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) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson'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.
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'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.
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.
2. Uptake Experiment
3. Data Analysis
4. High-performance Liquid Chromatography (Method 2)
5. Tissue Preparation
6. High-performance Liquid Chromatography Analysis
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.
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 μM. In addition, the varying concentrations of tritiated DA are added to a control sample containing 500 μM cocaine. Lastly, the samples are counted in a beta-counter and data analysis is performed revealing the saturation curve of DAT. Please click here to view a larger version of this figure.
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 ± 3.2 fmol/min/μg protein). Data are shown as mean ± SEM. (E) Histogram showing the KM for DAT to be 0.1 ± 0.03 μM corresponding well to the rotating disk voltammetry method depicting KM values of DAT to be 0.6 μM19. A KM value of 0.1 μM also corresponds well to the KM values of 0.22 μM that have been obtained by stimulation models of stimulated DA overflow in striatum20,21. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: Representative results of a high-performance liquid chromatography experiment. (A) HPLC chromatogram for 10 μL 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 ± SEM. Indicates a change in resistance, and has been added to the chromatogram manually. Please click here to view a larger version of this figure.
10 | 5 | 2.5 | 1.25 | 0.62 | 0.31 |
10 | 5 | 2.5 | 1.25 | 0.62 | 0.31 |
10 | 5 | 2.5 | 1.25 | 0.62 | 0.31 |
10+coc | 5+coc | 2.5+coc | 1.25+coc | 0.62+coc | 0.31+coc |
Table 1: Microcentrifuge tube layout for synaptosomal preparation.
Time | Range | Filter | Valve | Auto zero | Offset | E cell |
0 | 1nA | 0.5 Hz | load | not | 30% | 0.7 V |
0.2 | 1nA | 0.5Hz | load | set | 30% | 0.7 V |
5 | 500pA | 0.5Hz | load | not | 30% | 0.7 V |
5.2 | 500pA | 0.5Hz | load | set | 30% | 0.7 V |
9.4 | 200pA | 0.5Hz | load | not | 30% | 0.7 V |
9.6 | 200pA | 0.5Hz | load | set | 30% | 0.7 V |
12 | 100pA | 0.5Hz | load | not | 30% | 0.7 V |
12.2 | 100pA | 0.5Hz | load | set | 30% | 0.7 V |
16.2 | 50pA | 0.5Hz | load | not | 30% | 0.7 V |
16.4 | 50pA | 0.5Hz | load | set | 30% | 0.7 V |
End time 25 min |
Table 2: HPLC time program used in this study.
Peak # | Ret. Time | Area | Hauteur | Area % | Height % |
1 | 3,745 | 230451 | 18500 | 0.299 | 0.626 |
2 | 6,691 | 5573485 | 382143 | 7,228 | 12,922 |
3 | 8,336 | 13209342 | 510378 | 17,131 | 17,258 |
4 | 10,760 | 16443198 | 831182 | 21,325 | 28,106 |
5 | 15,344 | 7129795 | 282473 | 9,247 | 9,552 |
6 | 18,830 | 11279424 | 346248 | 14,628 | 11,708 |
7 | 20,846 | 23241754 | 586419 | 30,142 | 19,829 |
Total | 77107450 | 2957344 | 100,000 | 100,000 |
Table 3: Peak analysis showing area and height of the different peaks used to calculate the concentrations shown in Figure 4B.
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'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's patients, thereby founding the principle of treating patients suffering from Parkinson'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'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.
The authors have nothing to disclose.
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.).
COMT inhibitor | Sigma Aldrich, Germany | RO-41-0960 | For synaptosomal DA uptake protocol |
[3H]-Dopamine | Perkin-Elmer Life Sciences, Boston, MA, USA | NET67-3001MC | For synaptosomal DA uptake protocol |
Glass microfiber filters | GF/C Whatman, GE Healthcare Life Sciences, Buckinghamshire | 1822-024 | For synaptosomal DA uptake protocol |
HiSafe Scintillation fluid | Perkin Elmer | 1200-437 | For synaptosomal DA uptake protocol |
MicroBeta2 | Perkin Elmer | For synaptosomal DA uptake protocol | |
BCA Protein Assay kit | Thermo Scientific Pierce | 23225 | For synaptosomal DA uptake protocol |
HEPES | Sigma Life Science | H3375 | For synaptosomal DA uptake protocol |
Sucrose | Sigma Life Science | S7903 | For synaptosomal DA uptake protocol |
NaCl | Sigma Life Science | S3014 | For synaptosomal DA uptake protocol |
KCl | Sigma Life Science | P9541 | For synaptosomal DA uptake protocol |
CaCl2 | Merck KGaA | 10043-52-4 | For synaptosomal DA uptake protocol |
MgSO4 | Sigma Life Science | 63065 | For synaptosomal DA uptake protocol |
Ascorbic Acid | Sigma Life Science | A0278 | For synaptosomal DA uptake protocol |
D-Glucose | Sigma Life Science | G7021 | For synaptosomal DA uptake protocol |
Pargyline | Sigma Aldrich | P-8013 | For synaptosomal DA uptake protocol |
Desipramine | Sigma Aldrich | D3900 | For synaptosomal DA uptake protocol |
Dopamine | Sigma Life Science | H8502 | For synaptosomal DA uptake protocol |
Cocaine | Sigma Life Science | C5776 | For synaptosomal DA uptake protocol |
Brain matrix | ASI instruments | RBM2000C | For synaptosomal DA uptake protocol |
Cafano mechanical teflon disrupter | Buch & Holm | Discontinued | For synaptosomal DA uptake protocol (homogenization) |
Antec Decade (Amperometric detector) | Antec, Leiden, The Netherlands | Discontinued: new model DECADE Elite / Lite™ Electrochemical Detector type 175 and 176 | For HPLC protocol |
Avantec 0.22 μm glass filter | Frisenette ApS, Denmark | 13CP020AS | For HPLC protocol |
Column: Prodigy 3 μ ODS-3 C18 | Phenomenex, YMC Europe, Chermbeck, Germany | Part Number:00A-3300-E0 | For HPLC protocol |
LC solution software | Shimadzu | LabSolutions Series Workstation | For HPLC protocol |
Perchlor acid 0.1M | Fluka Analytical | 35418-500ml | For HPLC protocol (Tissue preparation) |
EDTA | Sigma | E5134-50g | For HPLC protocol |
Natriumdihydrogenphosphar | Bie&Berntsen | 1.06346 1000g | For HPLC protocol |
Sodium 1-octanesulfonate monohydrate | Aldrich | 74885 -10g | For HPLC protocol |
Acetonitrile, isocratic HPLC grade | Scharlau | AC03402500 | For HPLC protocol |
Filtre 0.22um | Frisenette ApS, Denmark | Avantec 13CP020AS | For HPLC protocol (Tissue preparation) |
ortho-Phosphoric acid 85% | Merck | 1.00563. 1000ml | For HPLC protocol |
Electrode | Antec, Leiden, The Netherlands | AN1161300 | For HPLC protocol (see manual online) |
Detector program on DECADE II electrochemical detector | Antec, Leiden, The Netherlands | Lite™ Electrochemical Detector type 175 and 176 | For HPLC protocol |