Transmitter contents of cells and fibers in the cephalic sensory organs of the gastropod mollusc<Emphasis Type="Italic"> Phestilla sibogae</Emphasis>

While the central ganglia of gastropod molluscs have been studied extensively, relatively little is known about the organization and functions of the peripheral nervous system in these animals. In the present study, we used immunohistochemical procedures to examine the innervation of the rhinophores, oral tentacles and region around the mouth of the aeolid nudibranch, Phestilla sibogae. Serotonin-like immunoreactivity was found in an extensive network of efferent projections apparently originating from central neurons, but was not detected within any peripheral cell bodies. In contrast, large numbers of peripheral, and presumably sensory, somata exhibited reactivity to an antibody raised against tyrosine hydroxylase (the enzyme catalyzing the initial step in the conversion of tyrosine into the catecholamines). Additional tyrosine hydroxylase-like immunoreactivity was detected in afferent fibers of the peripheral cells and in several cells within the rhinophoral ganglia. The presence of serotonin, dopamine and norepinephrine in the rhinophores, tentacles and central ganglia was confirmed using high-performance liquid chromatography. Finally, FMRFamide-like immunoreactivity was detected in cells and tangles of fibers found within the rhinophore, possibly revealing glomerulus-like structures along olfactory pathways. FMRFamide-like immunoreactivity was also found in somata of the rhinophoral ganglia, in a small number of cells located in the body wall lateral to the tentacles and in what appeared to be varicose terminals of efferent projections to the periphery. Together, these results indicate several new features of the gastropod peripheral nervous system and suggest future experiments that will elucidate the function of the novel cells and innervation patterns described here.This research was supported by Natural Sciences and Research Council of Canada Grant #OPG38863 to R.P.C. and Office of Naval Research Grant #N00014-94-1-0524 to M.G.H.     

GASTROPOD CHEMORECEPTION

(I). Gastropods use chemoreception for a wide variety of behaviours including feeding, homing, escape from predators and a variety of social and reproductive behaviours. Chemoreception is used to locate distant food sources, and to discriminate between potential foods. Responses to chemical food stimuli result from a combination of innate and experiential factors. Gastropods use chemical cues in mucus trails to home. They also home by direct olfactory orientation. Reproductive behaviour in a variety of gastropods appears to involve chemical cues. Evidence exists for pheromones controlling aggregation and mating. Numerous gastropods use chemical cues to avoid or escape from predators. (2). Amino acids appear as likely candidates for attractants and phagostimulants for gastropod feeding. Macromolecules are probably also involved. Amino acids have also been shown to stimulate reproductive behaviours in certain gastropods, thus suggesting a pheromonal function. However, the significance of this finding to the behaviour of the organisms in the field has yet to be evaluated. Saponins have been implicated as the active substances found in sea stars that elicit escape responses of marine gastropods. Choline esters may play a homologous role in gastropod—prey and gastropod-predator interactions. (3). Gastropods can apparently use a number of different methods to orient to olfactory cues. These include anemotaxis or rheotaxis, klinotaxis and tropotaxis. (4). The major chemosensory organs of gastropods have been identified. They include the anterior and posterior tentacles and lips of terrestrial pulmonates; the cephalic tentacles, the lips and buccal cavity lining, and possibly the osphradium of aquatic pulmonates; the cephalic and mantle tentacles, the anterior margin of the foot, the siphon tip, and the osphradium of prosobranchs; and the rhinophores, tentacles, oral veil and osphradium of opisthobranchs. (5). Many of the organs named above have been examined by both light and electron microscopy. The most common anatomical organization includes bipolar primary sensory cells with cell bodies located subepithelially, and a distal dendrite extending to the free surface. Often a peripheral ganglion is located deep to the sensory epithelium. It is unclear whether axons of the sensory cells project directly to the central ganglion or by way of interneurones located in the peripheral ganglia. (6). The dendritic specializations of the sensory cells vary considerably. Most bear cilia or a combination of cilia and microvilli. The functional significance of the variation in the types of sensory endings is unknown, although the chemosensory epithelia also respond to other sensory modalities, and it is often difficult to ascribe any one cell type to any one modality. Species-specific variations may also complicate the picture. (7). Prospects for and importance of future studies on gastropod chemoreception are discussed.     

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.     

Innervation patterns of the cerebral nerves in <Emphasis Type="Italic">Haminoea hydatis</Emphasis> (Gastropoda: Opisthobranchia): a test for intraspecific variability

This study describes the innervation patterns for the cerebral nerves which project to the cephalic sensory organs (CSOs) in the opisthobranch Haminoea hydatis (Linnaeus 1758) and uses axonal tracing techniques (backfilling) to reveal the central cellular origins for these cerebral nerves. Cell clusters projecting into the cerebral nerves can be defined by their positions in the ganglion relative to other clusters, nerve roots and lobes. The number of cell clusters and the relative sizes of somata are constant in a given cluster, whereas the absolute number of somata and absolute sizes of single somata in a given cluster increase with the size of the animal. Additionally, the invariable morphological characteristics of the cell clusters are used to define criteria for the assessment of possible homology for the clusters innervating the CSOs in Opisthobranchia. The data suggest two different strategies to accommodate the increasing body size; first, the additions of nerve cells and second, the growth of nerve cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aspects on the Ultrastructure of the Cephalic Region in Two Species of Acrobelinae (Nematoda,Cephalobidae)

The lip structures termed labial probolae, characteristic for the subfamily Acrobelinae, and the anterior sense organs have been studied by electron microscopy. The labial probolae consist of an amorphous tissue with denser strengthenings. They do not contain any sensory structures or musculature. The anterior sense organs consists of: (1) six inner labial sensilla, each with one receptor ending in a pore on a papilla; (2) six outer labial sensilla, each with one receptor ending in the cuticle; (3) four cephalic sensilla, each with two receptors, one ending in a pore on a papilla and the other in the cuticle; (4) two amphids, each with 12 sensory neurons basally; (5) accessory sensilla ventro-laterally. The combination of several chemoreceptive anterior sensilla and the morphological diversity of the labial probolae within the subfamily may aid in the utilization of different soil microhabitats.     

The central nervous system of Bulla gouldiana: Peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Histology and ultrastructure of the olfactory organ of the freshwater pulmonate Helisoma trivolvis

Summary The olfactory organ of Helisoma trivolvis is located on the surface of the body at the base of the cephalic tentacles. An evagination of skin, the olfactory plica, at the base of the tentacle extends over the olfactory organ dorsally. The epithelium of the olfactory organs contains unspecialized epithelial cells, ciliated epithelial cells, basal cells, mucous secretory cells, and sensory dendrites. The surface of the epithelium has a complex brush border of thick plasmatic processes, which branch to form several terminal microvillar twigs. Long slender cytoplasmic processes form a dense spongy layer among the plasmatic processes beneath the level of the terminal twigs. Bipolar primary sensory neurons clustered beneath the epithelium of the olfactory organ send dendrites through the epithelium to the free surface. Some sensory endings have a few short cilia, but most bear only microvilli. Cilia of sensory endings and epithelial cells extend beyond the brush border of the epithelium. Small axons arise from the perikarya of the sensory neurons and enter a branch of the olfactory nerve. HRP tracing indicates that the axons pass to the cerebral ganglion without interruption. Histochemical tests indicate that the sensory neurons are neither aminergic nor cholinergic.     

Fine Structure of Cephalic Sense Organs in Meloidogyne incognita Males

Amphids, and the cephalic and labial papillae of Meloidogyne incognita males were examined in detail by electron microscopy. Each amphid basically consists of an amphidial gland, a nerve bundle and an amphidial duct. The gland is a broad microvillous organ with a narrow anterior process, which is closely associated with the amphidial duct. A posterior process of the gland contains secretory organelles and proceeds along the esophagus with the lateral cephalic nerve bundle. The nerve bundle penetrates the broad portion of the gland and, subsequently, individual nerve processes (dendrites) separate from one another, thus forming the sensilla pouch which is enveloped by the gland. Anterior to the pouch, the dendrites converge as they enter and eventually terminate in the amphidial duct. The external opening of the duct is a broad slit which separates the cheek, the outermost part of the lateral lip, from the remainder of the lip region. M. incognita males have six inner labial papillae and four outer cephalic papillae which are each innervated by two and one cilia, respectively. In labial papillae, the cilia appear to terminate at the base of a pore opening, whereas in cephalic papillae each cilium terminates beneath the labial cuticle.     

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.     

Scanning Electron Microscope Studies of Steinernema anomali Kozodoi, 1984

This paper presents SEM micrographs of portions of the male, female, and infective-stage juvenile of Steinernema anomali. Included are micrographs of the cephalic and caudal region, spicules, and gubernaculum of the male, the cephalic and vulval region of the female, and the cephalic region of the infective-stage juvenile. Males have six labial and four prominent cephalic papillae and small amphids. There are 11-14 pairs and one single genital papillae; of these, 6-9 pairs are preanal and subventral, one pair preanal, lateral, one pair adanal, and three pairs postanal. Spicules have a short head, a long blade, and a reduced shaft. The distal end is enlarged and bears a dorsal aperture. Gubernaculum much shorter than spicules; cuneus of gubernaculum short and bifurcate anteriorly. Females have six labial and four cephalic papillae and small amphids. Vulva with a thickened posterior lip. Infective juveniles have a smooth head, prominent amphids, and four cephalic papillae. Labial papillae, if present, are not evident.     

FMRFamide is endogenous to the Aplysia heart

The presence of the molluscan neuropeptide FMRFamide was investigated in the heart of the sea hare, Aplysia californica. Immunohistochemical localization and high performance liquid chromatography (HPLC) coupled with radioimmunoassays of HPLC fractions were used to demonstrate the presence of FMRFamide and FLRFamide in the heart. FMRFamide-immunoreactive (FMRFamide-IR) nerve fibers, varicosities, and neuronal somata were observed in whole-mounts of the hearts. The atrium and atrioventricular (AV) valve regions contained significantly higher densities (P<0.05, ANOVA) of immunoreactive varicosities compared to the ventricle. The high density of FMRF-amide-IR varicosities in the atrium and the lack of sensitivity of this region to FMRFamide suggest that the atrium may be a neurohemal organ for the release of FMRF-amide. The presence of FMRFamide-IR somata in the Aplysia heart suggests that peripheral neurons may play a role in modifying heart activity, independent of the central nervous system.This work was supported by California State University, Fullerton intramural grants and NIH grant NS29750 to J.K.O., Departmental Associations Council student research grants to L.L.H., and NIH grant HL08371 to W.L.     

Monoamines in the nervous system of the tube-wormChaetopterus variopedatus (Polychaeta): Biochemical detection and serotonin immunoreactivity

Summary The presence and distribution of biogenic monoamines in the tube-wormChaetopterus variopedatus were investigated by a radioenzymatic method and HPLC with electrochemical detection, and the cellular localization of serotonin by peroxidase-antiperoxidase (PAP) immunohistochemistry with an antibody against serotonin-formaldehyde-protein conjugate. Dopamine, norepinephrine, epinephrine, serotonin (5-HT) and some of their metabolites were detectable, dopamine and norepinephrine being present in substantially larger amounts than 5-HT and epinephrine. With few exceptions, the largest amounts of amines were localized in the most nerve-rich tissues such as tentacles, and those containing cerebral ganglia and the ventral nerve cord. Serotonin-immunoreactive unipolar neurons were widely distributed in the dorso-lateral cerebral ganglia, the neurosecretory pharyngeal ganglion and the segmental ganglia of the anterior (dorsolateral) and posterior (medioventral) nerve cords. Some nerve-fiber tracts stained in the cerebral ganglia, but the neuropiles of segmental ganglia were the most intensely reactive CNS structures. Numerous reactive fibers were also present in connectives, commissures and segmental nerves. All peripheral sensory structures included serotonin-immunoreactive cells and neurites, especially the parapodial cirri and the bristle receptors of the setae. Trunk and parapodial muscles contained reactive varicose fibers and neuronal somata. These results suggest that monoamines are abundant and widespread in these worms and that 5-HT appears to have a key sensory role.     

Distribution of catecholamine-containing,serotonin-like and neuropeptide FMRFamide-like immunoreactive neurons and processes in the nervous system of the actinotroch larva ofPhoronis muelleri (Phoronida)

Summary Glyoxylic-acid-induced fluorescence of catecholamines and antibodies against serotonin and FMRFamide were used to study the distribution of putative neurotransmitters in the actinotroch larva ofPhoronis muelleri Selys-Longchamps, 1903. Catecholamines occur in the neuropile of the apical ganglion, in the longitudinal median epistome nerves, in the epistome marginal nerves, and in the nerve along the bases of the tentacles. The tentacles have laterofrontal and latero-abfrontal bundles of processes that form two minor nerves along the lateral ciliary band of the tentacles, and a medio-frontal bundle of processes. Monopolar cells are located on the ventro-lateral part of the mesosome. Processes are located along the posterior ciliary band and as a reticulum in the epidermis. Serotonin-like immunoreactive cells and processes are located in the apical ganglion, in the longitudinal median epistome nerves, and as a dorsal and ventral pair of bundles along the tentacle bases. Processes from the latter extend into the tentacles as the medioabfrontal processes. The latero-abfrontal processes form a minor nerve along the ciliary band. The dorsal bundles forms the major nerve ring along the tentacles and processes extend from it to the metasome. Processes are located along the posterior ciliary band. FMRFamide-like immunoreactive cells and processes are found in the apical ganglion, in the longitudinal median epistome nerves and as a pair of lateral epistome processes projecting towards the ring of tentacles. In the tentacles, a pair of latero-frontal processes are found; these form a minor nerve along the ciliary band. A band of cells can be seen along the tentacle ring.     

Comparative immunohistochemistry of the cephalic sensory organs in Opisthobranchia (Mollusca,Gastropoda)

The structure and function of the central nervous systems of opisthobranch gastropods have been studied extensively. However, the organisation and function of the peripheral nervous system are poorly understood. The cephalic sensory organs (CSOs) are known to be chemosensory structures in the head region of opisthobranchs. In the present study, we used immunohistochemical methods and confocal laserscanning microscopy to comparatively examine the CSOs of different opisthobranchs, namely Acteon tornatilis, Aplysia punctata, Archidoris pseudoargus and Haminoea hydatis. We wanted to characterise sensory epithelia in order to infer the function of sensory structures and the organs they constitute. Immunoreactivity against the three antigens tyrosine hydroxylase, FMRFamide and serotonin was very similar in the CSOs of all investigated species. Tyrosine hydroxylase-like immunoreactivity was detected primarily in subepidermal sensory cell bodies, which were much more abundant in the anteriorly situated CSOs. This observation indicates that these cells and the respective organs may be involved in contact chemoreception and mechanoreception. The dominant features of FMRFamide-like immunoreactivity, especially in the posterior CSOs, were tightly knotted fibres which reveal glomerulus-like structures. This suggests an olfactory role for these organs. Serotonin-like immunoreactivity was detected in an extensive network of efferent fibres, but was not found within any peripheral cell bodies. Serotonin-like immunoreactivity was found in the same glomerulus-like structures as FMRFamide-like immunoreactivity, indicating a function of serotonin in the efferent control of olfactory inputs. Besides this functional implication, this study could also add some knowledge on the doubtful homology of the CSOs in opisthobranch gastropods.     

Ganglionic distribution of inputs and outputs of C-PR, a neuron involved in the generation of a food-induced arousal state inAplysia

Cerebral neuron C-PR is thought to play an important role in the appetitive phase of feeding behavior ofAplysia. Here, we describe the organization of input and output pathways of C-PR. Intracellular dye fills of C-PR revealed extensive arborization of processes within the cerebral and the pedal ganglia. Numerous varicosities of varying sizes may provide points of synaptic inputs and outputs.Blocking polysynaptic transmission in the cerebral ganglion eliminated the sensory inputs to C-PR from stimuli applied to the rhinophores or tentacles, indicating that this input is probably mediated by cerebral interneurons. Identified cerebral mechanoafferent sensory neurons polysynaptically excite C-PR. Stimulation of the eyes and rhinophores with light depresses C-PR spike activity, and this effect also appears to be mediated by cerebral interneurons.C-PR has bilateral synaptic actions on numerous pedal ganglion neurons, and also has effects on cerebral neurons, including the MCC, Bn cells, CBIs and the contralateral C-PR. Although the somata of these cerebral neurons are physically close to C-PR, experiments using high divalent cation-containing solutions and cutting of various connectives indicated that the effects of C-PR on other cerebral ganglion neurons (specifically Bn cells and the MCC) are mediated by interneurons that project back to the cerebral ganglion via the pedal and pleural connectives. The indirect pathways of C-PR to other cerebral neurons may help to ensure that consummatory motor programs are not activated until the appropriate appetitive motor programs, mediated by the pedal ganglia, have begun to be expressed.     

Ultrastructure of aesthetasc innervation and external morphology of the lateral antennule setae of the spiny lobster Panulirus interruptus (Randall)

Summary Six types of setae and one type of cuticular depression were examined on the lateral antennule of the spiny lobster Panulirus interruptus using scanning electron microscopy. The organization and ultrastructure of the innervation of the most numerous setal type, the aesthetasc, were investigated using light-and transmission electron microscopy.Each aesthetasc is innervated by approximately 300 bipolar neurons whose sensory dendrites penetrate the hair and extend toward the tip, and whose axons project towards the central nervous system. The neuronal somata and two types of glia form a cluster within the antennular lumen. The inner sheath-cell somata encircle the dendritic tract distal to the sensory somata. These cells appear to extend distal processes which wrap the dendritic tract to the base of the aesthetasc. Elongate outer sheath cells are interposed between the glia-wrapped dendritic tract and the hypodermis which underlies the antennule cuticle. A continuous investment of neural lamella separates the hypodermis, the entire cluster of somata, and sensillar nerve from the antennule lumen. The organization of the neuronal somata and their association with outer and inner sheath cells in this marine species appear similar to those of crustaceans from freshwater and terrestrial habitats.     

Distribution of biogenic amines in the slug,Limax maximus

Summary The distribution of monoamines inLimax maximus was studied by the histochemical fluorescent method of Falck and Hillarp. The number of 5-HT-containing and catecholamine-containing perikarya in the central nervous system is small compared with the non-fluorescent perikarya. However, all the ganglia except the proto-cerebral ganglia have some amine-containing neurons. There are relatively larger numbers of fluorescent cells in the cerebral, visceral, pedal and right parietal ganglia than in the other ganglia. A single, giant 5-HT-containing neuron was observed in each meta-cerebral ganglion.Monoamine neurons are localised in a number of peripheral tissues (heart, integument, tentacles, penis retractor muscle, sole of foot, kidney, alimentary canal, reproductive organs and tentacular, pharyngeal and cephalic retractor muscles). Neurons containing catecholamine are mostly associated with sensory structures such as the statocysts, the retina of the eye and the integument of the tentacles, whereas 5-HT-containing nerve fibres are mainly observed in muscle tissues.We wish to thank the Wellcome Trust for financial support.     

Local nonpermissive and oriented permissive cues guide vestibular axons to the cerebellum

Information that originates from peripheral sensory organs is conveyed by axons of cephalic sensory cranial ganglia connecting the sensory organs to appropriate central targets in the brain. Thus, the establishment of correct axonal projections by sensory afferents is one of the most important issues in neural development. Previously, we examined the development of the vestibular nerve that originates from the VIIIth ganglion using a flat whole-mount preparation of the rat hindbrain and developed an in vitro, culture preparation that can recapitulate vestibular nerve development (Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M., Heizmann, C. W. and Murakami, F. (2000) J. Comp. Neurol. 417, 491-500). Both in vivo and in vitro, the ascending branch of the VIIIth ganglion projecting to the cerebellum reaches the base of the cerebellar primordium and starts to splay out towards the rhombic lip, apparently avoiding the ventral metencephalon. We now examine the nature of cues that guide vestibulocerebellar axons by applying various manipulations to the flat whole-mount in vitro preparation. Our observations suggest that local nonpermissive cues and oriented cues play a pivotal role in the guidance of vestibular axons to their central target.     

Candidate chemoreceptor subfamilies differentially expressed in the chemosensory organs of the molluscAplysia

Background  

Marine molluscs, as is the case with most aquatic animals, rely heavily on olfactory cues for survival. In the molluscAplysia californica, mate-attraction is mediated by a blend of water-borne protein pheromones that are detected by sensory structures called rhinophores. The expression of G protein and phospholipase C signaling molecules in this organ is consistent with chemosensory detection being via a G-protein-coupled signaling mechanism.