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1 Department of Biological Sciences, University of Southern Maine, Portland, Maine 04104
2 The Whitney Laboratory of the University of Florida, 9505 Ocean Shore Blvd., St. Augustine, Florida 32086
* To whom correspondence should be addressed. Department of Biological Sciences, University of Southern Maine, PO Box 9300, Portland, ME 04104-9300. E-mail: gainey{at}usm.maine.edu
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Abstract |
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The gills of the venerid clam Mercenaria mercenaria are eulamellibranch and plicate (Kellogg, 1892). That is, the filaments are connected to adjacent filaments via tissue junctions, and the descending and ascending lamellae are connected to each other and thrown into a series of folds (the plicae) by interlamellar septa (Fig. 1). The dorsoventral spaces within the gill, defined by adjacent septa and the intervening plicae, are the water tubes. The plicae exist in two conformations (Fig. 1C): either their contours are smooth—the "primary folds" of Kellogg, or the "major plicae" of Eble (2001)—or smaller depressions appear at the apexes of the plicae, giving rise to "secondary folds" (Kellogg), or "minor plicae" (Eble). We have seen the plicae alternate between these two conformations. Dorsoventral blood vessels lie at the apex of each plica, within each septum, and within each filament (Kellogg, 1892; Eble, 2001). The blood channels of the branchial filaments are connected with the dorsoventral and septal blood vessels by a meshwork of horizontal blood vessels that are actually interlamellar abfrontal extensions of the filaments (see fig. 34 of the venerid Tapes aureus in Ridewood, 1903; fig. 4.20 in Eble, 2001; fig. 1 in Medler and Silverman, 2001). The horizontal meshwork of vessels (collectively called the "subfilamentar tissue"; see Ridewood, 1903) lines the water tubes (Fig. 1D).
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Neural elements occur within the filaments of both filibranch and eulamellibranch gills (e.g., Setna, 1930; Aiello, 1990), but they also occur in the gills of eulamellibranchs (like Mercenaria) in association with the septa, blood vessels, and interfilamentar muscles; structures that, by definition, do not occur in filibranch gills. Indeed, neurons have been reported in association with the longitudinal muscles in a unionid mussel, Ligumia subrostrata (Dietz et al., 1985), in Mercenaria (Gainey et al., 1999a), and in an oyster, Crassostrea virginica (Nelson, 1960). Nerves have also been observed in the interlamellar septa in Solen marginatus and Ensis siliqua (Atkins, 1937), Mercenaria (Gainey et al., 1999a), and Crassostrea (Nelson, 1960, Galtsoff, 1964); and in the water-tube muscles and ostia of Mercenaria (Candelario-Martinez et al., 1993).
An extensive literature indicates that the flow of water through bivalve gills varies continuously within wide limits in response to both physical and biological factors (summarized by Dame, 1996; Jorgensen, 1996; Bayne, 1998). But the lateral cilia in both Mercenaria and Mytilus edulis beat only within a relatively narrow range of frequencies (about 10–25 beats/s) (Aiello, 1960; Catapane, 1983; Gainey et al., 1999a), so the stimulatory and inhibitory motor nerves to the cilia seem to be activating a simple on-off switch. Medler and Silverman (2001) found, in Mercenaria, that the geometry of the water tubes changed, and their diameters decreased, in response to 5-hydroxytryptamine (5HT; serotonin). Such changes would tend to modify flow (Grunbaum et al., 1998), so changes in the tone of the branchial musculature might well be participating in the continuously variable regulation of water flow through the gill.
Although the branchial muscles have the potential to modulate water flow through the gills, and neural elements are clearly present, the pharmacology and neural control of these muscles has received relatively little attention. In brief, acetylcholine (ACh) contracts the gill muscles in both Dreissena polymorpha and Corbicula fluminea (Snow et al., 1995; Medler and Silverman, 1997), whereas 5HT relaxes the gill muscles of Ligumia subrostrata (Gardiner et al., 1991) and contracts those of Mercenaria (Gainey et al., 1998; Medler and Silverman, 2001). In addition, the peptide FMRFamide contracts the gill muscles of Dreissena (Medler and Silverman, 1997). The relationships between the effects of possible neurotransmitters on gill muscles, the distribution of these agents in identifiable neural networks, and the interactions among the elements of the networks are at present unexplored.
We have been using the gill of the quahog Mercenaria mercenaria to study the neural control of branchial water flow. In a previous study, we found that 5HT and dopamine (DA), respectively, switch the activity of the lateral cilia on and off, and that YFAFPRQamide, an SCP-like peptide endogenous to Mercenaria, modulates the effects of DA (Gainey et al., 1999a). Now we report on the pharmacology of the branchial muscles, focusing especially on the actions of 5HT, DA, ACh, and their antagonists. We have also investigated the distribution of the branchial muscles and their innervation by immunoreactive serotonergic and dopaminergic nerves, expecting the findings to be consistent with our pharmacological observations. Preliminary results of this study have been presented to the Society for Integrative and Comparative Biology (Gainey et al., 1998, 2001).
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Materials and Methods |
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Gill preparation and apparatus
Gills were dissected away from the body wall and separated into demibranchs, and the branchial nerves removed (Fig. 1). Muscular contractions were recorded as changes in the length of the anterior-posterior axis of the isolated demibranchs.
Contractions of the branchial muscles were recorded in either of two ways: (1) Isolated demibranchs were suspended in organ baths and attached with thread to isometric force transducers (Grass FT03 and UFI 1030) equipped with springs; the resulting contractions were therefore semi-isotonic. The transducers were interfaced to Biopac DA 100 amplifiers and a Biopac MP100 analog-to-digital converter. (2) Ultratrasonic crystal transceivers (Sonometrics) were tied to the ends of demibranchs with thread. One end of the demibranch was pinned to a piece of rubber band that was glued with rubber cement to the bottom of a plastic petri dish (4.7-cm diameter); the petri dishes were placed on a cooling plate to maintain temperature. Under these conditions, the muscles were unrestrained and contracted against virtually no external load. The isotonic contractions were measured with a digital ultrasonic measurement system (Sonometrics TRX series 8). In both cases, the magnitude of the contractions was measured with AcqKnowledge version 3.5 (Biopac Systems).
All experiments were carried out at 10 °C in aerated artificial seawater (ASW; recipe in Welsh et al., 1968). To retard the oxidation of dopamine (DA), the water was buffered with an ascorbic acid buffer as described by Malanga (1975); this buffered seawater was used in all of the experiments.
Production and analyses of dose-response curves
Our initial experiments were performed with force transducers; but prolonged contraction against the load of the springs used with these devices caused the gill muscles to fatigue. Consequently, we exposed each demibranch only once to a single concentration of agonist, and the dose-response curve was constructed from these individual responses. In later experiments with the Sonometrics digital ultrasound measurement system, no external force was applied to the muscles. No evidence of fatigue was observed, so a single demibranch could be used to construct an entire dose-response curve. Because the response to serotonin (5HT) and DA has a seasonal component (Gainey, pers. obs.), the dose-response data reported here were collected between November and July.
All contractions and relaxations, measured in millimeters, were expressed as a percentage of the initial length of each demibranch. Regression lines were fitted with a logistic function of the form: response = 
/1 + exp(ß0 + ß1* log(agonist)), where is the asymptotic value of the maximal contraction, and ß0 and ß1 are intercept and slope parameters. Initially, all three parameters were estimated using nonlinear regression (Systat, v 9); later, was fixed in the model, reducing the error estimates of the remaining parameters. The concentrations of agonist giving half-maximal responses (EC50) were estimated according to the following formula: EC50 = 10(-ß0/ß1).
Effects of antagonists
Each of the four demibranchs from the same clam were suspended in an organ bath and attached to a force transducer. After 15 min of relaxation, each of the demibranchs was exposed to an agonist at a standard concentration: 5HT = 2 x 10-5 M; DA and acetylcholine (ACh) = 5 x 10-5 M. After the resulting contractions had stabilized, the baths were flushed, and an antagonist at 10-4 M was added to three of the four demibranchs. After 60 min, the standard dose of agonist was reapplied to all four demibranchs, with the antagonist still present on the three demibranchs. The total number of demibranchs treated with a specific antagonist is given in the data tables.
The effect of the antagonist was expressed as the ratio between the second and first agonist-induced contraction (contraction ratio). Analysis of the contraction ratios of untreated controls with a Kolmogorov-Smirnov one-sample test revealed that these data were not normally distributed (P < 0.001, two-tailed, n = 139). The contraction ratios were therefore normalized by a logarithmic transformation, and the normality of this transformation was checked as above (Kolmogorov-Smirnov; P = 0.614). The ln transformed ratios of the controls were tested against a mean of 0 (since ln 1 = 0) with a one-sample t test. This is mathematically equivalent to a paired t test because the contractions used to construct the ratios were from the same demibranch.
Since the contraction ratios of the controls for 5HT, DA, and ACh were all significantly greater than 1, the normalized contraction ratios of the antagonists were compared to the normalized contraction ratios of the appropriate agonist control using post hoc paired Tukey HSD tests after an initial one-way ANOVA. But some of the antagonist contraction ratios were 0, thus these ratios become undefined by a logarithmic transformation. To overcome this limitation, 0.1 was added to all contraction ratios prior to the logarithmic transformation. Although the statistical tests were performed on the ln-transformed data, tabular data are presented in the Results section untransformed for clarity. The P values reported for these tests are one-tailed probabilities; P values less than 0.05 were considered significant. In some of the experiments—e.g., ACh after exposure to cyproheptadine or mianserin—the gills relaxed rather than contracted; these data are coded in the tables as negative values.
The concentration of antagonist that produced 50% inhibition (IC50) was calculated using the experimental design described above, except that the demibranchs were exposed to lower concentrations of antagonists. Contraction ratios—i.e., the ratios of the second to the first contractions—were regressed against the log of the concentration of antagonist. Because the contraction ratios were significantly greater than 1 for all of the controls, the IC50 was calculated by solving the regression equation for a contraction ratio that was 50% of the mean contraction ratio of the control.
Branchial anatomy
For relaxed specimens, isolated demibranchs were kept overnight, at 5 °C, in isotonic MgCl2 in ASW (7.6% MgCl2 in distilled water added to an equal volume of ASW). For contracted specimens, the isolated demibranchs were placed in 10-4 M 5HT immediately after dissection. To observe the inner face of the water tubes, we cut dorsoventrally along several septa with fine scissors, separating a section of the demibranch into two layers. One of these was removed, and the remainder of the demibranch was then pinned to the bottom of a small petri dish, which had been coated with Sylgard. Fixation—always carried out at 5 °C—varied with the object to be observed (e.g., muscle, 5HT, DA) and is described below. Because mammalian antibodies were used for the immunohistochemistry, subsequent rinses and solutions were made with mammalian phosphate-buffered saline (PBS).
Crysostat sections were prepared as follows. After fixation and a 15-min rinse in PBS (0.1 M sodium phosphate, 140 mM NaCl; pH 7.3), the demibranchs were placed in a solution of 30% sucrose/PBS overnight at 5 °C. Pieces of demibranch were then placed in Tissue Tek OCT compound, frozen, and sectioned at 12 µm. Sections were placed on gel-coated slides and stored at -20 °C until used.
Thick sections were prepared as follows. After fixation and three 15-min rinses in PBS, pieces of demibranch were placed in a plastic mold and covered with 12% Type A pigskin gelatin in 0.1 M PBS that had been heated to 50 °C. After the gelatin had cooled, the tissue was sectioned at 100 µm with a vibratome. The sections were heated briefly at 50 °C on gel-coated slides to melt the excess gelatin.
We usually processed whole mounts and sections simultaneously and therefore followed a schedule designed for whole mounts. All of the steps in this protocol were carried out at 5 °C.
Muscle.
The branchial muscles were visualized with phalloidin conjugated to the fluorescent probe Alexa Fluor 488 (Molecular Probes, Eugene, Oregon), the conjugate used in a concentration of 1 unit/100 µl in 0.1 M PBS. For single-stained preparations, whole mounts were fixed for 1 h in 4% formaldehyde with 0.01 M PBS (pH 7.3; 530 mM NaCl), rinsed twice, and then stained overnight. To double-label immunochemically stained preparations, the phalloidin was added to the tissues at the same time as the secondary antibody.
5HT and YFAFPRQamide.
Pieces and sections of demibranch were fixed overnight in 4% paraformaldehyde in 0.01 M PBS (pH 7.3; 530 mM NaCl); the fixative was prepared as described in Gainey et al. (1999a). For 5HT, the primary polyclonal antiserum was raised in rabbit to 5HT conjugated to BSA with paraformaldehdye (Diasorin, Stillwater, Minnesota). For YFAFPRQamide, the primary polyclonal antiserum was raised in rabbit to the peptide conjugated to thyroglobulin (custom synthesis, etc., by SynPep, Dublin, California). In both cases, the secondary antibody was raised in goat to rabbit IgG conjugated to Alexa Fluor 594 (Molecular Probes).
Dopamine.
Pieces and sections of demibranch were fixed for 2 h in 5% glutaraldehyde/1% sodium metabisulfite/PBS (0.01 M; pH 7.3; 530 mM NaCl). The primary polyclonal antiserum was raised in rabbit to DA conjugated to BSA with glutaraldehyde (Diasorin). The secondary antibody was raised in goat to rabbit IgG and conjugated to Alexa Fluor 594 (Molecular Probes). For negative controls, the primary antibodies were omitted from a slide in each series of preparations.
Confocal images of 5HT distribution were made with a Leica LSCM SP2 microscope at the Whitney Laboratory, St. Augustine, Florida. Fluorescent images were made with a Nikon Eclipse TE200 microscope equipped with a Spot RT digital color camera (Diagnostic Instruments). Images were prepared for publication with Adobe Photoshop.
Drugs
All chemicals were purchased from Sigma-Aldrich, St. Louis, Missouri, or ICN Pharmaceuticals, Costa Mesa, California. The specificities of the antagonists listed in the tables were obtained from the Cell Signaling & Neuroscience catalog (2000/2001 ed.) of Sigma/RBI.
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Results |
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Previously, we found that YFAFPRQamide modulates the effects of DA on the lateral cilia and those of 5HT on the frontal cilia (Gainey et al., 1999a). Therefore, to determine whether the peptide would modulate the effects of 5HT or DA on the muscle, we applied YFAFPRQamide to the demibranchs before exposing them to 2 x 10-5 M 5HT or DA. At concentrations ranging from 10-9 to 10-6 M (5HT) or 10-8 to 10-6 M (DA), and exposures ranging from 15 min to 1 h (5HT) or 1 h (DA), the peptide had no effect upon contractions induced by either 5HT or DA.
Antagonists
Because the three effective agonists contract the gill, and since the mechanical responses to 5HT, DA, and ACh are indistinguishable, we asked whether the muscles have receptors for each of the agonists, or whether one or more of the agonists are acting indirectly by stimulating the release of another agonist from motor nerves. To test these possibilities, antagonists were sought for each agonist, and these agents were cross-tested against the other agonists.
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The water-tube muscles form a complex, but regular, lattice-like network associated with the horizontal blood vessels of the subfilamentar tissue (Fig. 6A). Because most of these muscles cross the septa—from one side of a water tube to the other—and appear to be continuous with the network of muscle fibers within the blood vessels, they form a mesh of circular muscles within the inner wall of each water tube. In cross section, at least some of these muscles also run diagonally from the abfrontal face of the gill filaments towards the septa and blood vessels (Fig. 6C).
Finally, comparison of relaxed and contracted gills revealed the following: (1) the distance between adjacent septa decreased; (2) the vertical spacing between the longitudinal muscle bands decreased, as well as the vertical spacing between the water-tube muscles; and (3) the interfilament space decreased (Fig. 6D). As a result of muscle contraction, the outer, frontal, faces of the gills take on a zigzag appearance. In addition, in freshly dissected gills in which the water tube had been cut open, we observed that if the blood vessel was gently pinched, contraction of the water-tube muscles brought the blood vessel towards the center of the water tube. This resulted in the formation of secondary folds (plicae) and decreased the cross-sectional area of the water tube (Fig. 1C). The blood vessel also constricted in response to the mechanical stimulation.
Distribution of 5HT in the gill.
Two distinct networks of immunoreactive serotonergic varicose nerve fibers are observable in the gill. Each gill filament contains two dorsoventral nerves that run under the epithelium near the lateral cilia and parallel to the dorsoventral muscle fibers (Fig. 6B); we observed no cross connections between the two fibers within a filament.
The second, more complex, network of serotonergic fibers is associated with the gill musculature; the unit of this network is a water tube. First, two large bundles of nerves run dorsoventrally within the septa, and another large bundle runs the length of each of the blood vessels. In addition to these large dorsoventral bundles, four finer ones run parallel to the septa, and two run parallel to the large nerve bundle in the blood vessel (Fig. 6E). These fine dorsoventral nerves are all associated with the subfilamentar tissue. Longitudinally disposed nerves run at right angles to and between the septa and the blood vessels in each water tube; these cross connectives are located within the interfilament tissue junctions adjacent and parallel to the longitudinal muscles (Fig. 6B). We also observed what appear to be fine branches of this longitudinal nerve running into the dorsoventral muscles of the filament (see the filament just above the measurement bar in Fig. 6B). Other longitudinal nerves are associated with the water-tube muscles (Fig. 6E). Finally, we observed connections between the dorsoventral nerves within the gill filaments and those associated with the longitudinal muscles. The primary antibody was omitted from the control sections (not shown), which had minimal background fluorescence comparable to the background in Figure 6E.
Distribution of DA in the gill.
Immunoreactive dopamine was found only in the gill filaments and the septa. Within the gill filament epithelium, there are several pairs of dorsoventral cords (Fig. 6F). When viewed from the outer face of the gill, these cords have a granular, rather than a smooth, appearance. The dopaminergic fibers that are confined to the septal epithelium are a pair of bundles connecting the ascending and descending lamellae (Fig. 6F, G). Thus, each septum contains paired cross connectives that are stacked vertically in the gill and spaced at about the same distance as the longitudinal muscles. No dopaminergic fibers are associated with either the longitudinal or water-tube muscles. Moreover, control sections prepared without the primary antibody (not shown) had minimal background fluorescence, comparable to the background in Figure 6F.
Distribution of YFAFPRQamide in the gill.
Immunoreactive YFAFPRQmide was concentrated in the outer half of the gill filament epithelium (Fig. 7). Weak fluorescence was found in the subfilamentar tissue (the abfrontal face of the filaments) and the blood vessel epithelium. The level of fluorescence in the musculature was comparable to background fluorescence in negative controls.
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Innervation and function of the branchial musculature
Our morphological studies indicate that the serotonergic and dopaminergic innervations of the Mercenaria gill are distinct and that the musculature lacks dopaminergic innervation. Furthermore, the motor innervation of the gill muscles and cilia seems to be organized in three divisions: the serotonergic neural network associated with the water-tube muscles; the serotonergic innervation of the longitudinal and dorsoventral muscles; and the innervation of the cilia on the surface of the gill filaments, including serotonergic, dopaminergic, and peptidergic elements. When these findings are considered together with our pharmacological data and behavioral observations, a picture of integrated gill function begins to emerge.
The water-tube muscles and their neural network.
The water-tube muscles occur in the walls of the major dorsoventral blood vessels, the subfilamentar horizontal vessels, and in the septa. When relaxed gills are exposed to 5HT, the plicae narrow, and secondary folds appear (see Fig. 1C; also fig. 4 in Medler and Silverman, 2001). We have also observed that if a dorsoventral blood vessel is gently pinched, the adjacent water-tube muscles contract, bringing the vessel inwards towards the center of the water tube, producing the secondary plical fold. This response decreases the cross-sectional area and modifies the shape of the water tubes, thus reducing the flow of water through the gill. In addition, pinching the blood vessel causes it to constrict locally, suggesting that the water-tube muscles also regulate the diameter of the blood vessels and thus the flow of hemolymph through them.
The longitudinal and dorsoventral muscles and their innervation.
The longitudinal muscles run along the inside of the interfilament tissue junctions, perpendicular to the gill filaments and to the dorsoventral muscles, which lie within the filaments. However, the two sets of muscles are closely apposed, and more important, the dorsoventrals appear to be composed of branches of the longitudinals (Fig. 6B, C). Thus, we speculate that this orthogonal net of muscle acts as a unit. The longitudinal muscle is accompanied by a serontonergic neural plexus, and we have observed serotonergic varicosities among the longitudinal muscle fibers. Moreover, the dorsoventral muscles appear to be innervated by branches of the neural plexus (Fig. 6B).
In response to 5HT, the overall length of the gill decreases, as does the spacing between individual filaments, and thus the diameter of the ostia; this is the action of the longitudinal muscle. In addition, however, the 5HT-treated gill decreases in height in a dose-dependent manner (Gainey, pers. obs.)—the action of the dorsoventral muscles. When gills are exposed to 10-4 M 5HT, the dorsoventral contraction of the gill is very strong and produces a zigzag formation of the filaments on the frontal face of the gill. This phenomenon has also been observed in contracted gills of Corbicula fluminea (Medler and Silverman, 2001, fig. 2).
Innervation of the branchial filaments.
The gill filaments bear the functionally and morphologically distinct tracks of cilia for which the bivalves are well known. Among these effectors, the lateral cilia—which constitute the branchial pump and thus produce the water current—have been studied best. In Mercenaria, the beat of these cilia is stimulated by 5HT and inhibited by DA (Aiello, 1970; Gainey et al., 1999a), results that are consistent with our identification of both serotonergic and dopaminergic fibers within the gill filaments. Furthermore, an identical pattern of structure and function has been demonstrated in a filibranch, the blue mussel Mytilus edulis (reviewed by Aiello, 1990), so we presume that these fibers, in fact, innervate the lateral cilia in Mercenaria. The frontal cilia, which move food particles along the gill, are slowed only weakly by 5HT (Gainey et al., 1999a). But the function of the innervation is not clear, for the frontal cilia continue beating even when the clam is closed (Shirley Baker, University of Florida, pers. comm.).
An endogenous SCP-like peptide, YFAFPRQamide, modulates the effects of DA on the lateral cilia and those of 5HT on the frontal cilia (Gainey et al., 1999a); but this peptide has no effect—direct or indirect—on the branchial musculature. YFAFPRQamide-related immunoreactivity occurs almost exclusively in the region under the epithelium bearing the gill cilia, and in nerves running out to that region (Gainey et al., 1999a). We have certainly not identified all of the transmitters in the innervation of the filaments, but the morphological restriction of YFAFPRQamide to the filaments, and its physiological restriction to effects on cilia, suggests that innervation of the branchial filaments may be exclusively in the service of the cilia, and that the remaining two neural divisions regulate the muscles. These considerations also support our hypothesis that the proposed synapses of dopaminergic and cholinergic neurons onto serotonergic neurons will be found in the septa.
Coordination between the ciliary pump and the branchial muscles.
Two video endoscopic observations suggest that the lateral cilia and the gill muscles act in a coordinated fashion. First, when the gills of a unionid, Pyganodon cataracta, stop pumping, the water tubes constrict, but re-open when pumping resumes (Tankersley, 1996). Second, when the valves of Mercenaria are closed, the lateral cilia are immobile, and the gills are tonically contracted, both longitudinally and dorsoventrally (Baker, pers. comm.).
When the clam is actively pumping, we expect that serotonergic stimulation of the muscles is reduced and the muscles are relaxed. Under these circumstances, the ostia, water tubes, and blood vessels would be open, so the flow of water and hemolymph would be maximized. When the clam closes, the dopaminergic innervation would become active, switching the lateral cilia off and stimulating the serotonergic plexus. The longitudinal and dorsoventral muscles and the water-tube muscles would then constrict, closing the ostia and constricting the water tubes and blood vessels.
Comparative aspects of branchial muscle pharmacology
Although there is an extensive literature on the pharmacology of bivalve muscles, it is largely focused on the anterior byssus retractor muscle of Mytilus and isolated ventricles of a variety of bivalves including that of Mercenaria. In contrast, the pharmacology of branchial muscles has been studied in relatively few species of bivalves, in part because the branchial musculature is not an advantageous model for the study of muscle cells per se. Branchial muscles are small and are embedded in a complex organ; thus they cannot be directly attached to a recording apparatus. Furthermore, their neural supply is complex, and the innervation of specific muscles is not readily accessible. However, the pharmacology of these muscles has been studied by those interested in the physiology of bivalve gills; the available data are summarized in Table 4.
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ACh had a net excitatory effect on five of the six species on which it was tested; one species (Ligumia subrostrata) showed no effect, but the gills were not pretreated with eserine. The effect of ACh on the gills of Mercenaria was revealed only after pretreating them with eserine; in addition, the inhibitory effect of ACh became evident only when the gills were exposed to a 5HT antagonist and eserine. The relaxing effect of ACh has not been seen in any of the other gills tested, but then the pharmacological analysis reported here was not used in the other studies.
Painter and Greenberg (1982) examined the effects of 5HT and FMRFamide on the ventricles of 50 species of bivalves and remarked that "the responses were strikingly diverse, varying qualitatively with dose as well as species." In their analysis, however, clear taxonomic relationships were discernable. In comparison to ventricles, gills are much more complex and interact directly with the environment. For example, sodium transport in the gills of freshwater mussels appears to be regulated by a serotonergic neural mechanism (data summarized in Dietz et al., 1985).
The odd response of the control gills in experiments with antagonists
When two successive equal doses of any agonist (i.e., 5HT, DA, or ACh) were applied to the control gills, the second contraction was typically larger than the first, and this result was initially inexplicable. Later, however, we discovered that the gills produce nitric oxide (NO) in response to 5HT, and that NO potentiates gill muscle contractions (Gainey et al., 1999b). This mechanism may also explain another experimental observation: that ultrasonic transducers record higher maximal contractions than force transducers, and they produce dose-response curves with lower EC50s. Thus, when force transducers were used, demibranchs could be exposed only to a single dose of agonist, so the individual contractions constituting the dose-response curves were not potentiated by NO. In contrast, when ultrasonic transducers were used, the demibranchs could be exposed to a set of increasing doses of agonist, so NO was produced, the contractions were potentiated, and the resulting dose-response curves were steeper.
Summary
The gills of Mercenaria are equipped with an array of muscles and four distinct sets of cilia, and the activity of these effectors—coordinated by a complex neural network—transports water and particles in support of respiration and feeding. This paper and a previous one on the modulation of ciliary activity (Gainey et al., 1999a) lay the groundwork for studies of the integrated control of gill function.
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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