Nitric oxide generation around buccal ganglia accompanying feeding behavior in the pond snail, Lymnaea stagnalis.

Although there are many lines of evidence for both the presence of nitric oxide synthase (NOS) in the central nervous system (CNS) and the effects of NO on activating and modulating the feeding circuit in Lymnaea stagnalis, there has been no direct evidence that NO generation in the CNS accompanies feeding behavior. In the present study, we used a NO specific electrode to measure the increase in NO concentration around the buccal ganglia when the lips of semi-intact preparations of L. stagnalis were stimulated by sucrose. The NO concentration of the buccal ganglia was significantly increased by an application of sucrose to the lips. A NO scavenger and a NOS inhibitor suppressed this increase in NO concentration. A pair of putative NO-generative neurons in the buccal ganglia, the B2 cells, are active during the inter-feeding phase, and the bursting of the B2 cell elicited by sucrose application starts simultaneously with the feeding response. The rhythmic pulses of NO generation corresponded well with the rhythmic bursting of the B2 cells, which itself corresponds to the 'fictive feeding response'. The present data provide the first direct evidence that NO is generated in the buccal ganglia of L. stagnalis and is involved in a specific behavior such as feeding.

Development of key neurons for learning stimulates learning ability in Lymnaea stagnalis.

The pond snails, Lymnaea stagnalis, change their ability of conditioned taste aversion (CTA) during their development, for example, stage 29 embryos can acquire the CTA, whereas immature snails come to use a long-term memory to maintain the conditioned response. We thus examined the relationships between the learning ability and the development of key neurons (cerebral giant cells: CGCs) for this CTA. The immunoreactivity of serotonin, which is a main neurotransmitter employed in the feeding circuitry, was first observed in the CGCs at the stage 29. After hatching, the CGCs developed their neuropile faster than other cells in the buccal and cerebral ganglia, resulting in their early innervation at the immature stage. The present results, therefore, indicate that the development of key neurons for learning stimulates the developmental changes in learning ability.

Histochemical study on the relation between NO-generative neurons and central circuitry for feeding in the pond snail, Lymnaea stagnalis.

To examine whether nitric oxide (NO)-generative neurons are included in the central circuitry for generation of feeding pattern in the pond snail, Lymnaea stagnalis, two staining techniques for NADPH diaphorase and serotonin (5-HT) were applied for its central nervous system (CNS). The former technique is known to show localization of NO synthase; the latter is well employed as a marker for the feeding circuitry because 5-HT is a main transmitter in it. In the buccal ganglion, B2 motoneuron was found to be a putative NO-generative neuron. This motoneuron is not involved directly in the coordination of feeding pattern but is activated simultaneously with the feeding to control the oesophageal and gut tissues for the digestion. Taking account of the diffusion effects of NO, the NO released from B2 motoneuron, when the feeding is started, is thought to sufficiently modulate the feeding circuitry. In the cerebral ganglion, the superior lip nerve, the median lip nerve and the tentacle nerve included both putative NO-generative fibers and serotonergic fibers. These fibers are not identical, but the NO released in the nerves may activate the serotonergic fibers, resulting in the influence upon the initiation of the feeding. Therefore, our present findings clearly showed that NO is not involved in transmission within the central circuitry for the feeding, but suggested that NO can crucially affect the feeding behavior, such as initiation and modulation of the feeding pattern.

Sensory preconditioning for feeding response in the pond snail, Lymnaea stagnalis.

We demonstrated a sensory preconditioning in the pond snail, Lymnaea stagnalis. An appetitive sucrose solution (a conditioned stimulus: CS1) and weak vibration (another conditioned stimulus: CS2) were first associated, and then the CS2 and an aversive KCl solution (an unconditioned stimulus: UCS) were done. To build the conditioning, two different training procedures, spaced and massed, were examined. After the both training, the sensory preconditioning was built: significantly fewer feeding response to the CS1 became elicited; slower latency to the first bite to the CS1 was induced. No significant differences on the memory retention between these training procedures were found in the sensory preconditioning.