Assessment of the Serotonin System
The Neurobehavioral Research Laboratory and Clinic has an active program of inquiry on the role of the neurotransmitter serotonin in behavioral and mental health. The examination of the serotonin system is important because serotonin is thought to be critical in the regulation of mood and behavior. We are examining serotonin (5-HT) transporter genetics, expression rate, and function to determine if stressful life events, impulsivity, serotonin dysregulation, and their interaction contribute to suicidal and drug-use behaviors observed longitudinally among a group of high-risk adolescents. Using the platelet model, we are studying 5-HT transporter function and expression rate in a given state (e.g., current mood, stress, medications). We are currently examining ways to allow us to observe the underlying innate cell biology of serotonin function and expression rate (i.e., without the influence of variable state influences).
Serotonin transporter function is assessed by measuring 5-HT uptake and 5-HT transporter expression rate is assessed by measuring paroxetine binding. First, blood is separated into platelet rich plasma, buffy coat (containing leucocytes), and red blood cells by centrifugation. The DNA is isolated from leucocytes for genotyping the 5-HT transporter. The platelet rich plasma is used to study 5-HT uptake (function of 5-HT transporter) and paroxetine binding (density of 5-HT transporter). Radiolabeled [3H]-5HT uptake into platelets is measured according to the procedure described by Greenberg et al. (1999). Tubes containing assay buffer, pargyline, and [3H]-5-HT (65.2 to 2000 nM; NEN, Boston) are preincubated for 10 min at 37oC. Immediately afterwards, 3X107 cells are added to each tube and the tubes are incubated for an additional 2 min. A second set of tubes containing fluoxetine is tested in an identical manner to assess non-specific uptake. Cells are collected by rapid filtration through Whatman GF/B filters and washed three times with washing buffer using a Brandel Cell Harvester. Filters are placed in scintillation vials containing Beckman Redi-Solv scintillation fluid and immediately counted on a Beckman Coulter LS 6500 Scintillation Counter. Specific 5-HT uptake is calculated by subtracting non-specific binding (with fluoxetine) from total binding (without fluoxetine) and are expressed as fmoles/107 cells*min. Km is expressed in nM. Km and Vmax are determined using Prism 5.0 software by GraphPad.
A [3H]-paroxetine binding assay is used to determine the density of 5-HT transporter (Greenberg et al., 1999). Briefly, the platelet suspensions are centrifuged at 20,000 g for 30 min at 4oC to obtain membrane pellets. Each pellet is washed once with washing buffer, resuspended in incubation buffer, and then sonicated to homogenize the suspension. Platelet membrane homogenates are incubated with 6 concentrations of [3H]-paroxetine (0.005 to 2.175 nM) in incubation buffer. A second set of tubes containing fluoxetine is tested in an identical manner to assess non-specific binding. After incubation for 60 min at 22oC, ice cold incubation buffer is added to each tube and the samples filtered through Whatman GF/B filters using a Brandel Cell Harvester The filters are washed three times with ice cold incubation buffer, air dried, then placed in scintillation vials with Beckman Redi-Solv scintillation fluid for counting. Amount of membrane protein is determined using the BioRad Protein Assay. Specific [3H]-paroxetine binding is calculated by subtracting non-specific binding (with fluoxetine) from total binding (without fluoxetine) and is expressed as fmoles/mg protein. Kd is expressed as nM. Kd and Bmax of [3H]-paroxetine binding are determined using Prism 5.0 software by GraphPad.
This work is performed by Nathalie Hill-Kapturczak, Ph.D., Pei Yu Tian, and David Galindo in the Biological Psychiatry Laboratory directed by Martin A. Javors, Ph.D. The Biological Psychiatry Laboratory is located in the medical school within the Department of Psychiatry. This laboratory conducts a full array of bioassays including platelet; lymphoblastoid cultured cells, DNA, and L-tryptophan markers of serotonin, as well as markers for metabolites of neurotransmitters, medication, and drugs of abuse. The BPAL has 3,000 square feet of space, which is equipped with a UV-VIS spectrophotometer, a scintillation counter, seven HPLC systems, four UV detectors, three coulometric detectors, an API Triple Quadrupole mass spectrophotometer, three tabletop refrigerated centrifuges, two high speed centrifuges, an ultra centrifuge, a Milli-Q water system, two Brandell cell harvesters, an analytic balance, a top loading balance, two shaking water baths, a sonicator, a whole blood lumi aggregometer with AGG RO/LINK data reduction system, a Perkin Elmer Thermal Cycler Model 9600, a BRL gel sequencing apparatus, a gel dryer, a vacuum pump, 6 separate –80° freezers, an autoclave, refrigerators, an ABI oligonucleotide synthesizer, incubators, an ABI DNA sequencing apparatus with peripherals needed for automated genotyping analyses, Deltascan Dual Wavelength Fluorometer, a Beckman Coulter Z2 Coulter Counter, and a humidified 5% CO2 incubator (NuAire). This laboratory is fully accredited by the College of American Pathologists and certified by the Health Care and Financing Administration, the government agency that oversees clinical labs in the United States.
Greenberg, B.D., Tolliver, T.J., Huang, S.J., Bengel, Q.L., Murphy, D.L. (1999). Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets. American Journal of Medical Genetics, 88, 83-87.