Brain Structure and Function

, Volume 218, Issue 2, pp 477–490 | Cite as

Organization of the procerebrum in terrestrial pulmonates (Helix, Limax) reconsidered: cell mass layer synaptology and its serotonergic input system

  • Károly Elekes
  • Izabella Battonyai
  • Suguru Kobayashi
  • Etsuro Ito
Original Article


The synaptology of the cell body layer of the olfactory center, procerebrum, was investigated in two prominent terrestrial pulmonate gastropod species, Helix pomatia and Limax valentianus. In addition, the analysis of the 5-HT-immunoreactive innervation, including ultrastructural level, was performed at high resolution in H. pomatia. A highly complex system of synaptic and non-synaptic connections was found in the procerebrum of both species connected to local neuropil areas of different size. The procerebral (globuli) cell perikarya were richly innervated by varicosities meanwhile the axon profiles also established contacts with each other. Synaptic configurations including convergence, divergence and presynaptic modulation were also revealed. The frequent occurrence of unspecialized but close axo-somatic and axo-axonic membrane contacts referring to the modulatory forms of transmitter release were also accompanied by membrane configurations indicative of active exocytosis. In H. pomatia, the cell mass layer was shown to receive a rich 5-HT-immunoreactive innervation, forming a dense network around the cell bodies. At ultrastructural level, 5-HT-immunoreactive varicosities contacted both cell bodies and different unlabeled axon profiles. Our results suggest that the local neuropil regions in the cell body layer are site of local circuits, which may play a decisive role in olfactory integrative processes bound to the procerebrum. The pattern and form of the 5-HT-immunoreactive innervation of extrinsic origin suggest an overall modulatory role in the cell body layer. The results may serve a basis for considering the role of local intercellular events, connected to microcircuits, within the procerebrum cell body layer involved in oscillation activities.


Procerebrum Axo-somatic contacts Local neuropils 5-HT Ultrastructure Immunocytochemistry Helix Limax Gastropoda Mollusca 



Authors’ thanks are due to the skillful technical assistance of Ms. Zsuzsanna N. Fekete and Mr. Boldizsár Balázs. We are grateful to Dr. Anna Kiss, Dr. József Kiss (both at the Semmelweis University Medical School, Budapest), and Professor László Seress (University of Pécs Medical School, Pécs) providing the possibility to use the Hitachi H-7650 and the JEOL 1200 EXII electron microscopes, respectively. This work was supported by a Grant from the Hungarian Scientific Research Fund (OTKA, No. 78224 to K.E.), and Grants-in-Aid for KAKENHI from the Japan Society for the Promotion of Science (No. 21657022 to E.I., and No. 23570099 to S.K.).


  1. Bailey CH, Thompson EB, Castellucci VF, Kandel ER (1979) Ultrastructure of the synapses of sensory neurons that mediate the gill-withdrawal reflex in Aplysia. J Neurocytol 8:415–444PubMedCrossRefGoogle Scholar
  2. Bullock TH, Horridge GA (1965) Structure and function in the nervous system of invertebrates. Freeman and Co., San FranciscoGoogle Scholar
  3. Chase R (2000) Structure and function in the cerebral ganglion. Microsc Res Tech 49:511–520PubMedCrossRefGoogle Scholar
  4. Chase R (2002) Behavior and neuronal control in gastropod mollusks. Oxford University Press, New YorkGoogle Scholar
  5. Cooke IRC, Gelperin A (1988a) Distribution of FMRFamide-like immunoreactivity in the nervous system of the slug Limax maximus. Cell Tissue Res 253:69–76PubMedGoogle Scholar
  6. Cooke IRC, Gelperin A (1988b) Distribution of GABA-like immunoreactive neurons in the slug Limax maximus. Cell Tissue Res 253:77–81Google Scholar
  7. Elekes K (1978) Ultrastructure of synapses in the central nervous system of lamellibranch molluscs. Acta Biol Hung 29:139–154Google Scholar
  8. Elekes K (1991) Serotonin-immunoreactive varicosities in the cell body layer and neural sheath of the snail, Helix pomatia, ganglia. An electron microscopic immunocytochemical study. Neuroscience 42:583–591PubMedCrossRefGoogle Scholar
  9. Elekes K, Nässel DR (1990) Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia. Cell Tissue Res 262:177–190CrossRefGoogle Scholar
  10. Elekes K, Vehovszky Á, Salánki J (1983) Ultrastructure of synaptic connections of a bimodal pacemaker giant neuron in the central nervous system of Helix pomatia L. Neuroscience 8:617–629PubMedCrossRefGoogle Scholar
  11. Elekes K, S-Rózsa K, Vehovszky Á, Hernádi L, Salánki J (1985) Ultrastructural organization of nerve cells and synaptic connections in the intestinal nerve of the snail Helix pomatia L. Cell Tissue Res 239:611–620PubMedCrossRefGoogle Scholar
  12. Elekes K, Kiss T, Fujisawa Y, Hernádi L, Erdélyi L, Muneoka Y (2000) Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods Lymnaea stagnalis and Helix pomatia: distribution and physiological actions. Cell Tissue Res 302:115–134PubMedCrossRefGoogle Scholar
  13. Gelperin A (1999) Oscillatory dynamics and information processing in olfactory systems. J Exp Biol 202:1855–1864PubMedGoogle Scholar
  14. Gelperin A, Tank DW (1990) Odour-modulated collective network oscillations of olfactory interneurons in a terrestrial mollusc. Nature 345:437–440PubMedCrossRefGoogle Scholar
  15. Gelperin A, Rhines LD, Flores J, Tank DW (1993) Coherent network oscillations by olfactory interneurons: modulation by endogenous amines. J Neurophysiol 69:1930–1939PubMedGoogle Scholar
  16. Glanzman DL (2007) Simple minds: the neurobiology of invertebrate learning and memory. In: North G, Greenspan RJ (eds) Invertebrate neurobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 347–380Google Scholar
  17. Hanström B (1925) Über die sogennanten Intelligenzsphären des Molluskengehirns und die Innervation des Tentakels von Helix. Acta Zool Stockh 6:183–215CrossRefGoogle Scholar
  18. Hernádi L, Terano Y, Muneoka Y, Kiss T (1995) Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia. Cell Tissue Res 280:335–348PubMedCrossRefGoogle Scholar
  19. Ierusamlimsky VN, Balaban PM (2010) Two morphological sub-systems within the olfactory organs of a terrestrial snail. Brain Res 326:68–74CrossRefGoogle Scholar
  20. Inoue T, Watanabe S, Kirino Y (2001) Serotonin and NO complementarily regulate generation of oscillatory activity in the olfactory CNS of a terrestrial mollusk. J Neurophysiol 85:2634–2638PubMedGoogle Scholar
  21. Inoue T, Inokuma Y, Watanabe S, Kirino Y (2004) In vitro study of odor-evoked behavior in a terrestrial mollusk. J Neurophysiol 9:372–381Google Scholar
  22. Kandel ER (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294:1030–1038PubMedCrossRefGoogle Scholar
  23. Kasai Y, Watanabe S, Kirino Y, Matsuo R (2006) The procerebrum is necessary for odor-aversion learning in the terrestrial slug Limax valentianus. Learn Mem 13:482–488PubMedCrossRefGoogle Scholar
  24. Kimura T, Suzuki H, Kono E, Sekiguchi T (1998) Mapping of interneurons that contribute to food aversive conditioning in the slug brain. Learn Mem 4:376–388PubMedCrossRefGoogle Scholar
  25. Kleinfeld D, Delaney KR, Fee MS, Flores JA, Tank DW, Gelperin A (1994) Dynamics of propagating waves in the olfactory network of a terrestrial mollusk: an electrical and optical study. J Neurophysiol 72:1402–1419PubMedGoogle Scholar
  26. Kobayashi S, Hattori M, Ito E (2008) The effects of GABA on the network oscillations of the procerebrum in Limax valentianus. Acta Biol Hung 59:77–79Google Scholar
  27. Kobayashi S, Hattori M, Elekes K, Ito E, Matsuo R (2010) FMRFamide regulates oscillatory activity of the olfactory center of the slug. Eur J Neurosci 32:1180–1192PubMedCrossRefGoogle Scholar
  28. Matsuo R, Ito E (2008) Recovery of learning ability after the ablation of the procerebrum in the terrestrial slug Limax valentianus. Acta Biol Hung 59 (Suppl.): 73–76Google Scholar
  29. Matsuo R, Ito E (2009) A novel nitric oxide synthase expressed specifically in the olfactory center. Biochem Biophys Res Commun 386:724–728PubMedCrossRefGoogle Scholar
  30. Matsuo R, Kobayashi S, Watanabe S, Namiki S, Iinuma S, Sakamoto H, Hirose K, Ito E (2009) Glutamatergic neurotransmission in the procerebrum (olfactory center) of a terrestrial mollusk. J Neurosci Res 87:3011–3023Google Scholar
  31. Matsuo R, Kobayashi S, Murakami J, Ito E (2010) Spontaneous recovery of the injured olfactory center in the terrestrial slug Limax. PLoS One 5:e9054PubMedCrossRefGoogle Scholar
  32. McCarragher G, Chase R (1985) Quantification of ultrastructural symmetry at molluscan chemical synapses. J Neurobiol 16:69–74CrossRefGoogle Scholar
  33. Nikitin ES, Balaban PM (2000) Optica recording of odor-evoked responses in the olfactory brain of the naïve and aversively trained terrestrial snails. Learn Mem 7:422–432PubMedCrossRefGoogle Scholar
  34. Osborne NN, Cottrell GA (1971) Distribution of biogenic amines in the slug Limax maximus. Z Zellforsch 112:15–30PubMedCrossRefGoogle Scholar
  35. Ratté S, Chase R (1997) Morphology of interneurons in the procerebrum of the snail Helix aspersa. J Comp Neurol 384:359–372PubMedCrossRefGoogle Scholar
  36. Ratté S, Chase R (2000) Synapse distribution of olfactory interneurons in the procerebrum of the snail Helix aspersa. J Comp Neurol 417:366–384PubMedCrossRefGoogle Scholar
  37. Roubos EW, Moorer-Van Delft CM (1979) Synaptology of the central nervous system of the fresh-water snail Lymnaea stagnalis (L.), with particular reference to neurosecretion. Cell Tissue Res 198:217–235PubMedCrossRefGoogle Scholar
  38. Samarova E, Balaban P (2009) Changes in frequency of spontaneous oscillations in procerebrum correlate to behavioral choice in terrestrial snails. Front. Cell. Neurosci 3:8Google Scholar
  39. Schürmann F-W, Geffard M, Elekes K (1989) Dopamine-like immunoreactivity in the bee brain. Cell Tissue Res 256:399–410CrossRefGoogle Scholar
  40. Shirahata T, Tsunoda M, Santa T, Kirino Y, Watanabe S (2006) Depletion of serotonin selectively impairs short-term memory without affecting long-term memory in odor learning in the terrestrial slug Limax valentianus. Learn Mem 13:267–270PubMedCrossRefGoogle Scholar
  41. 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 201:925–930PubMedGoogle Scholar
  42. Watanabe S, Inoue T, Kirino Y (2003) Contribution of excitatory chloride conductance in the determination of the direction of traveling waves in an olfactory center. J Neurosci 23:2932–2938PubMedGoogle Scholar
  43. Zaitseva OV (2000) Structural organization of procerebrums of terrestrial mollusks: characteristics of neuronal pattern, plasticity, and age peculiarities. J Evol Biochem Physiol 3:324–333CrossRefGoogle Scholar
  44. Zaitseva OV, Ivanova IP, Luk’yanova EL (2000) Ultrastructure of the area of procerebrum cell bodies in snails and slugs. J Evol Biochem Physiol 36:421–431CrossRefGoogle Scholar
  45. Zs-Nagy I, Sakharov DA (1970) The fine structure of the procerebrum of pulmonate mollusks, Helix and Limax. Tissue Cell 2:399-411Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Károly Elekes
    • 1
  • Izabella Battonyai
    • 1
  • Suguru Kobayashi
    • 2
  • Etsuro Ito
    • 2
  1. 1.Department of Experimental ZoologyBalaton Limnological Institute, Centre for Ecological Research, Hungarian Academy of SciencesTihanyHungary
  2. 2.Laboratory of Functional Biology, Kagawa School of Pharmaceutical SciencesTokushima Bunri UniversitySanukiJapan

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