Immunodetection and Localization of Nitric Oxide Synthase in the Olfactory Center of the Terrestrial Snail, Helix pomatia

Abstract

The procerebrum of stylommatophoran snails produces nitric oxide (NO)-modulated oscillatory local field potentials which are considered the basis of olfactory information processing. Although the function of NO is well characterized in the PC, the identification and distribution of NO synthase (NOS) has not known completely. In the present study, applying a mammalian anti-NOS antibody, a 170 kDa molecular weight NOS-like protein was demonstrated in the procerebrum homogenate of Helix pomatia. NOS-like immunolabeling of the globuli cells, the internal and terminal neuropils displayed an identical distribution compared to that of NADPH-diaphorase reactive material, confirming the specificity of immunohistochemistry. The detailed characteristics of the immunostaining (different intensity of the neural perikarya, a gradual appearance in the terminal neuropil and in the axon bundles of the tentacular nerve, as well as an intense, homogeneous distribution of NOS-like immunoreactivity in the internal neuropil) suggest that NOS is expressed constitutively, maintaining a high level of the enzyme in neuropil areas. NOS accumulation in the internal neuropil suggests that NO plays an important role in delivering olfactory signals extrinsic to the procerebrum, and integrating them with other sensory modalities, respectively. Our results are the first, demonstrating unequivocally the presence of NOS and resolving its differential distribution in the Helix procerebrum.

References

  1. 1.

    Blottner, D., Grozdanovic, Z., Gossrau, R. (1995) Histochemistry of nitric oxide synthase in the nervous system. Histochem. J. 27, 785–811.

    CAS  Article  Google Scholar 

  2. 2.

    Cooke, I. R., Edwards, S. L., Anderson, C. R. (1994) The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa. Cell Tissue Res. 277, 565–572.

    CAS  Article  Google Scholar 

  3. 3.

    Di Cosmo, A., Di Cristo, C., Palumbo, A., d’Ischia, M., Messenger, J. B. (2000) Nitric oxide synthase (NOS) in the brain of the cephalopod Sepia officinalis. J. Comp. Neurol. 428, 411–427.

    Article  Google Scholar 

  4. 4.

    Fujie, S., Aonuma, H., Ito, I., Gelperin, A., Ito, E. (2002) The nitric oxide/cyclic GMP pathway in the olfactory processing system of the terrestrial slug Limax marginatus. Zool. Sci. 19, 15–26.

    CAS  Article  Google Scholar 

  5. 5.

    Fujie, S., Yamamoto, T., Murakami, J., Hatakeyama, D., Shiga, H., Suzuki, N., Ito, E. (2005) Nitric oxide synthase and soluble guanylyl cyclase underlying the modulation of electrical oscillations in a central olfactory organ. J. Neurobiol. 62, 14–30.

    CAS  Article  Google Scholar 

  6. 6.

    Gelperin, A. (1994) Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc. Nature 369, 61–63.

    CAS  Article  Google Scholar 

  7. 7.

    Gelperin, A., Kao, J. P. Y., Cooke, I. R. C. (2001) Gaseous oxides and olfactory computation. Amer. Zool. 41, 332–345.

    CAS  Google Scholar 

  8. 8.

    Gervais, R., Kleinfeld, D., Delaney, K. R., Gelperin, A. (1996) Central and reflux neuronal responses elicited by odour in a terrestrial mollusk. J. Neurophysiol. 76, 1327–1339.

    CAS  Article  Google Scholar 

  9. 9.

    Huang, S., Kerschbaum, H. H., Engel, E., Hermann, A. (1997) Biochemical characterisation and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia. J. Neurochem. 69, 2516–2528.

    CAS  Article  Google Scholar 

  10. 10.

    Hurst, W. J., Moroz, L. L., Gillette, M. U., Gillette, R. (1999) Nitric oxide synthase immunolabeling in the molluscan CNS and peripheral tissues. Biochem. Biophys. Res. Commun. 262, 545–548.

    CAS  Article  Google Scholar 

  11. 11.

    Ierusalimsky, V. N., Balaban, P. M. (2010) Two morphological sub-systems within the olfactory organs of a terrestrial snail. Brain Res. 1326, 68–74.

    CAS  Article  Google Scholar 

  12. 12.

    Inoue, T., Watanabe, S., Kawahara, S., Kirino, Y. (2000) Phase-dependent filtering of sensory information in the oscillatory olfactory center of a terrestrial mollusk. J. Neurophysiol. 84, 1112–1115.

    CAS  Article  Google Scholar 

  13. 13.

    Kimura, T., Toda, S., Sekiguchi, T., Kawahara, S., Kirino, Y. (1998) Optical recording analysis of olfactory response of the procerebral lobe int he slug brain. Learn. Mem. 4, 389–400.

    CAS  Article  Google Scholar 

  14. 14.

    Knowles, R. G., Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249–258.

    CAS  Article  Google Scholar 

  15. 15.

    Matsuo, R., Ito, E. (2009) A novel nitric oxide synthase expressed specifically in the olfactory center. Biochem. Biophys. Res. Com. 386, 724–728.

    CAS  Article  Google Scholar 

  16. 16.

    Matsuo, R., Misawa, K., Ito, E. (2008) Genomic structure of nitric oxide synthase in the terrestrial slug is highly conserved. Gene 415, 74–81.

    CAS  Article  Google Scholar 

  17. 17.

    Meulemans, A., Mothet, J. P., Schirar, A., Fossier, P., Tauc, L., Baux, G. (1995) A nitric oxide synthase activity is involved in the modulation of acetylcholine release in Aplysia ganglion neurons: a histological, voltammetric and electrophysiological study. Neuroscience 69, 985–995.

    CAS  Article  Google Scholar 

  18. 18.

    Moroz, L. L., Winlow, W., Turner, R. W., Bulloch, A. G. M., Lukowiak, K., Syed, N. I. (1994) Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea. NeuroReport 5, 1277–1280.

    CAS  Article  Google Scholar 

  19. 19.

    Peruzzi, E., Fontana, G., Sonetti, D. (2004) Presence and role of nitric oxide in the central nervous system of the freshwater snail Planorbarius corneus: possible implication in neuron-microglia communication. Brain Res. 1005, 9–20.

    CAS  Article  Google Scholar 

  20. 20.

    Pisu, M. B., Conforti, E., Fenoglio, C., Necchi, D., Scherini, E., Bernocchi, G. (1999) Nitric oxidecontaining neurons in the nervous ganglia of Helix aspersa during rest and activity: immunocytochemical and enzyme histochemical detection. J. Comp. Neurol. 409, 274–284.

    CAS  Article  Google Scholar 

  21. 21.

    Ratté, S., Chase, R. (2000) Synapse distribution of olfactory interneurons in the procerebrum of the snail Helix aspersa. J. Comp. Neurol. 417, 366–384.

    Article  Google Scholar 

  22. 22.

    Scherer-Singler, U., Vincent, S. R., Kimura, H., McGeer, E. G. (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J. Neurosci. Meth. 9, 229–234.

    CAS  Article  Google Scholar 

  23. 23.

    Shimozono, S., Watanabe, S., Inoue, T., Kirino, Y. (2001) Identification and characterization of an output neuron from the oscillatory molluscan olfactory network. Brain Res. 921, 98–105.

    CAS  Article  Google Scholar 

  24. 24.

    Thavaradhara, K., Leise, E. M. (2001) Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleata. J. Neurocytol. 30, 449–456.

    CAS  Article  Google Scholar 

  25. 25.

    Watanabe, S., Kawahara, S., Kirino, Y. (1998) Morphological characterization of the bursting and nonbursting neurons in the olfactory centre of the terrestrial slug Limax marginatus. J. Exp. Biol. 201, 925–930.

    PubMed  Google Scholar 

  26. 26.

    Watanabe, S., Kirino, Y., Gelperin, A. (2008) Neural and molecular mechanisms of microcognition in Limax. Learn. Mem. 15, 633–642.

    Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Z. Serfőző.

Additional information

Presented during the 12th ISIN Symposium on Invertebrate Neurobiology, August 31–September 4, 2011, Tihany, Hungary.

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Cite this article

Nacsa, K., Elekes, K. & Serfőző, Z. Immunodetection and Localization of Nitric Oxide Synthase in the Olfactory Center of the Terrestrial Snail, Helix pomatia. BIOLOGIA FUTURA 63, 104–112 (2012). https://doi.org/10.1556/ABiol.63.2012.Suppl.2.14

Download citation

Keywords

  • Nitric oxide synthase
  • immunohistochemistry
  • NADPH-diaphorase
  • procerebrum
  • Helix pomatia