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Fluorescence-Based Neurotransmitter Transporter Uptake Assay in Primary Neuronal Cultures

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Fluorescence-Based Neurotransmitter Transporter Uptake Assay in Primary Neuronal Cultures Fluorescence-based Neurotransmitter Transporter Uptake Assay in Primary Neuronal Cultures. Parsa Safa, MDS Analytical Technologies, Sunnyvale, CA 94089 & Lesley Radov, AstraZeneca Pharmaceuticals, CNSP Discovery, Wilmington, DE 19803 Abstract Results The use of a fluorescence-based assay kit for the measurement of neurotransmitter Fig. 1. Dye loading occurs in a time and Fig. 4. Cultures from several but not all brain regions show significant Table 2. IC50 values are reproducible in primary transporter (NT) uptake activity in cell lines stably expressing the human DAT, NET and SERT gene has been previously reported. We describe here the application of this temperature dependent manner in primary dye uptake. neuronal cultures of the same age. fluorescence-based method to measure NT uptake activity in primary E-18 rat neuronal neuronal cultures and is sensitive to external Nucleus accumbens 7000 Striatum cultures. Real-time changes in NET, DAT & SERT transport activity were monitored in 7000 2 sodium and chloride concentrations. 6000 Compound Confidence R Days in these neurons. Since these 3 biogenic amine transporters show overlapping localization and 6000 37°C 5000 IC50 (nM) Interval (nM) Culture expression in the brain, NT uptake in sister cultures derived from the hippocampus, cortex, a) Time and Temperature 5000 midbrain, striatum and nucleus accumbens were compared.. Hippocampal cultures were 4000 4000 Week 1 Day 1 10.5 5.8 - 58.9 0.9863 Day 7 used to assess NET uptake activity; nucleus accumbens cultures were used to assess DAT 7000 3000 RFU uptake activity; and midbrain cultures were used to assess SERT uptake activity. Reference RFU 3000 Day 2 8.6 6.1 - 12.1 0.9272 Day 8 37°C 2000 NET, DAT & SERT inhibitors from various pharmacological classes were evaluated for their 6000 2000 RT 37°C ability to inhibit transport in these neuronal cultures and the IC50 values generated 1000 1000 compared to literature values. 5000 0 0 500 1000 1500 2000 2500 3000 3500 4000 Week 2 Day 1 11.3 4.8 - 26.7 0.9130 Day 7 Time (seconds) RT RFU -1000 4000 RT -1000 Day 2 6.8 2.4 - 19.0 0.9914 Day 8 1000 2000 3000 4000 5000 6000 7000 8000 Time (seconds) 3000 Week 3 Day 1 15.7 11.1 - 22.3 0.9852 Day 7 2000 Hippocampus Midbrain 7000 Day 2 6.8 3.0 - 15.0 0.9113 Day 8 7000 6000 1000 37°C 0 500 1000 1500 2000 2500 3000 3500 4000 6000 This table illustrates that the results are reproducible from week to week when Time (seconds) 5000 5000 cultures of the same age are used. The confidence limits overlap between all 6 4000 4000 studies, showing that there is no significant difference among the 6 the IC50 RT RFU 3000 values. Introduction RFU 3000 37°C 2000 b) Replacing Sodium or Chloride in the Buffer 2000 1000 1000 RT 6500 Depression is a common metal disorder. It is estimated that the lifetime prevalence of major Normal Table 3. Comparison of IC results from primary 6000 0 0 50 1 500 1000 1500 2000 2500 3000 3500 4000 depressive disorder (MDD) is ~7%. Approximately two dozen marketed antidepressants are 5500 Time (seconds) -1000 -1000 500 1000 1500 2000 2500 3000 3500 4000 neuronal cultures with reported K values. currently available worldwide. Many of these drugs increase the concentration of the 3 5000 i Time (seconds) biogenic amine neurotransmitters most closely linked to depression (serotonin, dopamine, 4500 4000 The dye uptake signal/noise window in primary cultures of hippocampus (NET), midbrain (SERT), and norephinephrine/noradrenaline) in the synaptic cleft. This is accomplished by inhibiting their 3500 reuptake back into the presynaptic neuron. Drugs targeting a single neurotransmitter 3000 nucleus accumbens (DAT) is sufficient and reliable enough for these tissues to be used for transporter Drug Syn 1° Cell Syn 1° Cell Syn 1° Cell RFU 2500 studies. The dye uptake signal/noise window in primary cultures of striatal cultures is NOT large enough transporter, such as the selective serotonin reuptake inhibitors (SSRIs) or selective 2000 NET NET SERT SERT DAT DAT noradrenalin reuptake inhibitors (SRNIs), were developed to limit the untoward side effects of 1500 Chloride Replacement to allow these cultures to be used on a routine basis – especially with the variability inherent in the system. nM nM nM nM nM nM the tricyclic antidepressants while mimicking their efficacy. The next step forward in 1000 500 depression treatment was the development of dual uptake inhibitors (SSRI/SSNI). These 0 Sodium Replacement agents have gained acceptance in the clinic, but are not totally satisfactory due to delayed onset, -500 Duloxetine 15.6 22.8 4.6 4.3 369.2 496 -1000 low response rates and their side effect profile. The latest advance in depression therapy has 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 4000 Fig. 5. The pharmacology of a triple uptake inhibitor: McN5652 Time (seconds) Fluoxetine 281 306 12 11.6 1600 2961 been the development of the triple uptake inhibitors (SSRI/SSNI/selective dopamine uptake IC50 Curve for McN5652 in the NT Dye Uptake IC50 Curve for McN5652 in the NT Dye Uptake Inhibition Citalopram 4000 6147 1.3 2.8 28000 33970 inhibitor). Inhibition Assay in Prim ary Cortical Cultures Assay in Primary Hippocampal (NET) Cultures Top: uptake proceeds faster at 37°C than at room temperature. This Fluvoxamine 620 709 7 3.3 5000 5293 is consistent with the ‘rule of thumb’ that rate of uptake 9000 The most common technique used to estimate biogenic amine transport has been monitoring 8500 14000 Paroxetine 81 121 0.29 0.47 5100 5800 approximately doubles for every increase of 10°C. the accumulation of radiolabeled substrate or inhibitor. One major drawback to the use of 8000 Bottom: sodium and chloride are essential for transport into 12500 Sertraline 160 197 0.19 0.11 48 66.1 these radioactive methods is the difficulty in monitoring real-time changes. A second 7500 primary neurons confirming that transport is via Na+ /Cl- coupled GBR12909 440 242 170 301 1 1.6 consideration is the increased pressure to minimize the use of radiolabeled materials due to 7000 11000 transporters 6500 Nisoxetine 1.3 1.7 310 351 510 1000 public safety concerns and increasing disposal costs. An alternative, non-radioactive approach RFU RFU 9500 for determining biogenic amine transport is available. The use of a fluorescence-based assay 6000 Nomifensine 5 15.5 1280 1660 51 65.1 5500 kit for the measurement of neurotransmitter transporter uptake activity in cell lines stably 8000 Desipramine 0.9 0.24 340 632 5200 6700 expressing the human DAT, NET and SERT gene has been previously reported. A question 5000 6500 that constantly arises is the relevance of the use of cell lines over-expressing the recombinant 4500 Bupropion 2300 2200 15600 11540 630 826 4000 10 -1 0 10 -9 10 -8 10 -7 10 -6 5000 SERT, DAT or NET vs. the use of native tissue protein to determine uptake. We describe here Fig. 2. The Neurotransmitter Transporter Uptake 10 -12 .5 10 -12.0 10 -11.5 10-1 1.0 10 -1 0.5 10 -10 .0 10 -9.5 10 -9.0 10-8.5 10-8 .0 10-7 .5 10-7 .0 10 -6 .5 Reboxetine 8.2 15.8 1070 1636 >100000 Concentration [M] the application of this fluorescence-based method to measure NT uptake activity in a native Concentration [M] Assay is rapid and robust. McN5652: IC = 27.8nM; CI: 16.6 - 46.8nM; R 2 = 0.9607 2 Imipramine 13 26 42 33.8 5110 8238 50 McN5652: IC 50 = 2.8nM; CI: 1.5 - 5.4nM; R = 0.9429 tissue system. The native tissue system we have chosen to employ is the primary E-18 rat Control Control neuronal culture model. Data in Excel Spreadsheet McN5652 2.9 2.8 0.68 0.68 36.8 44.9 Plate 1° neuronal cells 5-9 [Conc] 60' Pre-dye Difference [Conc] 60' Pre-dye Difference [Conc] 60' Pre-dye Difference 1.67E-07 9493.9 9167.7 326.1 6.17E-09 10476.0 8490.4 1985.7 2.28624E-10 12115.6 9452.4 2663.2 IC50 Curve for McN5652 in the NT Dye Uptake Inhibition IC50 Curve for McN5652 in the NT Dye Uptake Inhibition days before assay 10755.3 10215.3 540.0 10849.3 9008.8 1840.5 11312.5 8588.7 2723.7 There was excellent agreement between published literature Ki values for 11342.8 10998.0 344.9 10035.7 7958.9 2076.8 10938.9 8298.8 2640.1 Assay in Primary Midbrain (SERT) Cultures Assayin Primary Nucleus Accumbens (DAT) Cultures 10667.7 10152.4 515.3 10804.5 8723.9 2080.6 10671.4 8021.8 2649.6 10945.8 10755.7 190.1 10770.8 9085.6 1685.1 10964.4 8356.3 2608.1 the 14 drugs evaluated and the IC values generated in the primary 10314.7 9896.7 418.0 11108.3 9048.4 2059.8 10069.3 7442.2 2627.1 1200 12000 50 5.56E-08 10346.6 9103.2 1243.4 11361.2 9269.3 2091.8 7.62079E-11 10350.1 7553.9 2796.1 1100 neuronal culture system. The Spearman r for each transporter is as Materials & Methods 12881.1 12305.8 575.3 2.06E-09 10479.5 8338.9 2140.6 11179.8 8435.0 2744.8 10263.6 9138.8 1124.8 10136.4 8037.3 2099.0 10712.6 7801.7 2910.9 Take initial reading 1000 11000 follows: NET = 0.9801; SERT = 0.9956; & DAT = 0.9835.
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