Skip to main content

Flow Cytometry to Determine Serotonin Transporter Function in Human Peripheral Blood Cells

  • 779 Accesses

Part of the Neuromethods book series (NM,volume 95)

Abstract

The serotonin transporter (SERT) is highly studied for its role in affective disorders and is the primary target of the first-line selective serotonin (5-HT) reuptake inhibitor (SSRI) antidepressant medications. While a number of methods exist for studying SERT function, most of these lack the ability to differentiate uptake with respect to individual cell types within heterogeneous cell populations such as those in the blood. Here, we describe a flow cytometry-based method that uses the fluorescent substrate APP+ to assess SERT function. The substrate APP+ is an MPP+ analog that is readily transported by SERT. Additionally, APP+ has the advantage of primarily fluorescing in hydrophobic environments, and, thereby, nonspecific fluorescence in aqueous environments prior to transport into cells is greatly reduced. Flow cytometry is a high-throughput technique often used in immunology to study and to characterize blood cell subtypes. Overall, the use of APP+ in combination with flow cytometry provides a readily available method for assessing and discerning SERT function in mixed cell populations.

Keywords

  • Serotonin transporter
  • Uptake
  • Platelets
  • Lymphocytes
  • Flow cytometry
  • Blood

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-2187-4_8
  • Chapter length: 17 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   89.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-2187-4
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   119.00
Price excludes VAT (USA)
Hardcover Book
USD   139.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Stein M, Seedat S, Gelernter J (2006) Serotonin transporter gene promoter polymorphism predicts SSRI response in generalized social anxiety disorder. Psychopharmacology 187:68–72

    CAS  PubMed  CrossRef  Google Scholar 

  2. Vaidya VA, Duman RS (2001) Depression—emerging insights from neurobiology. Br Med Bull 57:61–79

    CAS  PubMed  CrossRef  Google Scholar 

  3. Sinyor M, Schaffer A, Levitt A (2010) The sequenced treatment alternatives to relieve depression (STAR*D) trial: a review. Can J Psychiatry 55:126–135

    PubMed  Google Scholar 

  4. Rush AJ, Warden D, Wisniewski S, Fava M, Trivedi M, Gaynes B, Nierenberg A (2009) STAR*D. CNS Drugs 23:627–647

    PubMed  Google Scholar 

  5. Leuchter A, Cook I, Hamilton S, Narr K, Toga A, Hunter A, Faull K, Whitelegge J, Andrews A, Loo J, Way B, Nelson S, Horvath S, Lebowitz B (2010) Biomarkers to predict antidepressant response. Curr Psychiatry Rep 12:553–562

    PubMed Central  PubMed  CrossRef  Google Scholar 

  6. Chen JJ, Li Z, Pan H, Murphy DL, Tamir H, Koepsell H, Gershon MD (2001) Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affinity serotonin transporter: abnormal intestinal motility and the expression of cation transporters. J Neurosci 21:6348–6361

    CAS  PubMed  Google Scholar 

  7. Lesch K-P, Wolozin BL, Murphy DL, Riederer P (1993) Primary structure of the human platelet serotonin uptake site: identity with the brain serotonin transporter. J Neurochem 60:2319–2322

    CAS  PubMed  CrossRef  Google Scholar 

  8. Ramamoorthy S, Bauman AL, Moore KR, Han H, Yang-Feng T, Chang AS, Ganapathy V, Blakely RD (1993) Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc Natl Acad Sci U S A 90:2542–2546

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  9. Murphy DL, Lesch KP (2008) Targeting the murine serotonin transporter: insights into human neurobiology. Nat Rev Neurosci 9:85–96

    CAS  PubMed  CrossRef  Google Scholar 

  10. Kraft JB, Peters EJ, Slager SL, Jenkins GD, Reinalda MS, McGrath PJ, Hamilton SP (2007) Analysis of association between the serotonin transporter and antidepressant response in a large clinical sample. Biol Psychiatry 61:734–742

    CAS  PubMed  CrossRef  Google Scholar 

  11. Rausch J, Johnson M, Li J, Hutcheson J, Carr B, Corley K, Gowans A, Smith J (2005) Serotonin transport kinetics correlated between human platelets and brain synaptosomes. Psychopharmacology 180:391–398

    CAS  PubMed  CrossRef  Google Scholar 

  12. Arora RC, Tong C, Jackman HL, Stoff D, Meltzer HY (1983) Serotonin uptake and imipramine binding in blood platelets and brain of Fawn-hooded and Sprague Dawley rats. Life Sci 33:437–442

    CAS  PubMed  CrossRef  Google Scholar 

  13. Chou DT, Cuzzone H, Hirsh KR (1983) Assessment of blood platelets as a model for CNS response: comparative effects of caffeine on 5-HT uptake and release mechanisms in rat platelets and rat brain serotonin neurons. Life Sci 33:1149–1156

    CAS  PubMed  CrossRef  Google Scholar 

  14. Greenberg BD, Tolliver TJ, Huang S-J, Li Q, Bengel D, Murphy DL (1999) Genetic variation in the serotonin transporter promoter region affects serotonin uptake in human blood platelets. Am J Med Genet 88:83–87

    CAS  PubMed  CrossRef  Google Scholar 

  15. Lesch K-P, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Müller CR, Hamer DH, Murphy DL (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 274:1527–1531

    CAS  PubMed  CrossRef  Google Scholar 

  16. Singh YS, Sawarynski LE, Michael HM, Ferrell RE, Murphey-Corb MA, Swain GM, Patel BA, Andrews AM (2009) Boron-doped diamond microelectrodes reveal reduced serotonin uptake rates in lymphocytes from adult rhesus monkeys carrying the short allele of the 5-HTTLPR. ACS Chem Neurosci 1:49–64

    PubMed Central  CrossRef  Google Scholar 

  17. Singh YS, Altieri SC, Gilman TL, Michael HM, Tomlinson ID, Rosenthal SJ, Swain GM, Murphey-Corb MA, Ferrell RE, Andrews AM (2012) Differential serotonin transport is linked to the rh5-HTTLPR in peripheral blood cells. Transl Psychiatry 2:e77

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  18. Jayanthi LD, Samuvel DJ, Blakely RD, Ramamoorthy S (2005) Evidence for biphasic effects of protein kinase C on serotonin transporter function, endocytosis, and phosphorylation. Mol Pharmacol 67:2077–2087

    CAS  PubMed  CrossRef  Google Scholar 

  19. Lampugnani MG, Buczko W, Ceci A, Mennini A, de Gaetano G (1986) Normal serotonin uptake by blood platelets and brain synaptosomes but selective impairment of platelet serotonin storage in mice with Chediak-Higashi syndrome. Life Sci 38:2193–2198

    CAS  PubMed  CrossRef  Google Scholar 

  20. Annamalai B, Mannangatti P, Arapulisamy O, Shippenberg TS, Jayanthi LD, Ramamoorthy S (2012) Tyrosine phosphorylation of the human serotonin transporter: a role in the transporter stability and function. Mol Pharmacol 81:73–85

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  21. Beikmann BS, Tomlinson ID, Rosenthal SJ, Andrews AM (2013) Serotonin uptake is largely mediated by platelets versus lymphocytes in peripheral blood cells. ACS Chem Neurosci 4:161–170

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  22. Mercado CP, Kilic F (2010) Molecular mechanisms of SERT in platelets: regulation of plasma serotonin levels. Mol Interv 10:231–241

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  23. Paul WE (2008) Fundamental immunology. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia

    Google Scholar 

  24. Yang GB, Qiu CL, Aye P, Shao Y, Lackner AA (2007) Expression of serotonin transporters by peripheral blood mononuclear cells of rhesus monkeys (Macaca mulatta). Cell Immunol 248:69–76

    CAS  PubMed  CrossRef  Google Scholar 

  25. Rudd ML, Nicolas AN, Brown BL, Fischer-Stenger K, Stewart JK (2005) Peritoneal macrophages express the serotonin transporter. J Neuroimmunol 159:113–118

    CAS  PubMed  CrossRef  Google Scholar 

  26. Faraj BA, Olkowski ZL, Jackson RT (1994) Expression of a high-affinity serotonin transporter in human lymphocytes. Int J Immunopharmacol 16:561–567

    CAS  PubMed  CrossRef  Google Scholar 

  27. Chamba A, Holder MJ, Jarrett RF, Shield L, Toellner KM, Drayson MT, Barnes NM, Gordon J (2010) SLC6A4 expression and anti-proliferative responses to serotonin transporter ligands clomipramine and fluoxetine in primary B-cell malignancies. Leuk Res 34:1103–1106

    CAS  PubMed  CrossRef  Google Scholar 

  28. Marazziti D, Baroni S, Masala I, Giannaccini G, Mungai F, Di Nasso E, Cassano GB (2003) Decreased lymphocyte [3H]-paroxetine binding in obsessive-compulsive disorder. Neuropsychobiology 47:128–130

    CAS  PubMed  CrossRef  Google Scholar 

  29. Urbina M, Pineda S, Piñango L, Carreira I, Lima L (1999) [3H]Paroxetine binding to human peripheral lymphocyte membranes of patients with major depression before and after treatment with fluoxetine. Int J Immunopharmacol 21:631–646

    CAS  PubMed  CrossRef  Google Scholar 

  30. Rivera-Baltanas T, Olivares JM, Calado-Otero M, Kalynchuk LE, Martinez-Villamarin JR, Caruncho HJ (2012) Serotonin transporter clustering in blood lymphocytes as a putative biomarker of therapeutic efficacy in major depressive disorder. J Affect Disord 137:46–55

    CAS  PubMed  CrossRef  Google Scholar 

  31. O'Connell PJ, Wang X, Leon-Ponte M, Griffiths C, Pingle SC, Ahern GP (2006) A novel form of immune signaling revealed by transmission of the inflammatory mediator serotonin between dendritic cells and T cells. Blood 107:1010–1017

    PubMed Central  PubMed  CrossRef  Google Scholar 

  32. Fazzino F, Urbina M, Cedeño N, Lima L (2009) Fluoxetine treatment to rats modifies serotonin transporter and cAMP in lymphocytes, CD4+ and CD8+ subpopulations and interleukins 2 and 4. Int Immunopharmacol 9:463–467

    CAS  PubMed  CrossRef  Google Scholar 

  33. Meredith EJ, Holder MJ, Chamba A, Challa A, Drake Lee A, Bunce CM, Drayson MT, Pilkington G, Blakely RD, Dyer MJS, Barnes NM, Gordon J (2005) The serotonin transporter (SLC6A4) is present in B-cell clones of diverse malignant origin: probing a potential antitumor target for psychotropics. FASEB J 19:1187–1189

    CAS  PubMed  Google Scholar 

  34. Mössner R, Daniel S, Albert D, Heils A, Okladnova O, Schmitt A, Lesch K-P (2000) Serotonin transporter function is modulated by brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF). Neurochem Int 36:197–202

    PubMed  CrossRef  Google Scholar 

  35. Mössner R, Daniel S, Schmitt A, Albert D, Lesch K-P (2001) Modulation of serotonin transporter function by interleukin-4. Life Sci 68:873–880

    PubMed  CrossRef  Google Scholar 

  36. Prasad HC, Zhu C-B, McCauley JL, Samuvel DJ, Ramamoorthy S, Shelton RC, Hewlett WA, Sutcliffe JS, Blakely RD (2005) Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase. Proc Natl Acad Sci U S A 102:11545–11550

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  37. Givan A (2011) Flow cytometry: an introduction. In: Hawley TS, Hawley RG (eds) Flow cytometry protocols. Humana Press, Totowa, NJ, pp 1–29

    CrossRef  Google Scholar 

  38. Solis E, Zdravkovic I, Tomlinson ID, Noskov SY, Rosenthal SJ, De Felice LJ (2012) 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter. J Biol Chem 287:8852–8863

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  39. Jørgensen S, Nielsen EØ, Peters D, Dyhring T (2008) Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity. J Neurosci Methods 169:168–176

    PubMed  CrossRef  Google Scholar 

  40. Wilson JN, Ladefoged LK, Babinchak WM, Schiøtt B (2014) Binding-induced fluorescence of serotonin transporter ligands: A spectroscopic and structural study of 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) and APP(+) analogues. ACS Chem Neurosci 5:296–304

    Google Scholar 

  41. Karpowicz RJ, Dunn M, Sulzer D, Sames D (2013) APP+, a fluorescent analogue of the neurotoxin MPP+, is a marker of catecholamine neurons in brain tissue, but not a fluorescent false neurotransmitter. ACS Chem Neurosci 4:858–869

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  42. Ahmed BA, Jeffus BC, Bukhari SIA, Harney JT, Unal R, Lupashin VV, van der Sluijs P, Kilic F (2008) Serotonin transamidates Rab4 and facilitates its binding to the C terminus of serotonin transporter. J Biol Chem 283:9388–9398

    CAS  PubMed  CrossRef  Google Scholar 

  43. Oz M, Isaev D, Lorke DE, Hasan M, Petroianu G, Shippenberg TS (2012) Methylene blue inhibits function of the 5-HT transporter. Br J Pharmacol 166:168–176

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

  44. Oz M, Libby T, Kivell B, Jaligam V, Ramamoorthy S, Shippenberg TS (2010) Real-time, spatially resolved analysis of serotonin transporter activity and regulation using the fluorescent substrate, ASP+. J Neurochem 114:1019–1029

    CAS  PubMed Central  PubMed  Google Scholar 

  45. Watts SW, Morrison SF, Davis RP, Barman SM (2012) Serotonin and blood pressure regulation. Pharmacol Rev 64:359–388

    CAS  PubMed Central  PubMed  CrossRef  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge the guidance of Dr. Elizabeth Breen regarding Ficoll separations. We would also like to acknowledge Drs. Sandra Rosenthal, Ian Tomlinson, and Jerry Chang for the APP+ synthesis and advice during assay development.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Anne M. Andrews .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Beikmann, B.S., Andrews, A.M. (2015). Flow Cytometry to Determine Serotonin Transporter Function in Human Peripheral Blood Cells. In: Blenau, W., Baumann, A. (eds) Serotonin Receptor Technologies. Neuromethods, vol 95. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2187-4_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2187-4_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2186-7

  • Online ISBN: 978-1-4939-2187-4

  • eBook Packages: Springer Protocols