Immunocytochemical localization of a putative strychnine-sensitive glycine receptor in Hydra vulgaris

  • Linda A. HufnagelEmail author
  • Paola Pierobon
  • Gabriele Kass-SimonEmail author
Regular Article


Previous biochemical studies have identified strychnine-sensitive glycine receptors in membrane preparations of Hydra vulgaris (Cnidaria: Hydrozoa). Electrophysiological and behavioral evidence has shown that these receptors play a role in modulating pacemaker activity and feeding behavior. Here, we present our genomic analysis that revealed hydra proteins having strong homology with the strychnine-binding region of the human receptor protein, GlyRα1. We further present immunocytochemical evidence for the specific labeling of cell and tissue preparations of hydra by a commercially available polyclonal anti-GlyRα1 antibody, selected through our genomic analysis. Tissue pieces and cell macerates from the upper and lower thirds of the body and ablated tentacles were double-labeled with this antibody and with an antibody specific for α-tubulin, to identify the glycine receptors and microtubules, respectively. Extensive receptor labeling was evident on the membranes, cell bodies and myonemes of endodermal and ectodermal epithelial cells, cell bodies and neurites of nerve cells, cnidocytes and interstitial cells. Labeling of the membranes of epithelial cells frequently corresponded to conspicuous varicosities (presumptive presynaptic sites) in the associated nerve net. Our findings support the idea that glycine receptors form an integral part of the nerve and effector systems that control hydra behavior.


Amino acid transmitters GABA Cnidocytes Nerve net Interstitial cells 



This research is based upon work conducted using the Rhode Island Genomics and Sequencing Center, which was supported in part by National Science Foundation EPSCoR grants. We thank Kathy Su for her help with immunochemical preparations.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

441_2019_3011_Fig18_ESM.png (7.8 mb)
Fig. S1

EMBOSS Needle pair-wise alignment of hydra protein #1 (XP_012560050) and #2 (XP_012560049), using default settings. Proteins #1 and #2 are identical, beginning with amino acid L5 in protein #1. Note that the GY motif necessary for strychnine binding lies far into the sequence of both proteins. Vertical lines = identity,: = strong functional similarity, . = functional similarity (PNG 7980 kb)

441_2019_3011_MOESM1_ESM.tif (6.2 mb)
High Resolution Image (TIF 6383 kb)
441_2019_3011_Fig19_ESM.png (11.5 mb)
Fig. S2

EMBOSS Needle pair-wise alignment of the human GlyRα1 protein (AAH 74980.1) and hydra protein #1, using default settings. Note the many regions of strong homology, including the predicted binding region of the antibody (Q170 to M221 in the hydra protein), containing groups of identical amino acids, together with amino acids of similar functionality. Note the shared presence of the GY sequence (Gly160, Tyr161, in the human protein in literature, and Gly188, Tyr189 in the hydra protein in the figure), requisite in the human protein for strychnine binding, within the beginning of a five-amino acid region of very strong homology (SFGYT in the human protein, overlined). Vertical lines = identity,: = strong functional similarity, . = functional similarity (PNG 11737 kb)

441_2019_3011_MOESM2_ESM.tif (9.6 mb)
High Resolution Image (TIF 9789 kb)
441_2019_3011_Fig20_ESM.png (15.9 mb)
Fig. S3

CLUSTAL Omega multiple sequence alignment of the human GlyRα1 protein (AAH 74980.1) and hydra proteins #1 (XP_012560050), #2 (XP_012560049), #9 (XP_012556838.1) and #18 (XP_012555765.1). Note that all proteins share the GY motif within a large region of strong homology (overlined). * = identity,: = strong functional similarity, . = functional similarity (PNG 16286 kb)

441_2019_3011_MOESM3_ESM.tif (13.7 mb)
High Resolution Image (TIF 14050 kb)


  1. Anctil M (2009) Chemical transmission in the sea anemone Nematostella vectensis: a genomic perspective. Comp Biochem Phys D 4:268–289Google Scholar
  2. Baer K, Waldvogel HJ, Faull RLM, Rees MI (2009) Localization of glycine receptors in the human forebrain, brainstem and cervical spinal cord: an immunohistochemical review. Front Mol Neurosci 2:25CrossRefGoogle Scholar
  3. Betz H, Kuhse J, Schmieden V, Laube B, Kirsch J, Harvey RJ (1999) Structure and functions of inhibitory and excitatory glycine receptors. Ann N Y Acad Sci 868:667–676CrossRefGoogle Scholar
  4. Chapman JA, Kirkness EF, Simakov O, Hampson SE, Mitros T, Weinmaier T, Rattei T, Balasubramanian PG, Borman J, Busam D et al (2010) The dynamic genome of hydra. Nature 464:592–596CrossRefGoogle Scholar
  5. Cleland T (1996) Inhibitory glutamate receptor channels. Mol Neurobiol 13:97–136CrossRefGoogle Scholar
  6. Cockroft VB, Osguthorpe DJ, Barnard EA, Friday AE, Lunt GG (1990) Ligand-gated ion channels. Homology and diversity. Mol Neurobiol 4:129–169CrossRefGoogle Scholar
  7. Concas A, Imperatore R, Santoru F, Locci A, Porcu P, Cristino L, Pierobon P (2016) Immunochemical localization of GABAA receptor subunits in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa). Neurochem Res 41:2914–2922CrossRefGoogle Scholar
  8. Corringer P-J, Poitevin F, Prevost MS, Sauguet L, Delarue M, Chanjeux J-P (2012) Structure and pharmacology of pentameric receptor channels: from bacteria to brain. Structure 20:941–956CrossRefGoogle Scholar
  9. Curtis DA, Game CJ, Lodge D, McCulloch RM (1976) A pharmacological study of Renshaw cell inhibition. J Physiol 258:227–242CrossRefGoogle Scholar
  10. Danysz W, Parsons CG (1998) Glycine and N-methyl-D-aspartate receptors: physiological significance and possible therapeutic applications. Pharmacol Rev 50:597–664Google Scholar
  11. David CN (1973) A quantitative method for maceration of hydra tissue. Roux Arch Dev Biol 171:259–268CrossRefGoogle Scholar
  12. Dunne JF, Javois LC, Huang LW, Bode HR (1985) A subset of cells in the nerve net of Hydra oligactis defined by a monoclonal antibody: its arrangement and development. Dev Biol 109:41–53CrossRefGoogle Scholar
  13. Dutertre S, Becker CM, Betz H (2012) Inhibitory glycine receptors: an update. J Biol Chem 287:40216–40223CrossRefGoogle Scholar
  14. Hadzi J (1909) Ueber das Nervensystem von Hydra. Arb Zool Inst Univ Wien 17:225–268Google Scholar
  15. Huettner JE (1989) Indole-2-carboxylic acid: a competitive agonist of potentiation by glycine at the NMDA receptor. Science 243:1611–1613CrossRefGoogle Scholar
  16. Hufnagel LA, Kass-Simon G, Lyon MK (1985) Functional organization of battery cell complexes in tentacles of Hydra attenuata. J Morphol 184:323–341CrossRefGoogle Scholar
  17. Kass-Simon G, Passano LM (1978) A neuropharmacological analysis of the pacemakers and conducting tissues of Hydra attenuata. J Comp Physiol A 128:71–79CrossRefGoogle Scholar
  18. Kass-Simon G, Scappaticci AA (2004) Glutamatergic and GABAnergic control in the tentacle effector systems of Hydra vulgaris. Hydrobiologia 530/531:67–71CrossRefGoogle Scholar
  19. Kass-Simon G, Pannaccione A, Pierobon P (2003) GABA and glutamate receptors are involved in modulating pacemaker activity in hydra. Comp Biochem Phys A 1136:329–342CrossRefGoogle Scholar
  20. Kass-Simon G, Zompa MA, Scappaticci AA, Zackroff RV, Hufnagel LA (2009) Nucleolar binding of an anti-NMDA receptor antibody in hydra: a non-canonical role for an NMDA receptor protein? J Exp Zool 311A:763–775CrossRefGoogle Scholar
  21. Kay JC, Kass-Simon G (2009) Glutamatergic transmission in hydra: NMDA/D-serine affects the electrical activity of the body and tentacles of Hydra vulgaris. Biol Bull 216:113–125CrossRefGoogle Scholar
  22. Kleckner NW, Dingledine R (1988) Requirement for glycine in activation of NMDA receptors expressed in Xenopus oocytes. Science 214:835–837CrossRefGoogle Scholar
  23. Langosch D, Becker CM, Betz H (1990) The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system. Eur J Biochem 194:1–8CrossRefGoogle Scholar
  24. Laughton DL, Amar M, Thomas P, Towner P, Harris P, Lunt GG, Wolstenholme AJ (1994) Cloning of a putative inhibitory amino acid receptor subunit from the parasitic nematode Haemonchus contortus. Recept Channels 2:155–163Google Scholar
  25. Laughton DL, Wheele SV, Lunt GG, Wolstenholme AJ (1995) The beta-subunit of Caenorhabditis elegans avermectin receptor responds to glycine and is encoded by chromosome 1. J Neurochem 64:2354–2357CrossRefGoogle Scholar
  26. Lauro BM, Kass-Simon G (2018) Hydra’s feeding response: effect of GABAB ligands on GSH-induced electrical activity in the hypostome of H. vulgaris. Comp Biochem Phys A 225:83–93CrossRefGoogle Scholar
  27. Liebeskind BJ, Hillis DM, Zakon HH (2015) Convergence of ion channel genome content in early animal evolution. Proc Natl Acad Sci U S A 112:E846–E851CrossRefGoogle Scholar
  28. Lynch JW (2004) Molecular structure and function of the glycine receptor chloride channel. Physiol Rev 84:1061–1095Google Scholar
  29. Mothet JP, Parent AT, Wolosker H, Brady RO, Linden DJ, Ferris CD, Rogawski MA, Snyder SH (2000) D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci U S A 97:4926–4931CrossRefGoogle Scholar
  30. Muscatine L, Lenhoff HM (1965) Symbiosis of hydra and algae I. Effects of some environmental cations on growth of symbiotic and aposymbiotic hydra. Biol Bull 128:415–424CrossRefGoogle Scholar
  31. Nishida T, Yoshimura R, Endo Y (2017) Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscopy tomography. Cell Tissue Res 396:467–476CrossRefGoogle Scholar
  32. Ortells MO, Lunt GG (1995) Evolutionary history of the ligand-gated ion channel superfamily of receptors. Trends Neurosci 18:121–127CrossRefGoogle Scholar
  33. Passano LM, McCullough CB (1965) Co-ordinating systems and behaviour in Hydra II. The rhythmic potential system. J Exp Biol 42:205–231Google Scholar
  34. Pierobon P (2015) Regional modulation of the response to glutathione in Hydra vulgaris. J Exp Biol 218:2226–2232CrossRefGoogle Scholar
  35. Pierobon P, Concas A, Santoro G, Marino G, Minei R, Pannaccione A, Mostallino MC, Biggio G (1995) Biochemical and functional identification of GABA receptors in Hydra vulgaris. Life Sci 56:1484–1497CrossRefGoogle Scholar
  36. Pierobon P, Minei R, Porcu P, Sogliano C, Tino A, Marino G, Biggio G, Concas A (2001) Putative glycine receptors in hydra: a biochemical and behavioural study. Eur J Neurosci 14:1659–1666CrossRefGoogle Scholar
  37. Pierobon P, Sogliano C, Minei R, Tino A, Porcu P, Marino G, Tortiglione C, Concas A (2004) Putative NMDA receptors in hydra: a biochemical and functional study. Eur J Neurosci 20:2598–2604CrossRefGoogle Scholar
  38. Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, Salamov A, Terry A, Shapiro H, Lindquis E, Kapitonov VV, Jurka J, Genikhovic G, Grigoriev IV, Lucas SM, Steele RE, Finnerty JR, Technau U, Martindale MQ, Rokhsar DS (2007) Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317:86–94CrossRefGoogle Scholar
  39. Ruggieri RD, Pierobon P, Kass-Simon G (2004) Pacemaker activity in hydra is modulated by glycine receptor ligands. Comp Biochem Phys A 138:193–202CrossRefGoogle Scholar
  40. Salvador-Martinez I, Salazar-Ciudad I (2015) How complexity increases in development: an analysis of the spatial–temporal dynamics of 1218 genes in Drosophila melanogaster. Dev Biol 405:328–339CrossRefGoogle Scholar
  41. Scappaticci AA, Kass-Simon G (2008) NMDA and GABAB receptors are involved in controlling nematocyst discharge in hydra. Comp Biochem Phys A 150:415–422CrossRefGoogle Scholar
  42. Scappaticci AA, Carroll JR, Hufnagel LA, Kass-Simon G (2004) Immunocytochemical evidence for an NMDAR1 receptor subunit in dissociated cells of Hydra vulgaris. Cell Tissue Res 316:263–270CrossRefGoogle Scholar
  43. Tasneem A, Iyer LM, Jakobsson E, Aravind L (2004) Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels. Genome Biol 6:R4CrossRefGoogle Scholar
  44. Vandenberg RJ, Handford CA, Schofield PR (1992) Distinct agonist- and antagonist-binding sites on the glycine receptor. Neuron 9:491–496CrossRefGoogle Scholar
  45. Young AB, Snyder SH (1973) Strychnine binding associated with glycine receptors of the central nervous system. Proc Natl Acad Sci U S A 70:2832–2836CrossRefGoogle Scholar
  46. Young AB, Snyder SH (1974) Strychnine binding in rat spinal cord membranes associated with the synaptic glycine receptor: cooperativity of glycine interactions. Mol Pharmacol 10:790–809Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cell and Molecular Biology & Interdisciplinary Neurosciences ProgramUniversity of Rhode IslandKingstonUSA
  2. 2.Institute of Applied Sciences and Intelligent Systems E. Caianiello, CNRNaplesItaly
  3. 3.Department of Biological Sciences & Interdisciplinary Neurosciences ProgramUniversity of Rhode IslandKingstonUSA

Personalised recommendations