Journal of Fluorescence

, Volume 26, Issue 3, pp 1029–1043 | Cite as

The Fluorescence Methods to Study Neurotransmitters (Biomediators) in Plant Cells



Fluorescence as a parameter for analysis of intracellular binding and localization of neurotransmitters also named biomediators (acetylcholine and biogenic amines such as catecholamines, serotonin, histamine) as well as their receptors in plant cells has been estimated basing on several world publications and own experiments of the author. The subjects of the consideration were 1. application of reagents forming fluorescent products (for catecholamines - glyoxylic acid, for histamine - formaldehyde or ortho-phthalic aldehyde) to show the presence and binding of the compounds in cells, 2. binding of their fluorescent agonists and antagonists with cell, 3. effects of the compounds, their agonists and antagonists on autofluorescence, 4. action of external factors on the accumulation of the compounds in cells. How neurotransmitters can bind to certain cellular compartments has been shown on intact individual cells (vegetative microspores, pollens, secretory cells) and isolated organelles. The staining with reagents on biogenic amines leads to the appearance blue or blue-green emission on the surface and excretions of intact cells as well in some DNA-containing organelles within cells. The difference between autofluorescence and histochemically induced fluorescence may reflect the occurrence and amount of biogenic amines in the cells studied. Ozone and salinity as external factors can regulate the emission of intact cells related to biogenic amines. After the treatment of isolated cellular organelles with glyoxylic acid blue emission with maximum 460–475 nm was seen in nuclei and chloroplasts (in control variants in this spectral region the noticeable emission was absent) and very expressive fluorescence (more than twenty times as compared to control) in the vacuoles. After exposure to ortho-phthalic aldehyde blue emission was more noticeable in nuclei and chloroplasts. Fluorescent agonists (muscarine, 6,7-diOHATN, BODIPY-dopamine or BODIPY-5HT) or antagonists (d-tubocurarine for acetylcholine, yohimbine for dopamine and norepinephrine, inmecarb for serotonin) of neurotransmitters that bound with animal receptors fluorescent in blue (460–480 nm) or blue-green (490–530 nm) and usually are bound with the plasmatic membrane of intact cells or with membrane of the isolated organelles studied. In some model cells autofluorescence (belonging to chlorophyll or not, for example secondary metabolites) may be stimulated by exogenous biogenic amines or their agonists and, on the contrary, be inhibited by certain antagonists. The fluorescence data may be applied for the testing in ecological monitoring, medicine and pharmacology.


Biogenic amines Agonists Antagonists Confocal microscopy Fluorescence microscopy Histochemisty Isolated organelles Microspectrofluorimetry Model systems 



Author thanks Mrs. V.A. Yashin and A. Kuchin for the assistance in the receiving of color images by confocal microscopy.

Supplementary material

10895_2016_1791_MOESM1_ESM.docx (169 kb)
ESM 1 (DOCX 168 kb)
10895_2016_1791_MOESM2_ESM.docx (227 kb)
ESM 2 (DOCX 226 kb)


  1. 1.
    Roshchina VV (2010) Evolutionary considerations of neurotransmitters in microbial, plant and animal cells. In: Lyte M, Freestone PPE (eds) Microbial Endocrinology. Interkingdom Signaling in Infectious Disease and Health. Springer, New York, Berlin, pp. 17–52CrossRefGoogle Scholar
  2. 2.
    Falck B (1962) Observation on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta Physiol Scand 56(suppl 197):5–25Google Scholar
  3. 3.
    Björklund A, Lindvall O, Svensson LA (1972) Mechanisms of fluorophore formation in the histochemical glyoxylic acid method for monoamines. Histochemistry 32(2):113–131CrossRefPubMedGoogle Scholar
  4. 4.
    Cross SAM, Even SWB, Rost FWD (1971) A study of the methods available for the cytochemical localization of histamine by fluorescence induced with o–phthalaldehyde or acetaldehyde. Histochem J 3(6):471–476CrossRefPubMedGoogle Scholar
  5. 5.
    Markova LN, Buznikov GA, Kovačević N, et al. (1985) Histochemical study of biogenic monoamines in early (Prenervous) and late embryos of sea urchins. Int J Dev Neurosci 3(5):493–499CrossRefPubMedGoogle Scholar
  6. 6.
    Bezuglov VV, Gretskaya NM, Esipov SS, et al. (2004) Fluorescent lipophilic analogs of serotonin, dopamine and acetylcholine: synthesis, mass-spectrometry and biological activity. Bioorganic Chemistry (Bioorg Khim, Russia) 30(5):512–519Google Scholar
  7. 7.
    Roshchina VV (2008) Fluorescing world of plant secreting cells. Science Publishers, Enfield, Plymouth, p. 338Google Scholar
  8. 8.
    Roshchina VV, Yashin VA, Kuchin AV (2015) Fluorescence in the study of neurotransmitters in plant cells and their reception. In: Zinchenko VP, Berezhnov AV (eds) Reception and Intracellular Signaling. Proceedings of the International Conference, 25–28 May 2015, Pushchino, Fix-Print, Pushchino, Vol. 1, p 364–369Google Scholar
  9. 9.
    Roshchina VV, Yashin VA, Vikhlyantsev IM (2012) Fluorescence of plant microspores as biosensors. Biochemistry (Moscow), Suppl ser. A: Membrane and Cell Biology 6(1): 105–112 (Biological Membranes 28 (6): 547–556)Google Scholar
  10. 10.
    Roshchina VV (2014) Model systems to study excretory function of higher plants. Heidelberg, Springer, Dordrecht, p. 220Google Scholar
  11. 11.
    Roshchina VV, Melnikova EV, Kovaleva LV, Spiridonov NA (1994) Cholinesterase of pollen grains. Dokl Biol Sci 337:424–427Google Scholar
  12. 12.
    Roshchina VV (2001) Neurotransmitters in plant life. Science PubL, Enfield, Plymouth, p. 283Google Scholar
  13. 13.
    Kimura M (1968) Fluorescence histochemical study on serotonin and catecholamine in some plants. Jap J Pharmac 18:162–168CrossRefGoogle Scholar
  14. 14.
    Kutchan TM, Rush M, Coscia CJ (1986) Subcellular localization of alkaloids and dopamine in different vacuolar compartment of Papaver bracteatum. Plant Physiol 81(1):161–166CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Barwell CJ (1979) The occurrence of histamine in the red alga of Furcellaria lumbricalis Lamour. Bot Mar 22:399–401Google Scholar
  16. 16.
    Barwell CJ (1989) Distribution of histamine in the thallus of Furcellaria lumbricalis. J Appl Phycol 1:341–344CrossRefGoogle Scholar
  17. 17.
    Roshchina VV, Yashin VA, Kuchin AV, Kulakov VI (2014) Fluorescent analysis of catecholamines and histamine in plant single cells. Int J Biochem, Photon 195:344–351Google Scholar
  18. 18.
    Roshchina VV, YashinVA (2014) Neurotransmitters catecholamines and histamine in allelopathy: plant cells as models in fluorescence microscopy. Allelopath J 34 (1):1–16Google Scholar
  19. 19.
    Roshchina VV, Yashin VA, Kuchin AV (2015) Fluorescent analysis for bioindication of ozone on unicellular models. J Fluoresc 25(3):595–601. doi: 10.1007/s10895-015-1540-2 CrossRefPubMedGoogle Scholar
  20. 20.
    Roshchina VV, Roshchina VD (2003) Ozone and plant cell. Kluwer Academic Publishers, Dordrecht, p. 240CrossRefGoogle Scholar
  21. 21.
    Roshchina VV, Yashin VA, Kuchin AV (2015) Microfluorescent analysis for bioindication of ozone on unicellular models. Physics of Wave Phenomena 23 (3): 192–198Google Scholar
  22. 22.
    Guidry G (1999) A method for Counterstaining tissues in Conjunction with the glyoxylic acid Condensation reaction for Detection of biogenic amines. J Histochem Cytochem 47(2):261–264CrossRefPubMedGoogle Scholar
  23. 23.
    Weber RW, Nelson HS (1985) Pollen allergens and their interrelationships. Clin Rev All 3:291–318CrossRefGoogle Scholar
  24. 24.
    Motta AC, Marliere M, Peltre G, et al. (2006) Traffic-related air pollutants induce the release of allergen-containing cytoplasm granules from grass pollen. Int Arch Allergy Immunol 139(2):294–298CrossRefPubMedGoogle Scholar
  25. 25.
    Roshchina VV, Yashin VA., Kuchin AV (2016) Fluorescence of neurotransmitters and their reception in plant cell. Biol Membr 33 (2): 105–112. DOI  10.1007/s10895-015-1540-2
  26. 26.
    Roshchina VV, Bezuglov VV, Markova LN et al (2003). Interaction of living cells with fluorescent derivatives of biogenic amines. Doklady Russian Academy of Sciences 393 (6): 832–835 (Dokl Biochem Biophys 393: 346–349).Google Scholar
  27. 27.
    Roshchina VV, Markova LN, Bezuglov VV et al (2005) Linkage of fluorescent derivatives of neurotransmitters with plant generative cells and animal embryos. In: Zinchenko VP (ed) Reception and Intracellular Signaling. 6–8 June 2005, Pushchino, Biological Center of Russian Academy of Sciences Pushchino p 399–402Google Scholar
  28. 28.
    Roshchina VV (2007) Luminescent cell analysis in allelopathy. In: Roshchina VV, Narwal SS (eds) Cell Diagnostics. Images, Biophysical and Biochemical Processes in Allelopathy. p. 103–115. Science Publisher: Enfield, Jersey (USA), PlymouthGoogle Scholar
  29. 29.
    Roshchina VV (2004) Сellular models to study the allelopathic mechanisms. Allelopath J 13(1):3–16Google Scholar
  30. 30.
    Roshchina VV, Yashina AV, Yashin VA, Gol’tyaev MV (2011) Fluorescence of Biologically active compounds in plant secretory cells. In: Narwal SS, Pavlovic P, John J (eds) Research methods in plant Science, Forestry and Agroforestry, vol 2. Studium Press, Houston, Texas, pp. 3–25Google Scholar
  31. 31.
    Roshchina VV (2012) Vital autofluorescence: application to the study of plant living cells. Int J spectroscopy. 2012-2013. ID 124672. pp. 1–14., doi: 10.1155/2012/124672.
  32. 32.
    Roshchina VV (2005) Allelochemicals as fluorescent markers, dyes and probes. Allelopath J 16(1):31–46Google Scholar
  33. 33.
    Arkhipova LV, Tretyak TM, Ozolin ON (1988) The influence of catecholamines and serotonin on RNA-synthesizing capacity of isolated nuclei and chromatin of brain and rat liver. Biochemistry (USSR) 53:1078–1081Google Scholar
  34. 34.
    Roshchina VV (1990) Biomediators in chloroplasts of higher plants. 4. Reception by photosynthetic membranes. Photosynthetica 24:539–549Google Scholar
  35. 35.
    Roshchina VV, Yashin VA, Yashina AV et al (2009) Microscopy of objects-models for the study of chemosignaling. In: Zinchenko VP, Kolesnikov SS, Berezhnov AV (eds) Reception and Intracellular Signalling, Poceedings of Int. Conference 2–4 June 2009, Pushchino, ONTI of Pushchino Science Center., Pushchino, p 699–703Google Scholar
  36. 36.
    Roshchina VV, Melnikova EV (1998) Chemosensory reactions at the interaction pollen-pistil. Biol Bull 6:678–685Google Scholar
  37. 37.
    Roshchina VV, Melnikova EV (1998) Allelopathy and plant generative cells. Participation of acetylcholine and histamine in a signalling at the interactions of pollen and pistil. Allelopath J 15(2):171–182Google Scholar
  38. 38.
    Roshchina VV, Roshchina VD (1993) The excretory function of higher plants. Springer, Berlin Heidelberg New York. p 334Google Scholar
  39. 39.
    Roshchina VV (2003) Autofluorescence of plant secreting cells as a Biosensor and bioindicator reaction. J Fluoresc 13(5):403–420CrossRefGoogle Scholar
  40. 40.
    Marquardt P, Vogg G (1952) Pharmakologische und chemische Untersuchungen uber Wirkstoffe in Bienenpollen. Arzneim Forsch 21(353):267–271Google Scholar
  41. 41.
    Emmelin N, Feldberg W (1947) The mechanism of the sting of the common nettle (Urtica urens). J Physiol 106:440–455CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Ekici K, Coskun H (2002) Histamine content of some commercial vegetable pickles. Proceedings of ICNP-2002 – Trabzon, Turkey 162–164.Google Scholar
  43. 43.
    Kuklin AI, Conger BV (1995) Catecholamines in plants. J Plant Growth Regul 14:91–97CrossRefGoogle Scholar
  44. 44.
    Kulma A, Szopa J (2007) Catecholamines are active compounds in plant. Plant Sci 172:433–440CrossRefGoogle Scholar
  45. 45.
    Guidotti BB, Gomes BR, Siqueira-Soares RD, Soares AR, Ferrarese-Filho O (2013) The effects of dopamine on root growth and enzyme activity in soybean seedlings. Plant Signal Behav 8:e25477CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Murch SJ, Alan AR, Cao J, Saxena PK (2009) Melatonin and serotonin in flowers and fruits of Datura metel L. J Pineal Res 47:277–283CrossRefPubMedGoogle Scholar
  47. 47.
    Ramakrishna A, Giridhar P, Ravishankar GA (2011) Phytoserotonin. Plant Signal Behav 6(6):800–809CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Pelagio-Flores R, Ortız-Castro R, Mendez-Bravo A, et al. (2011) Serotonin, a tryptophan-derived signal conserved in plants and animals, regulates root system architecture probably acting as a natural auxin inhibitor in Arabidopsis. Plant Cell Physiol 52(3):490–508CrossRefPubMedGoogle Scholar
  49. 49.
    Korobova LN, Beletskii YD, Karnaukhova TB (1988) The experiment of the selection of salt-tolerant forms of sunflower among the selection material based on the content of histamine in seeds. Fiziologiya and Biokhimiya Kulturnikh Rastenii (USSR) (Physiology and Biochemistry of Economic Plants) 20:403–406Google Scholar
  50. 50.
    Roshchina VV, Karnaukhov VN (2010) The fluorescence analysis of the medicinal drugs’ interaction with unicellular biosensors. Pharmacia (Russia) 3:43–46Google Scholar
  51. 51.
    Envionmental health criteria 89.
  52. 52.
    Sarkar B, Banerjee A, Kant Das A, Nag S, et al. (2014) Label-Free dopamine imaging in live rat brain slices. ACS Chem Neurosci 5(5):329–334CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Balaji J, Desai R, Maiti S (2004) Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation. Microsc Res Tech 63(1):67–71CrossRefPubMedGoogle Scholar
  54. 54.
    Kaushalya SK, Desai R, Arumugam S, Ghosh H, Balaji J, Maiti S (2008) Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain. J Neurosci Res 86:3469–3480CrossRefPubMedGoogle Scholar
  55. 55.
    Gubernator NG, Zhang H, Staal RG, Mosharov EV, et al. (2009) Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals. Science 324(5933):1441–1444CrossRefPubMedGoogle Scholar
  56. 56.
    Nikishin D, Milošević I, Gojković M, Rakić L, Bezuglov VV, Shmukler YB (2015) Expression and functional activity of neurotransmitter system components in sea urchins’ early development. Zygote 16(1):79–86Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Russian Academy of SciencesInstitute of Cell BiophysicsPushchinoRussia

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