Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

Presence of a Ciliary Patch in Preoral Epithelium of Sea Urchin Plutei

Removal of the hyaline layer from sea urchin embryos at the pluteus stage discloses a densely ciliated region in the preoral area of the ectodermal epithelium. In four-armed plutei, this ciliary path is located between the anterolateral arms and in eight-armed plutei it becomes surrounded by preoral and anterolateral arms. The area of the patch and the number of cilia increase with age. This patch is covered by cilia of unusual morphology and orientation. There are more than two cilia per cell which are coiled together several times around a small cone at the apical end of the cell. These coiled cilia run parallel to the surface of the cell but do not extend beyond the hyaline layer. The ciliary axoneme consists of a "9+2" microtubular structure, but no outer or inner dynein arms are observed. Although the cells with coiled cilia are present in a cluster constituting a part of the epithelium, they have axons that project from their basal (inner) ends. The structural characteistics of the ciliary patch suggest that it possesses a sensory function.     

"Light" epithelial cells of swine and bovine oviducts

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.     

An ultrastructural study of primary cilia,abnormal cilia and ciliary knobs from the ciliated cells of the guinea-pig trachea

Summary Single primary cilia are found in developing as well as mature ciliated cells of guinea-pig tracheal epithelium. A few biciliated cells were observed, and in a rare case one cell had developed four such processes. Primary cilia are characterized by a 9 + 0 microtubular arrangement near the base, while a transition to an 8 + 1 pattern occurs at a slightly more distal position. Spokes are lacking, and dynein arms are absent or incompletely developed. The function, if any, of primary cilia remains unknown.In the population of the motile 9 + 2 cilia atypical forms are very rare, i.e. <0.1%. Of the various abnormalities cilia with supernumary microtubules are most common. Only one atypical basal body was observed. Although some of the aberrant forms undoubtedly are non-motile, their very low number suggests that they have no practical influence on the muco-ciliary clearance.Local extrusions of the ciliary membrane, here named ciliary knobs, are believed to be fixation artefacts. It is suggested that they represent circumscribed regions of the ciliary membrane which are sensitive to changes in the environmental osmotic pressure.     

Scanning electron microscopy of Mycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth's MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the "parent" explant was removed. Monolayer cultures infected with Mycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments of M. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or "sperules" of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites for M. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas.     

Ultrastructure of rabbit tracheal epithelium after saline lavage of the airways

In experiment the effect of saline lavage on the ultrastructure of the tracheal epithelium was investigated. Immediately after this procedure the trachea was lined with markedly altered pseudostratified columnar epithelium with remnants of the ciliary border. The intercellular spaces were markedly dilated, but the apical junctional complexes were left intact. 99% of the goblet cells were exhausted degenerated and were gradually expelled from the epithelium. Damage to the ciliated cells was not so generalized but some of them displayed signs of vacuolar degeneration. The ciliary border was severely impaired with only 1.5 kinocilia/microns 2, but the majority of remaining cilia were intact. Compared with controls there was only mild, but significant increase in the number of degenerating and malformed cilia. Morphological signs of impaired self-cleaning ability of the epithelium were not noticed.     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Automated measurement of ciliary beat frequency

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.     

Ultrastructural study of the ciliated cyst in the human uterine tube epithelium

Ciliated cysts in the human uterine tube epithelium were investigated with the transmission electron microscope. The cysts were about 3-9 microns in diameter and were provided with many ciliary apparatuses and microvilli. Degenerative changes of these cilia, such as electron-dense round or irregular bodies and amorphous substance, were observed in many cysts, but complete disappearance of ciliary structures was not detected in any ciliated cysts. The ciliated cysts were mostly observed in basal cells and were occasionally found in ciliated cells bordering the tubal lumen. In the basal cells, these cysts distended with the increase in degenerated cilia. Distended ciliated-cyst-containing cells became exposed directly to the tubal lumen. U- or reverse omega-shaped deep indentations of the apical surface of ciliated cells confirmed the opening of ciliated cysts into the lumen. It was suggested that the ciliated cysts result from the premature differentiation of basal cells or disturbed migration of centrioles in ciliogenic cells.     

Association of SPARC (osteonectin, BM-40) with extracellular and intracellular components of the ciliated surface ectoderm of Xenopus embryos

SPARC (Secreted Protein, Acidic, Rich in Cysteine) was detected by immunohistochemistry in the sensorial layer of the bilayered embryonic epidermis of Xenopus laevis during neurulation, when a subset of the sensorial cells are selected to differentiate into ciliated cell precursors. After the ciliated cells had intercalated into the outer layer and had undergone ciliogenesis, intense SPARC immunostaining was associated with the cilia and remained associated with the cilia throughout their persistence on the epidermis. Circumferential SPARC immunostaining was also detected at the interface between surface epithelial cells. Animal cap explants indicated that the embryonic activation of SPARC expression in the dorsal ectoderm does not require signaling from factors secreted by the underlying mesoderm. Immunoelectron microscopy revealed that SPARC is intimately associated with the 9 + 2 microtubule arrays of cilia. Our data indicate that SPARC plays a role in the development and function of the surface ciliated epidermis of Xenopus embryos. We propose that the counter-adhesive activity of SPARC facilitates the intercalation of ciliary cell precursors to the surface epithelial layer, where its Ca(2+)-binding abilities promote cell-cell adhesion. Based on its association with ciliary microtubule arrays, we also propose that intracellular SPARC may play a role in regulating ciliary beat frequency and polarity.     

Electron microscopic study of intestinal epithelium of <Emphasis Type="Italic">Saccoglossus mereschkowskii</Emphasis> (Enteropneusta,Hemichordata)

The epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata), a group located at the base of Chordata, has been studied by using electron microscopy. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions are characterized. The data on the details of the structure of the ciliary apparatus and the system of ciliary rootlets are presented. Justification is provided for the presence of a complicated construction in the ciliated cells, a supportive carcass of cilia that performs a mechanical stabilizing function, and possibly the synchronization of the ciliary movement. The existence of cilia with two centrioles is considered as adaptation to the high functional load on the ciliary apparatus. Well-developed bundles of myofilaments have been revealed in the cytoplasm of the basal parts of ciliated cells, which characterizes these cells as epitheliomuscular. Peculiarities indicating the role of ciliated cells in absorption are described, as well as the capability of these cells for balloon-like secretion. Data are presented on the accumulation of reserved nutritional substances in the cell cytoplasm in the form of lipids and glycogen. With respect to their function, ciliated cells are determined as the ciliated secretory-absorptive epitheliomuscular cells. The location of secretory granules in both apical and basal parts of granular cells indicates the exocrine-endocrine function of these cells. There are no typical endocrine cells in the intestinal epithelium of S. mereschkowskii. Several types of granules are described in the cytoplasm of nerve fibers. Junctions between nerve fibers and basal parts of ciliated and granular epithelial cells have been revealed; the neural regulation of the contractile and secretory functions of epithelial cells is assumed. The intestinal epithelium of enteropneusts is presumed to contain a regulatory neuroendocrine system composed of receptor cells of the open type, secretory endocrine-like cells, and of nerve elements of the nervous layer.     

The sensory epithelium of the tentacles and the rhinophore of Nautilus pompilius L. (cephalopoda, nautiloidea)

Nine intraepithelial ciliated cell types that are presumed to be sensory cells were identified in the epithelium of the pre- and postocular tentacles, the digital tentacles, and the rhinophore of the juvenile tetrabranchiate cephalopod Nautilus pompilius L. The morphological diversity and specialization in distribution of the different ciliated cell types analyzed by SEM methods suggest that these cells include receptors of several sensory functions. Ciliated cell types in different organs that show similar surface features were combined in named groups. The most striking cell, type I, is characterized by a tuft of long and numerous cilia. The highest density of this cell type occurs in ciliary fields in the epithelium of the lamellae of the pre- and postocular tentacles, in the olfactory pits of the rhinophores, and in the lamellae of four pairs of lateral digital tentacles, but not in the epithelium of the medial digital tentacles. The similar morphological data, together with behavioral observations on feeding habits, suggest that this cell type may serve in long-distance chemosensory function. The other ciliated cell types are solitary cells with specific spatial distributions in the various organs. Cell types with tufts of relatively short, stiff cilia (types III, IV, VIII), which are distributed in the lateral and aboral areas of the tentacles and at the base of the tentacle-like process of the rhinophore, are considered to be employed in mechanosensory transduction, while the solitary cells with bristle-like cilia at the margin of the ciliary fields (type II) and at the base of the rhinophore (type IX) may be involved in chemoreception. Histological investigation of the epithelium and the nerve structures of the different organs shows the proportion and distribution of the sensory pathways. Two different types of digital tentacles can be distinguished according to their putative functions: lateral slender digital tentacles in four pairs, of which the lowermost are the so-called long digital tentacles, participate in distance chemoreception, and the medial digital tentacles, whose terminal axial nerve cord may represent a specialized neuromechanosensory structure, appear to have contact chemoreceptive abilities.     

Proliferation,differentiation and ciliary beating of human respiratory ciliated cells in primary culture

Summary The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29±5% to 37±6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51±5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3±1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P<0.01) beating frequency (10.7±1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.     

Electron microscopic observations on tracheal epithelia of mice infected with Bordetella bronchiseptica

To clarify the pathogenesis of Bordetella in vivo infection, the tracheal epithelia of mice were examined in detail by electron microscopy at various intervals after intranasal inoculation with graded doses of phase I Bordetella bronchiseptica. In mice infected with a lethal dose (6 to 7 x 10(7) CFU), a remarkable rupture of the cell membranes of cilia and microvilli of the middle trachea was found on day I postinfection. The rupture of the membrane was observed over the entire tracheal epithelia, on day 2 after infection. The affected cilia were constricted at the transitional region and were broken off. In the ciliated cells the adherence of organisms to ciliary apexes and colonization in the interciliary spaces were also remarkable. In both the ciliated and nonciliated epithelial cells, the cytoplasmic vacuolation and pyknosis or karyorrehexis were also notable. In mice infected with one-tenth of the lethal dose, similar findings were seen, but appeared more slowly and the bacteria were not seen attaching to ciliary apexes. In mice receiving one-hundredth of the lethal dose, only mild cilial abnormality such as aggregation of cilia, and slight cytoplasmic vacuolation were found 6 days postinfection. Based on these findings, a possible mechanism of the ciliary damages produced by B. bronchiseptica was postulated.     

Regulation of ciliary activity in the mammalian respiratory tract

A computer-assisted transillumination, photoelectronic technique has been used to measure the beat frequency of cilia of rabbit tracheal cells grown in culture. When ciliated cells are mechanically stimulated with a microprobe the cells respond rapidly by increasing the beat frequency of their cilia. This mechanosensitive response is not limited to the stimulated cell, but is communicated in all directions to neighboring cells. To characterize the progression of this communicated response we used an automated computer-assisted imaging system to examine high-speed films of responding cells. The time it takes for the response to be transmitted between cells is slow (1-3 sec) with each cell responding after a lag-time that is proportional to the distance of the cell from the stimulated cell. We have confirmed that gap junctions are present between cells and that adjacent or non-adjacent ciliated, as well as non-ciliated, cells are electrically coupled. To correlate the mechanosensitive response with intracellular calcium fluxes we have used fura-2, a calcium-specific fluorescent dye, and digital video microscopy. Mechanical stimulation of the cultured ciliated cells, in the presence of extracellular calcium, resulted in an initial increase in intracellular calcium, which was communicated to neighboring cells. Without extracellular calcium, mechanosensitivity of cultured cells was lost and a small decrease in intracellular calcium was observed in the stimulated cell. However, neighboring cells still displayed an increase in intracellular calcium. The time course and general pattern of calcium increase in adjacent cells was similar to the responses in ciliary activity produced by mechanical stimulation. Ciliary beat frequency is also elevated by beta-adrenergic drugs independently of mechanosensitivity. These responses are important because they could provide a dual regulatory mechanism for the control of mucus transport. Adrenergic agonists could provide non-specific control by increasing ciliary activity throughout the airways while mechanosensitivity could provide local control by increasing activity in those regions of heavy mucus load.     

Electron microscopic study of the intestinal epithelium of Saccoglossus mereschkowskii (Enteropneusta, Hemichordata)

Epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata) standing at the base of Chordata, has been investigated using electron microscope. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions have been characterized. The data concerning details of the organization of the ciliary apparatus and rootlets system are presented. It is justified the presence of complicated supporting construction of cilia which performs a mechanical stabilizing function and possibly also provide synchronization of ciliary movements. The presence of cilia with two centrioles is considered as an adaptation to high functional load on ciliary apparatus. Well developed bundles of myofilaments are found in the cytoplasm of the basal portions of ciliary cells that characterizes these cells as myoepithelial. The features indicating the role of ciliary cells in absorption are described. The capability of these cells to balloon-like secretion is considered. Data on the accumulation of food reserves in the form of lipid droplets and glycogen in the cell cytoplasm are presented. Ciliated cells are characterized by their function as ciliated secretory-absorptive myoepithelial cells. Based on the location of secretory granules both in the apical and basal portions of granular cells, an exocrine-endocrine function of these cells has been suggested. Typical endocrine cells in the intestinal epithelium of S. mereschkowskii are absent. Several types of granules in the nerve fibers cytoplasm are described. Junctions between the nerve fibers and basal portions of ciliary and granular epithelial cells are found. Nerve regulation of contractile and secretory functions of epithelial cells is supposed. The presence of the regulatory nerve-endocrine system that includes receptor cells of open type, secretory endocrine-like cells and nerve elements of nerve layer is supposed in the intestinal epithelium of enteropneusts.     

In vivo ciliogenesis in human fetal tracheal epithelium

Development of ciliated cells (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18-19 weeks (stage II), centriologenesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22-23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P less than .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.     

Paddle cilia (discocilia) in chemosensitive structures of the gastropod mollusk Pleurobranchaea californica

Summary Scanning electron microscopy of various regions of the body of the marine gastropod Pleurobranchaea californica (McFarland) has revealed a characteristic cell type that bears cilia with dilated discoid-shaped tips. The tips of the cilia consist of an expansion of the ciliary membrane around a looped distal extension of the axoneme. These kinocilia have been observed in numerous other marine invertebrates and are generally referred to as paddle cilia (Tamarin et al. 1974) or discocilia (Heimler 1978). Although many functions have been proposed for paddle cilia, little empirical evidence supports any of the proposals. In Pleurobranchaea we have found that the distribution of this ciliated cell type corresponds exactly to areas of the body known from behavioral studies (Lee et al. 1974; Davis and Matera 1981) to mediate chemoreception. Transmission electron microscopy of the epithelium lining the rhinophores and tentacles of Pleurobranchaea revealed details of the ultrastructure of these ciliated cells and showed that they are primary receptors. These ciliated receptors lie in a yellow-brown pseudostratified columnar epithelium that superficially resembles the olfactory mucosa of vertebrates.     

Determination of ciliary polarity precedes differentiation in the epithelial cells of quail oviduct.

In quail oviduct epithelium, as in all metazoan and protozoan ciliated cells, cilia beat in a coordinated cycle. They are arranged in a polarized pattern oriented according to the anteroposterior axis of the oviduct and are most likely responsible for transport of the ovum and egg white proteins from the infundibulum toward the uterus. Orientation of ciliary beating is related to that of the basal bodies, indicated by the location of the lateral basal foot, which points in the direction of the active stroke of ciliary beating. This arrangement of the ciliary cortex occurs as the ultimate step in ciliogenesis and following the oviduct development. Cilia first develop in a random orientation and reorient later, simultaneously with the development of the cortical cytoskeleton. In order to know when the final orientation of basal bodies and cilia is determined in the course of oviduct development, microsurgical reversal of a segment of the immature oviduct was performed. Then, after hormone-induced development and ciliogenesis, ciliary orientation was examined in the inverted segment and in normal parts of the ciliated epithelium. In the inverted segment, orientation was reversed, as shown by a video recording of the direction of effective flow produced by beating cilia, by the three-dimensional bending forms of cilia immobilized during the beating cycle and screened by scanning electron microscopy, and by the position of basal body appendages as seen in thin sections by transmission electron microscopy. These results demonstrate that basal body and ciliary orientation are irreversibly determined prior to development by an endogenous signal present early in the cells of the immature oviduct, transmitted to daughter cells during the proliferative phase and expressed at the end of ciliogenesis.     

The nervous system control of lateral ciliary activity of the gill of the bivalve mollusc, Crassostrea virginica

Lateral cilia of the gill of Mytilus edulis are controlled by a reciprocal serotonergic-dopaminergic innervation from their ganglia. Other bivalves have been studied to lesser degrees and lateral cilia of most respond to serotonin and dopamine when applied directly to the gill indicating a possible neuro or endocrine mechanism. Lateral cilia in Crassostrea virginica are affected by serotonin and dopamine, but little work has been done regarding ganglionic control of their cilia. We examined the role of the cerebral and visceral ganglia in innervating the lateral ciliated cells of the gill epithelium of C. virginica. Ciliary beating rates were measured in preparations which had the ipsilateral cerebral or visceral ganglia attached. Superfusion of the cerebral or visceral ganglia with serotonin increased ciliary beating rates which was antagonized by pretreating with methysergide. Superfusion with dopamine decreased beating rates and was antagonized by ergonovine. This study demonstrates there is a reciprocal serotonergic-dopaminergic innervation of the lateral ciliated cells, similar to that of M. edulis, originating in the cerebral and visceral ganglia of the animal and this preparation is a useful model to study regulatory mechanisms of ciliary activity as well as the pharmacology of drugs affecting biogenic amines in nervous systems.