Modulation of swimming in the gastropod Melibe leonina by nitric oxide
1 Zoology Department and Center for Marine Biology, University of New Hampshire, Durham, NH 03824, USA and
2 Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, USA
*Present address: Department of Biology, Georgia State University, PO Box 4010, Atlanta, GA 30302-4010, USA (e-mail: biojnn{at}langate.gsu.edu)
Accepted 15 November 2001
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Summary |
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Key words: nitric oxide, modulation, Melibe leonina, swimming, central pattern generator, cyclic GMP, gastropod, nudibranch.
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Introduction |
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NO appears to modulate rhythmic activity in various groups of animals. For example, in the crustacean Cancer productus, NO is required for functional division of the stomatogastric ganglion into the pyloric and gastric mill rhythms (Scholz et al., 2001) and also modulates the cardiac ganglion (N. L. Scholz, personal communication). NO also mediates excitatory input to the central pattern generator (CPG) controlling respiration in the amphibian Rana catesbeiana (Hedrick and Morales, 1999
), modulates locomotion in Xenopus laevis larvae (McLean and Sillar, 2000
) and controls the functional state of oscillatory activity in mammalian thalamocortical neurons (Pape and Mager, 1992
). In molluscs, NO is involved in feeding and locomotion in Clione limacina (Moroz et al., 2000
), in the regulation of feeding in Aplysia californica (Lovell et al., 2000
), in food-attraction conditioning in Helix pomatia (Teyke, 1996
), in chemosensory activation of feeding in Lymnaea stagnalis (Moroz et al., 1993
; Elphick et al., 1995
) and in the oscillation of olfactory neurons in the procerebral lobe in Limax maximus (Gelperin, 1994
). Since gastropods express a number of rhythmic behaviors (e.g. feeding, breathing, crawling and swimming) that are controlled by relatively simple CPGs, they offer very suitable model systems for investigating how NO modulates the rhythmic circuits underlying specific behaviors.
We have previously demonstrated the presence of two nitrergic cells in the CNS of the gastropod Melibe leonina, using both immunocytochemistry and NADPH-diaphorase histochemistry (Newcomb and Watson, 2001). These two bilaterally symmetrical cells are located in the cerebropleural ganglia and project into the ipsilateral pedal ganglia. While most of the interneurons involved in swim central pattern generators (sCPGs) in gastropods often reside in the cerebral ganglia, the swim motoneurons (SMNs) are usually situated in the pedal ganglia (Getting et al., 1980
; Arshavsky et al., 1985
; McPherson and Blankenship, 1991
; Gamkrelidze et al., 1995
; Jing and Gillette, 1999
). In Melibe leonina, the pedal ganglia contain two of the four identified sCPG interneurons and all the SMNs (Lawrence, 1997
; Newcomb and Watson, 2000
; Watson et al., 2001
). The fact that the axons of the two NOS-containing cells project into the pedal ganglia suggests that NO might modulate swimming or other forms of locomotion in Melibe leonina.
In Melibe leonina, isolated brains readily express a swim rhythm (hereafter referred to as fictive swimming). It is also possible to record from identified neurons in semi-intact preparations while the animals are exhibiting swimming behavior. These features make it possible to examine the modulation of fictive swimming in isolated brains and to gain insight into the effects of nitrergic neuromodulation on actual swimming behavior. In this paper, we report that NO donors decrease the rate of both fictive swimming and actual swimming and that this decrease can be eliminated in the presence of the NO scavenger reduced oxyhemoglobin. Furthermore, we found that a cGMP analogue mimics the effects of NO, indicating that NO is probably acting via a cGMP-dependent mechanism. These results suggest that NO is used in the CNS of Melibe leonina to modulate swimming.
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Materials and methods |
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Animal collection and maintenance
Adult Melibe leonina (Gould) were collected from subtidal eelgrass beds near the University of Washingtons Friday Harbor Laboratories (FHL) on San Juan Island, WA, USA, by the authors and David Duggins and also from Patricia Bay in Saanich Inlet, British Columbia, Canada, by WestWind SeaLab Supplies (Victoria, British Columbia, Canada). Experiments were carried out at FHL, where Melibe leonina were kept in flow-through seawater tables at ambient temperature (approximately 10°C), and at the University of New Hampshire, where they were maintained in recirculating seawater tanks at 10°C. All Melibe leonina were anesthetized by chilling prior to dissection.
Isolated brains
Brains (N=33) were removed from Melibe leonina by cutting all nerve roots except for the pedalpedal connectives that travel around the ventral surface of the esophagus. Isolated brains were pinned in a Sylgard-lined, 2-ml recording dish and perfused with seawater. Coolant or ambient seawater was circulated through a surrounding aluminum plate to keep the recording chamber at 812°C. Intracellular electrodes, filled with 2 mol l1 potassium acetate (2050 M), were used to record from SMNs in the pedal ganglia. These SMNs were identified by position (center of the dorsal surface of the pedal ganglia) and their characteristic rhythmic bursting activity (Watson et al., 2001
). Intracellular amplifiers (A-M Systems, Inc. Neuroprobe Amplifier, model 1600, and Dagan 8700 Cell Explorer) were used to obtain these recordings, and their output was viewed on an oscilloscope and recorded on an Astro-Med, Inc. Dash IV chart recorder.
Initial experiments were conducted with NO donors [sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP)] to determine the lowest dose that would yield consistent responses. The resulting concentration, 1 mmol l1, was used for all subsequent experiments. NO donors (SNP, N=12 brains; SNAP, N=9 brains) were added to the recording chamber while recording from SMNs. When the effects of the donors on the swim motor program had stabilized, preparations were washed with fresh seawater to determine whether the effects were reversible. Control experiments were also carried out for each NO donor (SNP, N=6 brains; SNAP, N=3 brains) by pre-incubating 200 µl of 0.75 mmol l1 reduced oxyhemoglobin with an equivalent volume of 1 mmol l1 SNP or SNAP for 15 min before applying the NO donor to the preparation. In separate experiments, the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP; N=3 brains) was added at a final concentration of 1 mmol l1 (after previous experiments had indicated that this was the lowest dose that yielded consistent responses).
Semi-intact preparations
Semi-intact preparations (N=3) were used to determine the behavioral correlates of fictive swimming both before and after the addition of NO donors. These preparations were carried out using a technique similar to that used by Willows to study swimming in Tritonia (Willows, 1967; Willows et al., 1973
). Animals were suspended in a 3-l chamber by hooks and threads attached to their dorsal integument, and the chamber was continuously perfused with natural seawater. A small opening was made in the skin just over the brain, and the brain was then immobilized by pinning it to a small wax-covered platform. This made it possible to obtain intracellular recordings from neurons in the brain while also allowing the animals to swim.
SNP (1 mmol l1) was used in the semi-intact preparations in the same manner as in the isolated brains to determine the effects of NO donors on swimming. In addition to recording the bursting activity of the SMNs, the movements of each animal were simultaneously recorded with Hi-8 videography or a photonic sensor. The period of the actual swim rhythm and the rhythm of the SMNs were calculated by averaging 10 swim cycles before, during and after the addition of SNP. A swim cycle was designated as the time from the initiation of one burst to the beginning of the next burst. All results are presented as means ± S.E.M.
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Results |
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Discussion |
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The concentrations of both NO donors (1 mmol l1) were comparable with those used in studies with other gastropods. Elphick et al. (1995) found that 1 mmol l1 SNAP stimulated chemosensory activation of feeding in Lymnaea stagnalis, and 1 mmol l1 SNP and SNAP were used by Huang et al. (1998
) to increase cGMP levels in target cells in neural ganglia of Helix pomatia. According to Ichimori et al. (1994
), the estimated concentration of NO in 1 mmol l1 SNAP is approximately 1 µmol l1, as measured by an NO-sensitive electrode. Moroz et al. (2000
), in a study examining the effects of NO on feeding and locomotor circuitry in the pteropod mollusc Clione limacina, measured the amount of NO released by another NO donor, diethylamine NO complex sodium salt, at 0.050.2 mmol l1. They found that threshold concentrations of NO proximal to neurons were 0.31 µmol l1. NO-sensitive electrodes have also been used to measure endogenous production of NO in the brain of two other gastropods, where concentrations were found to be 0.010.05 µmol l1 in the buccal ganglia of Lymnaea stagnalis and 0.030.1 µmol l1 in the protocerebrum of Limax limacina (Moroz et al., 1995
). All these studies indicate that millimolar concentrations of NO donors tend to release micromolar concentrations of NO at target sites and that these micromolar concentrations appear to be biologically relevant to the endogenous NO production from gastropod neurons.
In pharmacological studies with NO donors, it is important to be sure that the results are actually due to NO and not to other chemical properties or to breakdown products of the donor compounds. For example, SNP contains a ferricyanide group, which can interact with cellular components (Schuman and Madison, 1994). The heme group of reduced hemoglobin readily binds to paramagnetic molecules, such as oxygen, carbon monoxide and NO, and has been demonstrated to readily scavenge NO (Miki et al., 1977
; Knowles et al., 1989
). Hemoglobin has also been used in similar gastropod preparations to control for the effects of NO (Elphick et al., 1995
; Jacklet, 1995
). In the present study, reduced oxyhemoglobin eliminated the effects of NO donors in isolated brains (Figs 2B, 3B), confirming that the changes in the swim rhythm elicited by SNP and SNAP were the result of NO and not any breakdown products of these compounds.
One of the distinctive features of our results was the time delay between the application of an NO donor and the resulting effects. This delay was 1.53 min in isolated brains and 38 min in semi-intact preparations. Some of this delay may have been due to the time required for diffusion of the NO donor through the connective tissue surrounding the brain. However, some of the delay may also be due to the mechanism of action of NO. Modulatory neurons often produce slow-acting, dramatic effects that outlast the duration of modulatory neuron activation (McCrohan, 1988). For example, in the stomatogastric ganglion of the lobster Jasus lalandii, brief firing of the cholinergic anterior pyloric modulator (APM) interneuron, located in the esophageal ganglion, produces delayed and long-term alterations in the pyloric rhythm (Dickinson and Nagy, 1983
; Nagy and Dickinson, 1983
). Rather than acting at a single locus in the CPG, APM is thought to modify the properties of all the elements of the network (Nagy and Dickinson, 1983
). Interestingly, this might also be the method of modulation used by a non-specific gaseous messenger such as NO.
It is not uncommon for sensory and modulatory systems to operate via second-messenger systems, such as pathways involving cGMP, cAMP or inositol trisphosphate. The kinetics of these pathways usually dictate a slower-acting time course than direct modification of ion channels. NO usually affects target cells by interacting with cytosolic guanylate cyclase and raising levels of intracellular cGMP (Ignarro, 1990; Southam and Garthwaite, 1993
). However, NO can also act via cGMP-independent mechanisms, such as ADP-ribosylation of cytosolic proteins (Brune and Lapetina, 1989
), and by binding to ironsulfur centers of enzymes involved in the mitochondrial electron transport chain (Granger et al., 1980
), the citric acid cycle (Drapier and Hibbs, 1986
) and DNA synthesis (Nakaki et al., 1990
). In addition, Hatcher et al. (2000
) found that NO modulated cAMP-gated Na+ currents in buccal neurons of Pleurobranchaea californica. In our study, we used a membrane-permeable, non-hydrolyzable cGMP analogue, 8-Br-cGMP, to test for a cGMP-dependent mechanism of NO action. 8-Br-cGMP has been used in experiments with other molluscs, such as Clione limacina (Moroz et al., 2000
) and the pond snail Lymnaea stagnalis (Elphick et al., 1995
), in which it mimicked the effects observed with NO donors. Since our results with 8-Br-cGMP also mimic the effects seen with NO donors, they suggest that NO is also working via a cGMP-dependent mechanism in Melibe leonina. In addition, recent experiments indicate that the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) blocks effects of SNP on the swim motor program (Newcomb et al., 2001
).
Melibe leonina swim spontaneously, as well as when their foot is dislodged from the substratum, or to escape from noxious stimuli (Lawrence, 1997; Watson et al., 2001
). The presence of prey increases feeding activity (Watson and Trimarchi, 1992
; Watson and Chester, 1993
), but also inhibits swimming (J. M. Newcomb and W. H. Watson III, personal observation), suggesting that the feeding CPG or a related component of the CNS may inhibit swimming under these circumstances. Since NO modulates swimming by decreasing the rate and consistency of swim cycles, it is possible that feeding-induced inhibition of swimming may be due to the release of NO. Inhibition between mutually exclusive neural networks is not unusual. For example, in Clione limacina, an interneuron involved in retraction of the parapodial wings (local withdrawal) also inhibits swimming (Huang and Satterlie, 1990
), and in Pleurobranchaea californica, there is evidence suggesting that one of the functions of the A1 neurons of the sCPG, in addition to generating the swim pattern, may be suppression of feeding behavior in response to noxious stimulation (Jing and Gillette, 1995
). A1 spike activity in these animals suppresses fictive feeding in isolated brains as well as causing proboscis retraction in semi-intact animal preparations induced to feed (Jing and Gillette, 1995
). Inhibition between command systems for feeding and escape behavior has also been demonstrated in crayfish (Krasne and Lee, 1988
; Edwards, 1991
). These studies indicate a tendency for neurons involved in mutually exclusive behaviors to inhibit each other at the neural level.
Moroz et al. (2000) have recently found evidence suggesting that NO is used as a neuromodulator in the swimming and feeding networks of Clione limacina. However, in contrast to our results with Melibe leonina, NO seems to play an excitatory role by activating both the swimming and feeding CPGs. This is not surprising when the feeding habits of these animals are considered. Melibe leonina remain stationary when they feed, whereas Clione limacina feed in the water column, and swimming is therefore stimulated by the presence of prey. In fact, an essential component of feeding behavior in Clione limacina is acceleration of swimming (Norekian, 1995
). Melibe leonina and Clione limacina are the first two gastropods in which NO has been implicated as a modulator of locomotion, and these represent yet another example of modulation of a rhythmic behavior by NO.
In summary, SNP and SNAP decrease the rate of fictive swimming in isolated brains and actual swimming movements in semi-intact preparations. Experiments with 8-Br-cGMP yield results that mimic those seen with NO donors, suggesting that NO is working via a cGMP-dependent mechanism. This evidence suggests that NO is being used to modulate the sCPG of Melibe leonina, possibly to decrease the likelihood that swimming will occur during certain behavioral states such as feeding arousal. Future work involving the nitrergic cerebral neurons themselves and their interactions with the sCPG should further elucidate the role of NO in the CNS of Melibe leonina and shed light on the neuromodulatory functions of this unique gaseous intercellular messenger.
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Acknowledgments |
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