In bivalves neurotransmitters are involved in a variety of behaviors, but their diversity and distribution in the nervous system of these organisms remains somewhat unclear. Here, we first examined immunohistochemically the distributions of neurons containing different neurotransmitters, neuropeptides, and related enzymes, as well as the proliferative status of neurons in the ganglia of the mussel Crenomytilus grayanus. H-Phe-Met-Arg-Phe-NH2 (FMRFamide), choline acetyltransferase (ChAT), γaminobutyric acid (GABA) and tyrosine hydroxylase (TH) were found to be expressed by neurons in all the ganglia, whereas serotonin (5-HT) neurons were found only in the cerebropleural and pedal, but not visceral ganglia. Moreover, incubation of living mussels in the presence of a 5-HT precursor (5-HTP) confirmed the absence of 5-HT-containing neurons from the visceral ganglia, indicating that the "serotonin center" of the visceral nervous system is located in the cerebral ganglia. Furthermore, immunostaining of molecules related to neurotransmission together with α-acetylated tubulin demonstrated that this cytoskeletal protein may be a potential pan-neuronal marker in bivalves. Adult mussel neurons do not proliferate, but a population of proliferating PCNA-LIP cells which do not express any of the neurotransmitters examined, perhaps glia cells, was detected in the ganglia. These novel findings suggest that the nervous system of bivalves contains a broad variety of signal molecules most likely involved in the regulation of different physiological and behavioral processes. In addition, proliferating cells may maintain and renew glial cells and neurons throughout the lives of bivalves.

SUMMARY

NADPH-diaphorase (NADPH-d) is a histochemical marker for nitric oxide synthase (NOS) and is widely used to identify nitric oxide (NO) producing cells in the central nervous system (CNS) of both vertebrates and invertebrates. NADPH-d histochemistry was used to quantitatively characterize putative NO-producing neurons in the CNS of the Gray mussel Crenomytilus grayanus subjected to two kinds of stress, environmental pollution and hypoxia, the latter caused by the mollusk transportation in a small volume of water. Mussels were sampled from one relatively clean (reference) and four polluted sites in Amursky and Ussuriysky Bays (Peter the Great Bay, Sea of Japan) in August, 2003. The number of NADPH-d-positive neurons was estimated and enzyme activity was determined from the optical density of the formazan precipitate in the CNS ganglia at 0, 3, and 72 h after sampling. Just after sampling, NADPH-d-positive neurons were found in the cerebropleural, visceral, and pedal ganglia. The number and staining intensity of NADPH-d-positive neurons were significantly higher in the pedal ganglia than the other two ganglia. There were significant differences in the number of NADPH-d-positive neurons and enzyme activity between the mussels from the reference and heavily polluted stations. The proportion and staining intensity of NADPH-d-positive neurons were maximum in the pedal ganglia of the mussels from the heavily polluted station in Amursky Bay. Transportation of mussels in a limited volume of water for 3h resulted in a significant increase in the proportion and staining intensity of NADPH-d-positive neurons in all ganglia. In mollusks from all stations kept in aerated aquaria for 72 h, both the proportion and staining intensity of NADPH-d-positive neurons did not differ significantly from the initial level. However, the differences in the proportion and staining intensity of NADPH-d-positive neurons between the reference and heavily polluted stations were significant. The present results suggest that NO is involved in mollusk nerve cell adaptation to environmental changes.

The nervous system expresses neuromolecules that play a crucial role in regulating physiological processes. Neuromolecule synthesis can be regulated by oxygen-dependent enzymes. Bivalves are a convenient model for studying air exposure-induced hypoxia. Here, we studied the effects of hypoxia on the expression and dynamics of neurotransmitters, and on neurotransmitter enzyme distribution, in the central nervous system (CNS) of the scallop Azumapecten farreri. We analyzed the expression of the neurotransmitters FMRFamide and serotonin (5-HT) and the choline acetyltransferase (CHAT) and universal NO-synthase (uNOS) enzymes during air exposure-induced hypoxia. We found that, in early-stage hypoxia, total serotonin content decreased in some CNS regions but increased in others. CHAT-lir cell numbers increased in all ganglia after hypoxia; CHAT probably appears de novo in accessory ganglia. Short-term hypoxia caused increased uNOS-lir cell numbers, while long-term exposure led to a reduction in their number. Thus, hypoxia weakly influences the number of FMRFamide-lir neurons in the visceral ganglion and does not affect peptide expression in the pedal ganglion. Ultimately, we found that the localization and level of synthesis of neuromolecules, and the numbers of cells expressing these molecules, vary in the scallop CNS during hypoxia exposure. This indicates their possible involvement in hypoxia resistance mechanisms.

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