecules. Retention times; 3.7 min for Noradrenaline (NA), 6.7 min for dihydroxyphenylacetic acid (DOPAC), 8.3 min for DA, 10.7 min for 5-hydroxyindoleacetic acid (HIAA), 15.3 min for homovanillic acid (HVA), 18.8 min for 3-methoxytyamine (3-MT) and 20.8 min for 5-hydroxtryptamine (5-HT). Only values for the DA peaks are calculated in this study. (B) Histogram showing the concentration of DA in NAc and dStr based on chromatrogram. HPLC analysis of striatal areas including dStr and NAc of C57BL/6 mice (n = 7). Data are shown as mean &#177; SEM. <img alt="Up_arrow" src="/files/ftp_upload/56093/56093uparrow.jpg" /> Indicates a change in resistance, and has been added to the chromatogram manually. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56093/56093fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10</td>
			<td>5</td>
			<td>2.5</td>
			<td>1.25</td>
			<td>0.62</td>
			<td>0.31</td>
		</tr>
		<tr>
			<td>10</td>
			<td>5</td>
			<td>2.5</td>
			<td>1.25</td>
			<td>0.62</td>
			<td>0.31</td>
		</tr>
		<tr>
			<td>10</td>
			<td>5</td>
			<td>2.5</td>
			<td>1.25</td>
			<td>0.62</td>
			<td>0.31</td>
		</tr>
		<tr>
			<td>10+coc</td>
			<td>5+coc</td>
			<td>2.5+coc</td>
			<td>1.25+coc</td>
			<td>0.62+coc</td>
			<td>0.31+coc</td>
		</tr>
	</tbody>
</table>
Table 1: Microcentrifuge tube layout for synaptosomal preparation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>Range</td>
			<td>Filter</td>
			<td>Valve</td>
			<td>Auto zero</td>
			<td>Offset</td>
			<td>E cell</td>
		</tr>
		<tr>
			<td>0</td>
			<td>1nA</td>
			<td>0.5 Hz</td>
			<td>load</td>
			<td>not</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>1nA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>set</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>5</td>
			<td>500pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>not</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>5.2</td>
			<td>500pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>set</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>9.4</td>
			<td>200pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>not</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>9.6</td>
			<td>200pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>set</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>12</td>
			<td>100pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>not</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>12.2</td>
			<td>100pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>set</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>16.2</td>
			<td>50pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>not</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td>16.4</td>
			<td>50pA</td>
			<td>0.5Hz</td>
			<td>load</td>
			<td>set</td>
			<td>30%</td>
			<td>0.7 V</td>
		</tr>
		<tr>
			<td colspan="2">End time 25 min</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: HPLC time program used in this study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak #</td>
			<td>Ret. Time</td>
			<td>Area</td>
			<td>Height</td>
			<td>Area %</td>
			<td>Height %</td>
		</tr>
		<tr>
			<td>1</td>
			<td>3,745</td>
			<td>230451</td>
			<td>18500</td>
			<td>0.299</td>
			<td>0.626</td>
		</tr>
		<tr>
			<td>2</td>
			<td>6,691</td>
			<td>5573485</td>
			<td>382143</td>
			<td>7,228</td>
			<td>12,922</td>
		</tr>
		<tr>
			<td>3</td>
			<td>8,336</td>
			<td>13209342</td>
			<td>510378</td>
			<td>17,131</td>
			<td>17,258</td>
		</tr>
		<tr>
			<td>4</td>
			<td>10,760</td>
			<td>16443198</td>
			<td>831182</td>
			<td>21,325</td>
			<td>28,106</td>
		</tr>
		<tr>
			<td>5</td>
			<td>15,344</td>
			<td>7129795</td>
			<td>282473</td>
			<td>9,247</td>
			<td>9,552</td>
		</tr>
		<tr>
			<td>6</td>
			<td>18,830</td>
			<td>11279424</td>
			<td>346248</td>
			<td>14,628</td>
			<td>11,708</td>
		</tr>
		<tr>
			<td>7</td>
			<td>20,846</td>
			<td>23241754</td>
			<td>586419</td>
			<td>30,142</td>
			<td>19,829</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td>77107450</td>
			<td>2957344</td>
			<td>100,000</td>
			<td>100,000</td>
		</tr>
	</tbody>
</table>
Table 3: Peak analysis showing area and height of the different peaks used to calculate the concentrations shown in Figure 4B.

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) ...

assessment-dopaminergic-homeostasis-mice-use-high-performance-liquid

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

12.4K Views.  University of Copenhagen. 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.

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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavior

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Medicine

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Chemistry

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

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

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

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

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

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

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

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

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

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