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Comparison of control of pedal sole cilia in the snails Lymnaea stagnalis appressa and Helisoma trivolvis.

Ciliary locomotion initiated by the central nervous system has been reported in the freshwater snail families Lymnaeidae and Planorbidae (1, 2). In this paper I show that a homologous 5-HT-like immunoreactive nerve, the ventromedian pedal nerve (vpn), controls ciliary beating on the pedal sole in both the lymnaeid snail Lymnaea stagnalis appressa Say (1821) and the planorbid snail Helisoma trivolvis Say (1817). In L. s. appressa, the vpn from the pedal ventral commissure has two large axon pairs from the bilaterally symmetric PeV1 and PeD7 pedal ganglia neurons. Electrical coupling of the PeV1 and PeD7 axons in the vpn to their contralateral homologs causes synchronous action potentials in each neuron pair. Initiation of action potentials in PeV1 and PeD7 neurons also occurs in PeV1 and PeD7 axons in the vpn. In L. s. appressa, acceleration of ciliary beating on the pedal sole can occur either by unevoked bursting of the PeV1 axon pair in the vpn or by intracellulary stimulating a PeV1 neuron. In the isolated foot in L. s. appressa and H. trivolvis, stimulating the vpn also accelerates ciliary beating. This simple preparation can be effectively used to study the neural control, of pedal sole ciliary beating.

There are well-known homologies of ganglia and nerves in the gastropod central nervous system (3). On the basis of location and immunoreactivity, individual neurons have also been found to be homologous in gastropod nervous systems (4-10). In this paper, the ventromedian pedal nerve (vpn) in H. trivolvis is shown to be homologous to the vpn in L. s. appressa on the basis of axon distribution, immunoreactivity, and embryonic development.

In both L. s. appressa and H. trivolvis, axons in the vpn form a major part of the 5-HT-like immunoreactive (5-HT-LIR) plexus on the pedal sole. At about 50% of prehatch development, 5-HT-LIR axons in the vpn begin to grow out from the pedal ventral commissure (vc) in both species (Fig. 1A, B). Later in development, the vpn extends to the posterior part of the foot (Fig. 1C, D). At that time, only one pair of 5-HT-LIR axons was seen in the H. trivolvis vpn where it exits the vc (not shown). In embryonic specimens of L. s. appressa, this 5-HT-LIR axon pair in the vpn was from PeD7 neurons. PeD7 axons in the L. s. appressa embryo were intensely immunoreactive to 5-HT antibodies, whereas PeV1 axons had weak immunoreactivity to these antibodies (11). Putative PeV1 axons with weak immunoreactivity, which by homology would also be expected in the H. trivolvis vpn, were not confirmed to be present in the H. trivolvis embryo.

[FIGURE 1 OMITTED]

In Lymnaea, two axon pairs in the vpn come from the bilaterally symmetric PeD7 and PeV1 neuron pairs (11, 12). Activity in the vpn was seen only from these two large axon pairs and two much smaller axons during about 20 h of recording from the L. s. appressa pedal ganglia. Cobalt backfills (n = 5) of the vpn in L. s. appressa also showed the PeD7 and PeV1 neuron pairs and a much smaller neuron (unpubl., obs., R. D. Longley). In the adult H. trivolvis, leakage and damage to the small nerve prevented cobalt backfills of the vpn from successfully showing neurons in the pedal ganglia, but two large pairs of axons were seen in the vpn, which was consistent with results in L. s. appressa (Fig. 1E).

In recordings from the vpn of L. s. appressa specimens, axon spikes from PeV1 and PeD7 neurons were always paired with their contralateral homologs. Electrical coupling of PeD7 axons was weaker than in PeV1 axons, as shown by nerve recordings of the vpn in which PeD7 axon spikes were separated by a few milliseconds but PeV1 spikes were coincident (Fig. 2). This electrical coupling occurred in the vpn, since there was no contact of PeV1 and PeD7 neurons to their contralateral homologs before their axons reached the vpn.

[FIGURE 2 OMITTED]

The action potential initiation zone of the PeV1 and PeD7 neurons is in their axons in the vpn. This is indicated by the negative polarity of their spikes in suction electrode recordings of the vpn (Fig. 2). An action potential originating in the suction pipette causes positive ions to flow into the axon, which initially makes the pipette electrode more negative than the bath reference electrode (13).

When a PeD7 neuron was depolarized with a constant current, the action potential initiation zone initially moved out of the suction pipette toward the nerve cell. This was indicated by the positive spike entering the suction pipette (Fig. 2A, C1). Later in this recording, the action potential initiation zone of both PeD7 axons was in the suction pipette (Fig. 2C2).

The initial PeD7 action potential was delayed 3.4 ms after its nerve spike (Fig. 2C1). This delay reached 7.1 ms with the sixth action potential (Fig. 2C2). The opposite relation was found for the interval between the pair of PeD7 axon spikes in the vpn. Initially their separation was 7.0 ms (Fig. 2C1), but it decreased to 4.7 ms with the sixth action potential (Fig. 2C2). These delays are consistent with the action potential initiation zone being closer to the nerve cell with the initial depolarization of PeD7, while the action potential in the electrically coupled PeD7 contralateral homolog continued to be initiated in the suction pipette.

In the PeD7 intracellular and vpn recording (Fig. 2A), the PeV1 axon pair had coincident spikes (Fig. 2B). The PeV1 spikes could be separated when the nerve cell of a PeV1 neuron was depolarized intracellularly (Fig. 2D). In this instance the action potential initiation zone moved out of the suction pipette toward the depolarized neuron to produce an initially positive waveform followed by the spike from the PeV1 contralateral homolog.

In L. s. appressa, the axon spikes from PeV1 were much larger than PeD7 axon spikes (Fig. 2A, E), which allowed them to be separated for correlation analysis (14). No interaction was seen between the PeV1 and PeD7 axon pairs in the vpn during 5 h of activity recorded from the pedal ganglia.

Axon pairs in the vpn from PeV1 and PeD7 neurons burst independently of a connection to the pedal ganglia. In recordings from the vpn connected to the isolated foot, action potentials that originated in the suction pipette could be seen from both PeV1 and PeD7 neuron pairs (Fig. 3A). During this recording from the vpn, there was a period when only the electrically coupled PeV1 axons were bursting (Fig. 3B). Pinching the foot with forceps, brushing the foot as a tactile stimulus, or applying 1 mmol [1.sup.-1] 5-HT had no effect on the bursting of the PeV1 axons. Bursting activity in PeV1 axons alone caused saltatory movement of carbon grains on the pedal sole in L. s. appressa (Fig. 3C; see also supplemental video 1 available at http://www.biobull.org/supplemental/).

[FIGURE 3 OMITTED]

When a PeV1 neuron was stimulated intracellularly in L. s. appressa, carbon grains were accelerated by ciliary beating over the posterior pedal sole (Fig. 4A). The movement of carbon grains was similar when PeV1 neurons were stimulated with only the vpn connected to the foot or when both the vpn and the inferior pedal nerves were connected to the foot. This result was consistent in 11 experiments.

[FIGURE 4 OMITTED]

In addition to the anatomical similarity of the vpn in L. s. appressa and H. trivolvis, the behavioral effect of accelerating carbon grain movement on the pedal sole was similar in both species when the vpn was stimulated. In eight experiments in L. s. appressa, stimulating the vpn with the foot isolated from the central ganglia produced acceleration of carbon grains (supplemental video 2 available at http://www.biobull.org/supplemental/). A similar acceleration of carbon grains in three experiments was seen on the pedal sole in H. trivolvis when the vpn was stimulated (Fig. 2B). A video of this carbon grain acceleration in H. trivolvis showed that carbon grains could reliably be accelerated by repeated stimulation of the vpn (supplemental video 3 available at http://www.biobull.org/supplemental/).

Mapping of neurons in the adult H. trivolvis pedal ganglia by cobalt backfills and visual observation has shown pedal neurons and neuron groups homologous to those in Lymnaea (15). As shown here, there is a homologous development of the 5-HT-LIR innervation of the pedal sole by the vpn in H. trivolvis and L. s. appressa. The ciliary response from stimulation of the vpn in H. trivolvis was also similar to that seen in L. s. appressa, which suggests that homologous axons from the L. s. appressa neuron pairs in the vpn are also present in the vpn in H. trivolvis, but this has not been confirmed by identifying these neurons.

In L. s. appressa, the PeV1 axons in the vpn accelerated carbon grains on the pedal sole, while PeD7 axons that form the 5-HT-LIR pedal sole plexus were silent. In the adult L s. appressa, the PeV1 axons in the vpn do not show immunore-activity to 5-HT antibodies (unpubl. obs., R. D. Longley). Thus the 5-HT-LIR axons in the pedal plexus do not necessarily increase pedal sole ciliary beating. Although cobalt backfills of the vpn showed four axons in the adult H. trivolvis (Fig. 1E), immunoreactivity to 5-HT antibodies showed only one pair of 5-HT-LIR axons in the embryo. If PeV1 homologs are absent in H. trivolvis, then an increase in pedal sole ciliary beating could be caused by serotonin in this species.

Application of 5-HT to the pedal sole in Lymnaea and Planorbis corneus increases ciliary beating (2, 16; unpubl. obs., R. D. Longley). But since non-serotonergic axons rather than serotonergic axons apparently increase ciliary beating in L. s. appressa, additional research is required to confirm the neurotransmitter and mechanism of coupling to the ciliary cells in both L. s. appressa and H. trivolvis. The presence of action potentials generated in the vpn and the ability to control pedal sole ciliary beating by stimulating the vpn in the isolated foot provides a simple preparation for the study of this problem in lymnaeid and planorbid snails.

Acknowledgments

I thank the director of Friday Harbor Laboratories for use of the facilities during this work, and the Center for Cell Dynamics for use of the confocal microscope.

Literature Cited

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ROGER D. LONGLEY *

Friday Harbor Laboratories, 620 University Road, Friday Harbor, Washington 98250

Received 14 June 2010; accepted 12 September 2010.

* To whom correspondence should be addressed. E-mail: rlongley@rockisland.com

Abbreviation: 5-HT-LIR, 5-HT-like immunoreactive; cc, cerebral commissure; cg, cerebral ganglion; cpc, cerebropedal connective; dc, pedal dorsal commissure; PeD7, dorsal pedal ganglia bilaterally symmetric neurons; PeV1, ventral pedal ganglia bilaterally symmetric neurons; pg, pedal ganglion; vc, pedal ventral commissure; vpn, ventromedian pedal nerve.
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Author:Longley, Roger D.
Publication:The Biological Bulletin
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Geographic Code:1USA
Date:Dec 1, 2010
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