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

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.

1. Preparations
   1. 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
   2. Label 51 scintillation tubes #1-51.
   3. Place phosphate buffered saline (PBS) on ice. This will be used to keep brains chilled.
   4. Turn on the centrifuge to allow to pre-cool to 4 °C.
   5. Pre-weigh two micro-centrifuge tubes to get the exact tissue weight during the synaptosomal preparation (section 2.6).
   6. 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 °C and thawed the day of the experiment.
   7. 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 °C. Make 1x working solution freshly on the day, supplementing with ascorbic acid and glucose. Adjust pH with NaOH to 7.4.
   8. Prepare uptake buffer + ligand by combining the following: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaC_l2, 1.2 mM MgSO₄, 1 mM ascorbic acid (0.194 g/L), 5 mM D-glucose (0.991 g/L), 1 μM 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.
   9. 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 μM pargyline, 100 nM desipramine, 10 nM COMT inhibitor, 500 μM 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.
2. Synaptosomal preparation
   1. Sacrifice one mouse at a time by cervical dislocation and decapitation.
   2. 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.
   3. 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.
   4. 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.
   5. Keep the tissue on ice at all times moving forward.
   6. Transfer the tissue to a 1.5 mL micro-centrifuge tube for weighing.
      NOTE: This weight will be used in step 1.2.14.
   7. Repeat step 1.2.1-1.2.6 for the second brain.
   8. Once weight has been obtained for both brains, transfer the tissue to a homogenization glass containing 1 mL of ice-cold homogenization buffer.
   9. 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.
   10. 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.
   11. 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.
   12. Pellet the nuclei and cell debris by centrifugation (1,000 x g, 10 min, 4°C).
   13. 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°C.
   14. Discard the supernatant (containing cytoplasm) and resuspend the pellet (P2) (containing crude synaptosomes) in 40 μL 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 μL. If not, diluting with more than 40 μL 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 ice^6,15,16.

2. Uptake Experiment

1. Add 440 μL uptake buffer + ligand + cocaine to the designated 12 1.5 mL microcentrifuge tubes and 440 μL 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.
2. 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 μM)).
   1. Label six 2 mL microcentrifuge tubes as 10, 5, 2.5, 1.25, 0.62, and 0.31.
   2. Add 750 μL uptake buffer + ligand to the 5 microcentrifuge tubes with lowest number.
   3. Add 1,455.4 μL uptake buffer + ligand, 14.6 μL dopamine (1 mM), 30 μl 3-H dopamine (11 μM) to the tube labelled 10.
   4. Transfer 750 μL from the tube labelled 10 to the tube labelled 5. Mix well and transfer 750 μL from this tube to the 2.5 tube. Repeat this dilution with the remaining tubes.
3. Pre-rinse the glass microfiber filters with 4 mL uptake buffer after placing them on a 12 well filter bucket.
4. Add 10 μL of the synaptosomal membrane suspension to the first 24 1.5 mL micro-centrifuge tubes (see Table 1 for the layout) containing 440 μL 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.
5. Add 50 μL dopamine of concentration 10 and 5 μM (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 μM (section 2.2) and then with concentration 0.62 and 0.31 μM.
6. 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.
7. Repeat step 2.4-2.6 for the next brain.
8. Prepare 3 max-counting tubes by placing a microfiber filter in the bottom of scintillation tubes 49-51 and adding 25 µL of the maximum dopamine concentration on top (10 μM).
9. Leave all 51 scintillation tubes in a fume hood for 1 h.
10. Add 3 mL scintillation fluid to each scintillation tube and shake vigorous on a shaker for 1h.
11. Count [³H]-DA in a beta-counter for 1 min.
    1. 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.
12. Protein determination
    1. Use a standard BCA Protein Assay kit to determine protein concentrations of the synaptosomes for adjustments from counts per min (cpm) to fmol/min/μg and for proper comparison of uptake between samples.

3. Data Analysis

1. Use the data from the uptake experiment to make a saturation curve for each group and calculate the rate of the reaction (V_max) and the Michaelis Menten constant (K_M).
   NOTE: The K_M value reflects the substrate concentration required to reach half of V_max. Accordingly, V_max 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 K_M 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 K_M and V_max values have been reviewed by Schmitz et al.¹⁷.

4. High-performance Liquid Chromatography (Method 2)

1. Brain dissection
   1. Label and weigh microcentrifuge tubes; one per region per mouse.
   2. 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.
   3. 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.
   4. 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.
   5. Repeat steps 4.1.1-4.1.4 with the next mouse.
      NOTE: Place the tissue at 80^oC until further processing or immediately proceed to tissue processing.

5. Tissue Preparation

1. Turn on centrifuge to allow it to pre-cool to 4 °C
2. Prepare the homogenization solution (0.1 N perchloric acid (HClO₄)) and keep on ice.
3. Using homogenization solution, prepare a fresh 0.1 pmol/μL 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 °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.
4. Add 500 μL 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).
5. Centrifuge the samples for 30 min at 4 °C, 14,000 x g.
6. Transfer approximately 200 μL sample to a glass 0.22 μm 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 μm to remove the high molecular weight substances.
7. Analyze samples by electro-chemical detection (EC)-HPLC methodology¹⁸ as described in section 6.

6. High-performance Liquid Chromatography Analysis

1. 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.
2. Set-up of detector:
   1. Set volt output to 700 mV and temperature for 32 °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.
3. 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 μL).
   NOTE: Standard (10 μL (10 μL x 0.1 pmol/μL = 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).
   1. 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 -> save Batch file as -> Save).
   2. Make the system inject the first sample by pressing 'Start Batch'. This will result in the injection of 10 μL 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 µ ODS (3) C18 (DA 2 mm x 100 mm, particle size 3 µm) to achieve chromatographic separation.
   3. Extract the recorded and calculated peak areas.
      1. Go to data acquisition to follow the chromatogram when the samples have finished. Go to Lc post run analysis -> chose the file name (the chromatogram will open) -> click on view. On the manual integration bar, add all the peaks to be integrated -> go to data report and print the read out.
         NOTE: Here the LC solution software was used for data analysis and system control.
4. From the peak areas, calculate the concentration of dopamine in a sample, as follows by using the known concentration of the standard:
   1. Use the peak area of the standard (equals 1 pmol) to acquire factor A.
      
   2. Use factor A to get the concentration of the samples.
      Concentration of sample (in pmol per 10 μL) = Factor A × peak area of the sample
   3. Use this formula to get the sample concentration in ng.
      Sample concentration pmol / 10 µL x 500 µL x MW of dopamine = sample concentration in ng
      Sample concentration (in ng) = Sample concentration (in pmol per 10 μL)  x 500 μL x MW of dopamine
   4. Use the sample concentration in ng to get the sample concentration in pg/g tissue (sample concentration in pg / tissue g = pg/g tissue).