Cellular and Molecular Neurobiology

, Volume 28, Issue 3, pp 331–342 | Cite as

Stress Upregulates TPH1 but not TPH2 mRNA in the Rat Dorsal Raphe Nucleus: Identification of Two TPH2 mRNA Splice Variants

  • Nashat Abumaria
  • Adema Ribic
  • Christoph Anacker
  • Eberhard Fuchs
  • Gabriele Flügge
Original Paper


Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3′-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3′-untranslated regions (UTRs). TPH2b (with a short 3′-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3′-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.


Serotonin Tryptophan hydroxylase Splice variant Stress Affective disorders Pineal gland 



3′-Rapid amplification of cDNA ends


5-hydroxytryptamine or serotonin


Dorsal raphe nucleus


Pineal gland


Room temperature


Tryptophan hydroxylase


Untranslated region



Nashat Abumaria was in part funded by the DFG-Research Center Molecular Physiology of the Brain (CMPB) at the University of Göttingen. Adema Ribic is supported by a European Community fellowship for Neuroscience Early Stage Research Training (NEUREST), Göttingen, and is enrolled in the MSc/PhD Program for Molecular Biology at the University of Göttingen. Christoph Anacker is a fellow and grant recipient of the MSc/PhD Program Neurosciences at the University of Göttingen. We are grateful to Dr. B. Czeh for helpful discussions and to the excellent technical assistance of A. Hoffmann.


  1. Abumaria N, Rygula R, Havemann-Reinecke U, Rüther E, Bodemer W, Roos C, Flügge G (2006) Identification of genes regulated by chronic social stress in the rat dorsal raphe nucleus. Cell Mol Neurobiol 26:145–162PubMedCrossRefGoogle Scholar
  2. Abumaria N, Rygula R, Hiemke C, Fuchs E, Havemann-Reinecke U, Rüther E, Flügge G (2007) Effect of chronic citalopram on serotonin-related and stress-regulated genes in the dorsal raphe nucleus of the rat. Eur Neuropsychopharmacol 17:417–429PubMedCrossRefGoogle Scholar
  3. Arango V, Underwood MD, Mann JJ (2002) Serotonin brain circuits involved in major depression and suicide. Prog Brain Res 136:443–453PubMedGoogle Scholar
  4. Austin MC, O’Donnell SM (1999) Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland. J Neurochem 72:2065–2073PubMedCrossRefGoogle Scholar
  5. Azmitia EC Jr, McEwen BS (1974) Adrenalcortical influence on rat brain tryptophan hydroxylase activity. Brain Res 78:291–302PubMedCrossRefGoogle Scholar
  6. Azmitia EC, McEwen BS (1976) Early response of rat brain tryptophan hydroxylase activity to cycloheximide, puromycin and corticosterone. J Neurochem 27:773–778PubMedCrossRefGoogle Scholar
  7. Bach-Mizrachi H, Underwood MD, Kassir SA, Bakalian MJ, Sibille E, Tamir H, Mann JJ, Arango V (2006) Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide. Neuropsychopharmacol 31:814–824CrossRefGoogle Scholar
  8. Boadle-Biber MC, Corley KC, Graves L, Phan TH, Rosecrans J (1989) Increase in the activity of tryptophan hydroxylase from cortex and midbrain of male Fischer 344 rats in response to acute or repeated sound stress. Brain Res 482:306–316PubMedCrossRefGoogle Scholar
  9. Boularand S, Darmon MC, Mallet J (1995a) The human tryptophan hydroxylase gene. An unusual splicing complexity in the 5′-untranslated region. J Biol Chem 270:3748–3756PubMedCrossRefGoogle Scholar
  10. Boularand S, Darmon MC., Ravassard P, Mallet J (1995b) Characterization of the human tryptophan hydroxylase gene promoter. Transcriptional regulation by cAMP requires a new motif distinct from the cAMP-responsive element. J Biol Chem 270:3757–3764PubMedCrossRefGoogle Scholar
  11. Brown SM, Peet E, Manuck SB, Williamson DE, Dahl RE, Ferrell RE, Hariri AR (2005) A regulatory variant of the human tryptophan hydroxylase-2 gene biases amygdala reactivity. Mol Psychiatry 10: 805, 884–888Google Scholar
  12. Chamas F, Sabban EL (2002) Role of the 5′ untranslated region (UTR) in the tissue-specific regulation of rat tryptophan hydroxylase gene expression by stress. J Neurochem 82:645–654PubMedCrossRefGoogle Scholar
  13. Chamas F, Serova L, Sabban EL (1999) Tryptophan hydroxylase mRNA levels are elevated by repeated immobilization stress in rat raphe nuclei but not in pineal gland. Neurosci Lett 267:157–160PubMedCrossRefGoogle Scholar
  14. Chamas FM, Underwood MD, Arango V, Serova L, Kassir SA, Mann JJ, Sabban EL (2004) Immobilization stress elevates tryptophan hydroxylase mRNA and protein in the rat raphe nuclei. Biol Psychiatry 55:278–283PubMedCrossRefGoogle Scholar
  15. Chaouloff F (2000) Serotonin, stress and corticoids. J Psychopharmacol 14:139–151PubMedCrossRefGoogle Scholar
  16. Chen GL, Novak MA, Hakim S, Xie Z, Miller GM (2006) Tryptophan hydroxylase-2 gene polymorphisms in rhesus monkeys: association with hypothalamic-pituitary-adrenal axis function and in vitro gene expression. Mol Psychiatry 11:914–928PubMedCrossRefGoogle Scholar
  17. Christensen AK, Kahn LE, Bourne CM (1987) Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat 178:1–10PubMedCrossRefGoogle Scholar
  18. Clark JA, Pai LY, Flick RB, Rohrer SP (2005) Differential hormonal regulation of tryptophan hydroxylase-2 mRNA in the murine dorsal raphe nucleus. Biol Psychiatry 57:943–946PubMedCrossRefGoogle Scholar
  19. Clark JA, Flick RB, Pai LY et al (2007) Glucocorticoid modulation of tryptophan hydroxylase-2 protein in raphe nuclei and 5-hydroxytryptophan concentrations in frontal cortex of C57/Bl6 mice. Mol Psychiatry; doi:  10.1038/sj.mp.4002041
  20. Cote F, Thevenot E, Fligny C et al (2003) Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc Natl Acad Sci USA 100:13525–13530PubMedCrossRefGoogle Scholar
  21. de Kloet ER (1997) Why dexamethasone poorly penetrates in brain. Stress 2:13–20PubMedCrossRefGoogle Scholar
  22. de Kloet ER, Reul JM (1987) Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83–105PubMedCrossRefGoogle Scholar
  23. Dumas S, Darmon MC, Delort J, Mallet J (1989) Differential control of tryptophan hydroxylase expression in raphe and in pineal gland: evidence for a role of translation efficiency. J Neurosci Res 24:537–547PubMedCrossRefGoogle Scholar
  24. Dygalo NN, Shishkina GT, Kalinina TS, Yudina AM, Ovchinnikova ES (2006) Effect of repeated treatment with fluoxetine on tryptophan hydroxylase-2 gene expression in the rat brainstem. Pharmacol Biochem Behav 85:220–227PubMedCrossRefGoogle Scholar
  25. Geijer T, Frisch A, Persson ML, et al (2000) Search for association between suicide attempt and serotonergic polymorphisms. Psychiatr Genet 10:19–26PubMedGoogle Scholar
  26. Gizatullin R, Zaboli G, Jonsson EG, Asberg M, Leopardi R (2006) Haplotype analysis reveals tryptophan hydroxylase (TPH) 1 gene variants associated with major depression. Biol Psychiatry 59:295–300PubMedCrossRefGoogle Scholar
  27. Gundlah C, Alves SE, Clark JA, Pai LY, Schaeffer JM, Rohrer SP (2005) Estrogen receptor-beta regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe. Biol Psychiatry 57:938–942PubMedCrossRefGoogle Scholar
  28. Hamon M, Bourgoin S, Artaud F, Nelson D (1981) Regulatory properties of neuronal tryptophan hydroxylase. Adv Exp Med Biol 133:231–251PubMedGoogle Scholar
  29. Huh SO, Park DH, Cho JY, Joh TH, Son JH (1994) A 6.1 kb 5′ upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice. Mol Brain Res 24:145–152PubMedCrossRefGoogle Scholar
  30. Kendler KS, Karkowski LM, Prescott CA (1999) Causal relationship between stressful life events and the onset of major depression. Am J Psychiatry 156:837–841PubMedGoogle Scholar
  31. Lowry CA, Rodda JE, Lightman SL, Ingram CD (2000) Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbocortical serotonergic system. J Neurosci 20:7728–7736PubMedGoogle Scholar
  32. Magarinos AM, McEwen BS (1995) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: comparison of stressors. Neuroscience 69:83–88PubMedCrossRefGoogle Scholar
  33. Mayr B, Montminy M (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2:599–609PubMedCrossRefGoogle Scholar
  34. McEwen BS (1998) Stress, adaptation, and disease. Allostasis and allostatic load. Ann N Y Acad Sci 840:33–44PubMedCrossRefGoogle Scholar
  35. McKinney J, Knappskog PM, Haavik J (2005) Different properties of the central and peripheral forms of human tryptophan hydroxylase. J Neurochem 92:311–320PubMedCrossRefGoogle Scholar
  36. Meyer H, Palchaudhuri M, Scheinin M, Flügge G (2000) Regulation of alpha(2A)-adrenoceptor expression by chronic stress in neurons of the brain stem. Brain Res 880:147–158PubMedCrossRefGoogle Scholar
  37. Nakamura K, Sugawara Y, Sawabe K, et al (2006) Late developmental stage-specific role of tryptophan hydroxylase 1 in brain serotonin levels. J Neurosci 26:530–534PubMedCrossRefGoogle Scholar
  38. Nielsen DA, Virkkunen M, Lappalainen J, Eggert M, Brown GL, Long JC, Goldman D, Linnoila M (1998) A tryptophan hydroxylase gene marker for suicidality and alcoholism. Arch Gen Psychiatry 55:593–602PubMedCrossRefGoogle Scholar
  39. Palkovits M (1973) Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res 59:449–450PubMedCrossRefGoogle Scholar
  40. Patel PD, Pontrello C, Burke S (2004) Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55:428–433PubMedCrossRefGoogle Scholar
  41. Peirson SN, Butler JN, Foster RG (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 31:e73PubMedCrossRefGoogle Scholar
  42. Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160PubMedCrossRefGoogle Scholar
  43. Sachs AB, Buratowski S (1997) Common themes in translational and transcriptional regulation. Trends Biochem Sci 22:189–192PubMedCrossRefGoogle Scholar
  44. Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89PubMedCrossRefGoogle Scholar
  45. Singh VB, Corley KC, Phan TH, Boadle-Biber MC (1990) Increases in the activity of tryptophan hydroxylase from rat cortex and midbrain in response to acute or repeated sound stress are blocked by adrenalectomy and restored by dexamethasone treatment. Brain Res 516:66–76PubMedCrossRefGoogle Scholar
  46. Sugden D (2003) Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real-time PCR. J Neurochem 86:1308–1311PubMedCrossRefGoogle Scholar
  47. Tenner K, Walther D, Bader M (2007) Influence of human tryptophan hydroxylase 2 N- and C-terminus on enzymatic activity and oligomerization. J Neurochem 102:1887–1894PubMedCrossRefGoogle Scholar
  48. Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76PubMedCrossRefGoogle Scholar
  49. Wang GA, Coon SL, Kaufman S (1998) Alternative splicing at the 3′-cDNA of human tryptophan hydroxylase. J Neurochem 71:1769–1772PubMedCrossRefGoogle Scholar
  50. Wiklund L, Bjorklund A (1980) Mechanisms of regrowth in the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations. Brain Res 191:109–127CrossRefGoogle Scholar
  51. Zaboli G, Jonsson EG, Gizatullin R, Asberg M, Leopardi R (2006) Tryptophan hydroxylase-1 gene variants associated with schizophrenia. Biol Psychiatry 60:563–569PubMedCrossRefGoogle Scholar
  52. Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG (2004) Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217PubMedCrossRefGoogle Scholar
  53. Zhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, Schwartz DA, Krishnan KR, Caron MG (2005) Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45:11–16PubMedCrossRefGoogle Scholar
  54. Zhou Z, Roy A, Lipsky R, Kuchipudi K, Zhu G, Taubman J, Enoch MA, Virkkunen M, Goldman D (2005) Haplotype-based linkage of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4 populations. Arch Gen Psychiatry 62:1109–1118PubMedCrossRefGoogle Scholar
  55. Zill P, Buttner A, Eisenmenger W, Bondy B, Ackenheil M (2004a) Regional mRNA expression of a second tryptophan hydroxylase isoform in postmortem tissue samples of two human brains. Eur Neuropsychopharmacol 14:282–284PubMedCrossRefGoogle Scholar
  56. Zill P, Buttner A, Eisenmenger W, Möller HJ, Bondy B, Ackenheil M (2004b) Single nucleotide polymorphism and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene in suicide victims. Biol Psychiatry 56:581–586PubMedCrossRefGoogle Scholar
  57. Zill P, Buttner A, Eisenmenger W, Möller HJ, Ackenheil M, Bondy B (2007) Analysis of tryptophan hydroxylase I and II mRNA expression in the human brain: a post-mortem study. J Psychiatr Res 41:168–173PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Nashat Abumaria
    • 1
    • 2
  • Adema Ribic
    • 1
    • 3
  • Christoph Anacker
    • 4
  • Eberhard Fuchs
    • 1
    • 5
    • 6
  • Gabriele Flügge
    • 1
    • 5
  1. 1.Clinical Neurobiology Laboratory, German Primate CenterLeibniz Institute for Primate ResearchGöttingenGermany
  2. 2.Center for Learning and Memory, School of Medicine B.303Tsinghua UniversityBeijingChina
  3. 3.Department of Primate GeneticsGerman Primate CenterGöttingenGermany
  4. 4.International Master’s/PhD Program NeurosciencesUniversity of GöttingenGöttingenGermany
  5. 5.DFG Research Center for the Molecular Physiology of the BrainGöttingenGermany
  6. 6.Department of Neurology, University Medical CenterUniversity of GöttingenGöttingenGermany

Personalised recommendations