Neuroscience Bulletin

, Volume 35, Issue 2, pp 205–215 | Cite as

Quantification of Tyrosine Hydroxylase and ErbB4 in the Locus Coeruleus of Mood Disorder Patients Using a Multispectral Method to Prevent Interference with Immunocytochemical Signals by Neuromelanin

  • Lei Guo
  • Jochem Stormmesand
  • Zheng Fang
  • Qingbin Zhu
  • Rawien Balesar
  • Joop van Heerikhuize
  • Arja Sluiter
  • Dick Swaab
  • Ai-Min BaoEmail author


The locus coeruleus (LC) has been studied in major depressive disorder (MDD) and bipolar disorder (BD). A major problem of immunocytochemical studies in the human LC is interference with the staining of the immunocytochemical end-product by the omnipresent natural brown pigment neuromelanin. Here, we used a multispectral method to untangle the two colors: blue immunocytochemical staining and brown neuromelanin. We found significantly increased tyrosine hydroxylase (TH) in the LC of MDD patients—thus validating the method—but not in BD patients, and we did not find significant changes in the receptor tyrosine-protein kinase ErbB4 in the LC in MDD or BD patients. We observed clear co-localization of ErbB4, TH, and neuromelanin in the LC neurons. The different stress-related molecular changes in the LC may contribute to the different clinical symptoms in MDD and BD.


Major depressive disorder Bipolar disorder Tyrosine hydroxylase ErbB4 Locus coeruleus 



This work was supported by the National Key R&D Program of China (2016YFC1306700) and the National Natural Science Foundation of China (91332102 and 31271130). We thank Mr. Bart Fisser, Hugo McGurran, Ling Shan, and Michiel Kooreman for technical support, Prof. Xiao-Ming Li for insightful comments, and Wilma Verweij for secretarial assistance.


  1. 1.
    Kocsis RN. Book Review: Diagnostic and Statistical Manual of Mental Disorders: fifth edition (DSM-5). Int J Offender Ther Comp Criminol 2013, 57: 1546–1548.CrossRefGoogle Scholar
  2. 2.
    Wang N, Zhang GF, Liu XY, Sun HL, Wang XM, Qiu LL, et al. Downregulation of neuregulin 1-ErbB4 signaling in parvalbumin interneurons in the rat brain may contribute to the antidepressant properties of ketamine. J Mol Neurosci 2014, 54: 211–218.CrossRefGoogle Scholar
  3. 3.
    Zhang K, Zhu Y, Zhu Y, Wu S, Liu H, Zhang W, et al. Molecular, functional, and structural imaging of major depressive disorder. Neurosci Bull 2016, 32: 273–285.CrossRefGoogle Scholar
  4. 4.
    Bao AM, Ruhe HG, Gao SF, Swaab DF. Neurotransmitters and neuropeptides in depression. Handb Clin Neurol 2012, 106: 107–136.CrossRefGoogle Scholar
  5. 5.
    Chandley MJ, Szebeni K, Szebeni A, Crawford J, Stockmeier CA, Turecki G, et al. Gene expression deficits in pontine locus coeruleus astrocytes in men with major depressive disorder. J Psychiatry Neurosci 2013, 38: 276–284.CrossRefGoogle Scholar
  6. 6.
    Zhu MY, Klimek V, Dilley GE, Haycock JW, Stockmeier C, Overholser JC, et al. Elevated levels of tyrosine hydroxylase in the locus coeruleus in major depression. Biol Psychiatry 1999, 46: 1275–1286.CrossRefGoogle Scholar
  7. 7.
    Hercher C, Turecki G, Mechawar N. Through the looking glass: examining neuroanatomical evidence for cellular alterations in major depression. J Psychiatr Res 2009, 43: 947–961.CrossRefGoogle Scholar
  8. 8.
    Sigitova E, Fisar Z, Hroudova J, Cikankova T, Raboch J. Biological hypotheses and biomarkers of bipolar disorder. Psychiatry Clin Neurosci 2017, 71: 77–103.CrossRefGoogle Scholar
  9. 9.
    Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis. J Biol Chem 1964, 239: 2910–2917.Google Scholar
  10. 10.
    Wiste AK, Arango V, Ellis SP, Mann JJ, Underwood MD. Norepinephrine and serotonin imbalance in the locus coeruleus in bipolar disorder. Bipolar Disord 2008, 10: 349–359.CrossRefGoogle Scholar
  11. 11.
    Wang P, Kitayama I, Nomura J. Tyrosine hydroxylase gene expression in the locus coeruleus of depression-model rats and rats exposed to short-and long-term forced walking stress. Life Sci 1998, 62: 2083–2092.CrossRefGoogle Scholar
  12. 12.
    Serova LI, Nankova BB, Feng Z, Hong JS, Hutt M, Sabban EL. Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress. Biol Psychiatry 1999, 45: 853–862.CrossRefGoogle Scholar
  13. 13.
    Sved AF, Cano G, Passerin AM, Rabin BS. The locus coeruleus, Barrington’s nucleus, and neural circuits of stress. Physiol Behav 2002, 77: 737–742.CrossRefGoogle Scholar
  14. 14.
    Baumann B, Danos P, Diekmann S, Krell D, Bielau H, Geretsegger C, et al. Tyrosine hydroxylase immunoreactivity in the locus coeruleus is reduced in depressed non-suicidal patients but normal in depressed suicide patients. Eur Arch Psychiatry Clin Neurosci 1999, 249: 212–219.CrossRefGoogle Scholar
  15. 15.
    Gos T, Krell D, Bielau H, Brisch R, Trubner K, Steiner J, et al. Tyrosine hydroxylase immunoreactivity in the locus coeruleus is elevated in violent suicidal depressive patients. Eur Arch Psychiatry Clin Neurosci 2008, 258: 513–520.CrossRefGoogle Scholar
  16. 16.
    Del Pino I, Garcia-Frigola C, Dehorter N, Brotons-Mas JR, Alvarez-Salvado E, Martinez de Lagran M, et al. Erbb4 deletion from fast-spiking interneurons causes schizophrenia-like phenotypes. Neuron 2013, 79: 1152–1168.CrossRefGoogle Scholar
  17. 17.
    Fisahn A, Neddens J, Yan L, Buonanno A. Neuregulin-1 modulates hippocampal gamma oscillations: implications for schizophrenia. Cereb Cortex 2009, 19: 612–618.CrossRefGoogle Scholar
  18. 18.
    Tan GH, Liu YY, Hu XL, Yin DM, Mei L, Xiong ZQ. Neuregulin 1 represses limbic epileptogenesis through ErbB4 in parvalbumin-expressing interneurons. Nat Neurosci 2011, 15: 258–266.CrossRefGoogle Scholar
  19. 19.
    Woo RS, Lee JH, Yu HN, Song D, Baik TK. Expression of ErbB4 in the apoptotic neurons of Alzheimer’s disease brain. Anat Cell Biol 2010, 43: 332–339.CrossRefGoogle Scholar
  20. 20.
    Dang R, Cai H, Zhang L, Liang D, Lv C, Guo Y, et al. Dysregulation of Neuregulin-1/ErbB signaling in the prefrontal cortex and hippocampus of rats exposed to chronic unpredictable mild stress. Physiol Behav 2016, 154: 145–150.CrossRefGoogle Scholar
  21. 21.
    Gerecke KM, Wyss JM, Karavanova I, Buonanno A, Carroll SL. ErbB transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat central nervous system. J Comp Neurol 2001, 433: 86–100.CrossRefGoogle Scholar
  22. 22.
    Norton N, Moskvina V, Morris DW, Bray NJ, Zammit S, Williams NM, et al. Evidence that interaction between neuregulin 1 and its receptor erbB4 increases susceptibility to schizophrenia. Am J Med Genet B Neuropsychiatr Genet 2006, 141B: 96–101.CrossRefGoogle Scholar
  23. 23.
    Joshi D, Fullerton JM, Weickert CS. Elevated ErbB4 mRNA is related to interneuron deficit in prefrontal cortex in schizophrenia. J Psychiatr Res 2014, 53: 125–132.CrossRefGoogle Scholar
  24. 24.
    Chen P, Chen J, Huang K, Ji W, Wang T, Li T, et al. Analysis of association between common SNPs in ErbB4 and bipolar affective disorder, major depressive disorder and schizophrenia in the Han Chinese population. Prog Neuropsychopharmacol Biol Psychiatry 2012, 36: 17–21.CrossRefGoogle Scholar
  25. 25.
    Goes FS, Rongione M, Chen YC, Karchin R, Elhaik E, Potash JB. Exonic DNA sequencing of ERBB4 in bipolar disorder. PLoS One 2011, 6: e20242.CrossRefGoogle Scholar
  26. 26.
    Cao SX, Zhang Y, Hu XY, Hong B, Sun P, He HY, et al. ErbB4 deletion in noradrenergic neurons in the locus coeruleus induces mania-like behavior via elevated catecholamines. Elife 2018, 7.Google Scholar
  27. 27.
    Zhu M, Klimek V, Haycock JW, Ordway GA. Quantitation of tyrosine hydroxylase protein in the locus coeruleus from postmortem human brain. J Neurosci Methods 2000, 99: 37–44.CrossRefGoogle Scholar
  28. 28.
    van de Nes JA, Kamphorst W, Ravid R, Swaab DF. Comparison of beta-protein/A4 deposits and Alz-50-stained cytoskeletal changes in the hypothalamus and adjoining areas of Alzheimer’s disease patients: amorphic plaques and cytoskeletal changes occur independently. Acta Neuropathol 1998, 96: 129–138.CrossRefGoogle Scholar
  29. 29.
    Hagihara H, Catts VS, Katayama Y, Shoji H, Takagi T, Huang FL, et al. Decreased brain pH as a shared endophenotype of psychiatric disorders. Neuropsychopharmacology 2018, 43: 459–468.CrossRefGoogle Scholar
  30. 30.
    Karolewicz B, Johnson L, Szebeni K, Stockmeier CA, Ordway GA. Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics. J Psychiatr Res 2008, 42: 348–355.CrossRefGoogle Scholar
  31. 31.
    Snow DM, Carman HM, Smith JD, Booze RM, Welch MA, Mactutus CF. Cocaine-induced inhibition of process outgrowth in locus coeruleus neurons: role of gestational exposure period and offspring sex. Int J Dev Neurosci 2004, 22: 297–308.CrossRefGoogle Scholar
  32. 32.
    van der Loos CM. Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging. J Histochem Cytochem 2008, 56: 313–328.CrossRefGoogle Scholar
  33. 33.
    Aumann TD, Raabus M, Tomas D, Prijanto A, Churilov L, Spitzer NC, et al. Differences in number of midbrain dopamine neurons associated with summer and winter photoperiods in humans. PLoS One 2016, 11: e0158847.CrossRefGoogle Scholar
  34. 34.
    Foote SL, Bloom FE, Aston-Jones G. Nucleus locus ceruleus: new evidence of anatomical and physiological specificity. Physiol Rev 1983, 63: 844–914.CrossRefGoogle Scholar
  35. 35.
    Van Bockstaele EJ, Bajic D, Proudfit H, Valentino RJ. Topographic architecture of stress-related pathways targeting the noradrenergic locus coeruleus. Physiol Behav 2001, 73: 273–283.CrossRefGoogle Scholar
  36. 36.
    Fujii S, Asakura M, Kanai S, Tanaka D, Hishinumai T, Nagashima H. Effect of concurrent treatment of SSRI on the tyrosine hydroxylase immunoreactivity in the rat locus coeruleus treated with chronic variable stress. Nihon Shinkei Seishin Yakurigaku Zasshi 2004, 24: 21–27.Google Scholar
  37. 37.
    Ordway G, Szebeni K. Effect of repeated treatment with olanzapine or olanzapine plus fluoxetine on tyrosine hydroxylase in the rat locus coeruleus. Int J Neuropsychopharmacol 2004, 7: 321–327.CrossRefGoogle Scholar
  38. 38.
    Verma V, Rasmussen K, Dawe GS. Effects of short-term and chronic olanzapine treatment on immediate early gene protein and tyrosine hydroxylase immunoreactivity in the rat locus coeruleus and medial prefrontal cortex. Neuroscience 2006, 143: 573–585.CrossRefGoogle Scholar
  39. 39.
    Ferrie L, Young AH, McQuade R. Effect of lithium and lithium withdrawal on potassium-evoked dopamine release and tyrosine hydroxylase expression in the rat. Int J Neuropsychopharmacol 2006, 9: 729–735.CrossRefGoogle Scholar
  40. 40.
    Kurita M. Noradrenaline plays a critical role in the switch to a manic episode and treatment of a depressive episode. Neuropsychiatr Dis Treat 2016, 12: 2373–2380.CrossRefGoogle Scholar
  41. 41.
    Manji HK, Quiroz JA, Payne JL, Singh J, Lopes BP, Viegas JS, et al. The underlying neurobiology of bipolar disorder. World Psychiatry 2003, 2: 136–146.Google Scholar
  42. 42.
    Braak H, Braak E. Neuropathological staging of Alzheimer’s disease-related changes. Acta Neuropathol 1991, 82: 239–259.CrossRefGoogle Scholar

Copyright information

© Shanghai Institutes for Biological Sciences, CAS 2019

Authors and Affiliations

  • Lei Guo
    • 1
    • 2
  • Jochem Stormmesand
    • 2
  • Zheng Fang
    • 1
  • Qingbin Zhu
    • 1
    • 3
  • Rawien Balesar
    • 2
  • Joop van Heerikhuize
    • 2
  • Arja Sluiter
    • 2
  • Dick Swaab
    • 2
  • Ai-Min Bao
    • 1
    Email author
  1. 1.Department of Neurobiology and Department of Neurology of the Second Affiliated Hospital, Institute of Neuroscience, NHC and CAMS Key Laboratory of Medical NeurobiologyZhejiang University School of MedicineHangzhouChina
  2. 2.Netherlands Institute for NeuroscienceInstitute of the Royal Netherlands Academy of Arts and SciencesAmsterdamthe Netherlands
  3. 3.Department of Neurology, Sir Run Run Shaw HospitalZhejiang University School of MedicineHangzhouChina

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