Pedal sole immunoreactive axons in terrestrial pulmonates: Limax, Arion, and Helix.

A century ago histological techniques such as formic acid-gold chloride showed the nerve morphology of the pedal sole in Limax and Helix. There have been no similar descriptions since then of the central nervous system relevant to locomotory pedal waves in the foot of slugs and snails. Topical application of 5-HT affects locomotory waves, but the innervation of the pedal sole with 5-HT axons is not known. Three-dimensional morphology of pedal axons in terrestrial pulmonate embryos is shown herein with modern histological techniques using antibodies and the confocal microscope. In Limax maximus, pedal ganglia are shown with Tritonia pedal peptide (TPep) antibodies. Ladder-like cross bridges in the pedal sole are shown with antibodies to both TPep and 5-HT. In Arion ater, pedal ganglia neurons and their axons that form a plexus in the pedal sole are shown with 5-HT antibodies. In Helix aspersa, 5-HT immunoreactive pedal ganglia neurons and a developing pedal sole axon plexus are seen as in A. ater. Axons in this plexus that grow across the pedal sole can be seen growing into pre-existing nerves. No peripheral 5-HT neurons were identified in these three species. This immunoreactive plexus to 5-HT antibodies in A. ater and H. aspersa spreads over the pedal sole epithelium. Axons immunoreactive to 5-HT antibodies in A. ater and H. aspersa extend the length of the foot, primarily in the rim, so that activity in these axons cannot provide local patterned input to produce locomotory waves, but may provide modulatory input to pedal sole muscles.

Neuronal control of pedal sole cilia in the pond snail Lymnaea stagnalis appressa.

5-HT (serotonin) is a ubiquitous neurotransmitter that produces ciliary beating in gastropods when applied topically, but ciliary beating caused by gastropod serotonergic neurons has been described in only three neuron pairs. We extend these results to the North American Lymnaea stagnalis appressa, which is a different species from the European Lymnaea stagnalis. We describe a non-serotonergic neuron pair, PeV1, which accelerates pedal sole mucociliary transport and a serotonergic neuron pair, PeD7, which slows mucociliary transport. We compare and discuss development and identified neurons in L. s. appressa and in L. stagnalis, which have homologs to L. s. appressa PeD7 and PeV1 neurons. In addition to PeD7 and PeV1 neurons, we test neurons immunoreactive to Tritonia pedal peptide antibodies with negative results for mucociliary transport. In characterizing PeD7 and PeV1 neurons, we find that PeV1 does not excite PeD7. In semi-intact preparations, a strong increase in PeD7 neuron activity occurs during tactile stimulation, but V1 neurons are inhibited during tactile stimulation. Following tactile stimulation, PeV1 neurons show strong activity. This suggests a distinct difference in function of the two neuron pairs, which both have their axons overlying pedal sole ciliary cells. Application of 5-HT to the pedal sole initiates mucociliary transport in 1.4-1.9 s with a time course similar to that seen when stimulating a PeV1 neuron. This result appears to be through a 5-HT(1A)-like receptor on the pedal sole. We describe a possible external source of 5-HT on the pedal sole from 5-HT immunoreactive granules that are released with mucus.

Neurogenesis in the procerebrum of the snail Helix aspersa: a quantitative analysis.

The procerebrum, a specialized structure for olfaction in terrestrial pulmonate molluscs, contains 20,000 to 50,000 small, uniformly sized neurons that increase in number with age. Here I show the likely source of neurons added to the procerebrum of Helix aspersa and that the rate of neuron addition depends on snail weight. After hatching, during the initial exponential growth phase, H. aspersa adds neurons to the procerebral apex by mitosis and from a cerebral tube. In the logistic growth phase beginning 30-40 days post-hatch, neurons also seem to be added to the procerebrum from the peritentacular and olfactory nerves, causing the rate of neuron addition to approximately double; but as in the earlier exponential growth phase, this rate remains a function of snail weight. This neuron addition throughout the life of the snail can be predicted by snail weight. In the two growth phases, the number of neurons in the procerebrum is given by logarithmic functions of snail weight. The results here for H. aspersa provide the basis for experiments to determine the peripheral origin and destination of neuronal precursors that are added to the procerebrum and to determine how neuron addition affects the function of the procerebrum.

Development of the 5-HT-like immunoreactive pedal plexus in the pond snail Lymnaea stagnalis appressa.

In many gastropods, a serotonin-like immunoreactive axon plexus lies over ciliary cells on the pedal sole. The origin and function of axons in this plexus is uncertain. By using serotonin antibodies in the direct-developing embryo of the pond snail, the axons that initially form this plexus were traced from seven large neurons in each pedal ganglion. Soon after metamorphosis begins, the first immunoreactive pedal ganglion neuron sends multiple branched neurites to lie directly over pedal sole ciliary cells. By 70% of the 11 days required for hatching, axons from the seven neuron pairs form a plexus over ciliary cells in the whole sole. The axon from each of the seven neurons is guided to a specific area of the pedal plexus where ciliary cells are developing. Axons from two pairs of these neurons, which form the pedal plexus in the posterior part of the foot, are in the unpaired nerve that comes from the pedal ganglia ventral commissure. It is likely that these two developing neuron pairs are homologs of the two neuron pairs in Lymnaea stagnalis that have axons in this ventral commissure nerve. Identification of these neurons and the other five neuron pairs with axons in the pedal plexus will provide a basis for future studies of the relation between the plexus and pedal ciliary locomotion.