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1. Synaptosomal Dopamine Uptake
NOTE: This protocol is for parallel assessment of two brains, but it can also successfully be used to perform synaptosomal dopamine (DA) uptake experiments with four brains in parallel.
- Preparations
- Label 48 1.5 mL micro-centrifuge tubes per Table 1 (24 for each brain). Use different colors for the tubes belonging to each brain to prevent confusion between samples.
- Label 51 scintillation tubes #1-51.
- Place phosphate buffered saline (PBS) on ice. This will be used to keep brains chilled.
- Turn on the centrifuge to allow it to pre-cool to 4 °C.
- Pre-weigh two micro-centrifuge tubes to get the exact tissue weight during the synaptosomal preparation (section 2.6).
- 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 on the day of the experiment.
- 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.
- 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 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 dopamine transporter (DAT).
- Prepare uptake buffer + ligand + cocaine (coc) 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.
- Synaptosomal preparation
- Sacrifice one mouse at a time by cervical dislocation and decapitation.
- Cut the skin using scissors to reveal the skull and cut off any excess tissue posterior of the skull left from decapitation. Cut the skull with small straight scissors along the sutura sagittalis, ending up as anteriorly as possible.
- Place the two scissor tips in each eye of the mouse and cut through the 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.
- 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.
- Keep the tissue on ice at all times moving forward.
- Transfer the tissue to a 1.5 mL micro-centrifuge tube for weighing.
NOTE: This weight will be used in step 1.2.14.
- Repeat steps 1.2.1-1.2.6 for the second brain.
- Once the weight has been obtained for both brains, transfer the tissue to a homogenization glass containing 1 mL of ice-cold homogenization buffer.
- 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 synaptosomes. Using appropriate force and isotonic medium will allow the presynaptic terminals to reseal enclosing cytoplasm, synaptic vesicles, mitochondria and cytoskeleton.
- Transfer the homogenate to a 1.5 mL micro-centrifuge tube and collect the remaining homogenate from the homogenization glass by rinsing it with 0.5 mL additional homogenization buffer.
- 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.
- Pellet the nuclei and cell debris by centrifugation (1,000 x g, 10 min, 4°C).
- 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.
- 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.
2. Uptake Experiment
- 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.
- 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)).
- Label six 2 mL microcentrifuge tubes as 10, 5, 2.5, 1.25, 0.62, and 0.31.
- Add 750 μL uptake buffer + ligand to the 5 microcentrifuge tubes with the lowest number.
- 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.
- Transfer 750 μL from the tube labeled 10 to the tube labeled 5. Mix well and transfer 750 μL from this tube to the 2.5 tube. Repeat this dilution with the remaining tubes.
- Pre-rinse the glass microfiber filters with 4 mL uptake buffer after placing them on a 12 well filter bucket.
- 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.
- Add 50 μL of dopamine at concentrations 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 concentrations 2.5 and 1.25 μM (section 2.2) and then with concentrations 0.62 and 0.31 μM.
- 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.
- Repeat steps 2.4-2.6 for the next brain.
- 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).
- Leave all 51 scintillation tubes in a fume hood for 1 h.
- Add 3 mL scintillation fluid to each scintillation tube and shake vigorous on a shaker for 1 h.
- Count [3H]-DA in a beta-counter for 1 min.
- 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 overnight.
- Protein determination
- 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
- 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.
Table 1: Microcentrifuge tube layout for synaptosomal preparation.
| 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 |
