Tentacle Nerves Research Articles

Neuromuscular organization of the benthic ctenophore, Vallicula multiformis

Ctenophora is the earliest metazoan taxon with neurons and muscles. Recent studies have described genetic, physiological, and cellular characteristics of the neural and muscular systems of this phylogenically important lineage. However, despite the ecological diversity of ctenophore niches, including both pelagic and benthic forms, studies have focused predominantly on pelagic species. In the present study, we describe the neural and muscular architectures of the benthic ctenophore, Vallicula multiformis (Order Platyctenida), employing immunohistochemical analysis using antibodies against amidated neuropeptides with the C-terminal sequences VWYa, NPWa, FGLa, or WTGa to compare it to pelagic species. In V. multiformis, which lacks the characteristic comb rows seen in pelagic ctenophores, neural structures that develop beneath the comb were not detected, whereas the subepithelial and tentacle neural networks showed considerable similarity to those of pelagic species. Despite significant differences in morphology and lifestyle, muscle organization in V. multiformis closely resembles that of pelagic species. Detailed analysis of neurons that express these peptides unveiled a neural architecture composed of various neural subtypes. This included widely distributed subepithelial neural networks (SNNs) and neurosecretory cells located primarily in the peripheral region. The consistent distribution patterns of the VWYa-positive SNN and tentacle nerves between V. multiformis and the pelagic species, Bolinopsis mikado, suggest evolutionarily conserved function of these neurons in the Ctenophora. In contrast, NPWa-positive neurons, which extend neurites connecting the apical organ and comb rows in B. mikado, showed a neurite-less neurosecretory cell morphology in this flattened, sessile species. Evaluation of characteristics and variations in neural and muscular architectures shared by benthic and pelagic ctenophore species may yield valuable insights for unraveling the biology of this rapidly evolving yet enigmatic metazoan lineage. These findings also provide important insight into neural control modalities in early metazoan evolution.

Chemosensory afference in the tentacle nerve of Lymnaea stagnalis

AbstractAlthough the neural control of behavior has been extensively studied in gastropods, basic gaps remain in our understanding of how sensory stimuli are processed. In particular, there is only patchy evidence regarding the functional roles of sense organs and the extensive peripheral nervous system they contain. Our goal was to use extracellular electrophysiological recordings to confirm the chemosensory role of the tentacles in the great pond snail, Lymnaea stagnalis. Employing a special twin‐channel suction electrode to improve signal‐to‐noise ratio, we applied three food odors (derived from earthworm‐based food pellets, algae‐based pellets, and fresh lettuce) to a reduced preparation of the tentacle while recording neuronal activity in the tentacle nerve. Responses were assessed by comparing average spike frequencies produced in response to saline flow with and without odors. We report stronger neuronal responses to earthworm‐based food odors and weaker responses to algae‐based food odors. There were no clear neuronal responses produced when lettuce food odor or control saline was applied to the tentacle. Overall, our results provide strong evidence for the chemosensory role of the tentacles in navigation behavior by L. stagnalis. Although it is unclear whether the differences in neuronal responses to different odors are a technical consequence of our recording system or a genuine feature of the snail sensory system, these results are a useful foundation for further study of peripheral nervous system function in gastropods.

Unusual lophophore innervation in ctenostome Flustrellidra hispida (Bryozoa).

Since ctenostomes are traditionally regarded as an ancestral clade to some other bryozoan groups, the study of additional species may help to clarify questions on bryozoan evolution and phylogeny. One of these questions is the bryozoan lophophore evolution: whether it occurred through simplification or complication. The morphology and innervation of the ctenostome Flustrellidra hispida (Fabricius, 1780) lophophore have been studied with electron microscopy and immunocytochemistry with confocal laser scanning microscopy. Lophophore nervous system of F. hispida consists of several main nerve elements: cerebral ganglion, circumoral nerve ring, and the outer nerve ring. Serotonin-like immunoreactive perikarya, which connect with the circumoral nerve ring, bear the cilium that directs to the abfrontal side of the lophophore and extends between tentacle bases. The circumoral nerve ring gives rise to the intertentacular and frontal tentacle nerves. The outer nerve ring gives rise to the abfrontal neurites, which connect to the outer groups of perikarya and contribute to the formation of the abfrontal tentacle nerve. The outer nerve ring has been described before in other bryozoans, but it never contributes to the innervation of tentacles. The presence of the outer nerve ring participating in the innervation of tentacles makes the F. hispida lophophore nervous system particularly similar to the lophophore nervous system of phoronids. This similarity allows to suggest that organization of the F. hispida lophophore nervous system may reflect the ancestral state for all bryozoans. The possible scenario of evolutionary transformation of the lophophore nervous system within bryozoans is suggested.

The nervous system of the most complex lophophore provides new insights into the evolution of Brachiopoda

The lophophore is a tentacle organ unique to the lophophorates. Recent research has revealed that the organization of the nervous and muscular systems of the lophophore is similar in phoronids, brachiopods, and bryozoans. At the same time, the evolution of the lophophore in certain lophophorates is still being debated. Innervation of the adult lophophore has been studied by immunocytochemistry and confocal laser scanning microscopy for only two brachiopod species belonging to two subphyla: Linguliformea and Rhynchonelliformea. Species from both groups have the spirolophe, which is the most common type of the lophophore among brachiopods. In this study, we used transmission electron microscopy, immunocytochemistry, and confocal laser scanning microscopy to describe the innervation of the most complex lophophore (the plectolophe) of the rhynchonelliform species Coptothyris grayi. The C. grayi lophophore (the plectolophe) is innervated by three brachial nerves: the main, second accessory, and lower. Thus, the plectolophe lacks the accessory brachial nerve, which is typically present in other studied brachiopods. All C. grayi brachial nerves contain two types of perikarya. Because the accessory nerve is absent, the cross nerves, which pass into the connective tissue, have a complex morphology: each nerve consists of two ascending and one descending branches. The outer and inner tentacles are innervated by several groups of neurite bundles: one frontal, two lateral, two abfrontal, and two latero-abfrontal (the latter is present in only the outer tentacles). Tentacle nerves originate from the second accessory and lower brachial nerves. The inner and outer tentacles are also innervated by numerous peritoneal neurites, which exhibit acetylated alpha-tubulin-like immunoreactivity. The nervous system of the lophophore of C. grayi manifests several evolutionary trends. On the one hand, it has undergone simplification, i.e., the absence of the accessory brachial nerve, which is apparently correlated with a reduction in the complexity of the lophophore’s musculature. On the other hand, C. grayi has a prominent second accessory nerve, which contains large groups of frontal perikarya, and also has additional nerves extending from the both ganglia to the medial arm; these features are consistent with the complex morphology of the C. grayi plectolophe. In brachiopods, the evolution of the lophophore nervous system apparently involved two main modifications. The first modification was the appearance and further strengthening of the second accessory brachial nerve, which apparently arose because of the formation of a double row of tentacles instead of the single row of the brachiopod ancestor. The second modification was the partial or complete reduction of some brachial nerves, which was correlated with the reduced complexity of the lophophore musculature and the appearance of skeletal structures that support the lophophore.

Peculiarities of Tentacle Innervation of Flustrellidra hispida and Evolution of Lophophore in Bryozoa.

The study of the lophophore organization is of great importance for the reconstruction of lophophorate phylogeny and for understanding the evolutionary transformation in each phylum of Lophophorata. The innervation of the lophophore in ctenostome bryozoan Flustrellidra hispida was studied using immunocytochemistry and confocal laser scanning microscopy. It has been demonstrated that this species has an outer nerve ring giving rise to the tentacle nerves. The outer nerve ring was earlier described in some ctenostomates and cyclostomates, but not as connected with nerves. The discovered feature of lophophore innervation in F. hispida suggests the evolutionary transformation from a hypothetical phoronida-like ancestor lophophore bearing a prominent outer nerve ring with numerous tentacle nerves emanating from it, to the complex bell-shaped lophophore of F. hispida with a well-pronounced outer nervous ring bearing a few tentacle nerves. The next one in this hypothetical row is the lophophore of the other ctenostomates and some cyclostomates with no ring-nerve connection and cheilostomates lophophore with no outer nerve ring at all.

Morphology of Stephanella hina (Bryozoa, Phylactolaemata): common phylactolaemate and unexpected, unique characters

Stephanella hina is a little studied freshwater bryozoan belonging to Phylactolaemata. It is currently the only representative of the family Stephanellidae, which in most reconstructions is early branching, sometimes even sister group to the remaining phylactolaemate families. The morphological and histological details of this species are entirely unknown. Consequently, the main aim of this study was to conduct a detailed morphological analysis of S. hina using histological serial sections, 3D reconstruction, immunocytochemical staining and confocal laser scanning microscopy techniques. The general morphology is reminiscent of other phylactolaemates; however, there are several, probably apomorphic, details characteristic of S. hina. The most evident difference lies in the lophophoral base, where the ganglionic horns/extensions do not follow the traverse of the lophophoral arms but bend medially inwards towards the mouth opening. Likewise, the paired forked canal does not fuse medially in the lophophoral concavity as found in all other phylactolaemates. Additional smaller differences are also found in the neuro-muscular system: the rooting of the tentacle muscle is less complex than in other phylactolaemates, the funiculus lacks longitudinal muscles, the caecum has smooth muscle fibres, latero-abfrontal tentacle nerves are not detected and the medio-frontal nerves mostly emerge directly from the circum-oral nerve ring. In the apertural area, several neurite bundles extend into the vestibular wall and probably innervate neurosecretory cells surrounding the orifice. These morphological characteristics support the distinct placement of this species in a separate family. Whether these characteristics are apomorphic or possibly shared with other phylactolaemates will require the study of the early branching Lophopodidae, which remains one of the least studied taxa to date.

Novel data on the innervation of the lophophore in adult phoronids (Lophophorata, Phoronida)

The structure of the lophophore nervous system may help clarify the status of the clade Lophophorata, whose monophyly is debated. In the current study, antibody labeling and confocal laser scanning microscopy revealed previously undescribed main nerve elements in the lophophore in adult phoronids: Phoronis australis and Phoronopsis harmeri. In both species, the nervous system includes a dorsal ganglion, a tentacle nerve ring, an inner nerve ring, intertentacular groups of perikarya, and tentacle nerves. The dorsal ganglion and tentacle nerve ring contain many serotonin-like immunoreactive perikarya of different sizes. The inner nerve ring is described for the first time in adult phoronids with complex lophophore. It contains a thin bundle of serotonin-like immunoreactive neurites. The tentacles possess abfrontal, frontal, and laterofrontal nerves. The abfrontal nerves originate from the tentacle nerve ring; the frontal tentacle nerves extend from the inner nerve ring in P. harmeri and from the intertentacular frontal nerves in P. australis. The intertentacular groups of perikarya are found in phoronids for the first time. These small nerve centers connect with neither the tentacle nerve ring nor the inner nerve ring, giving rise to the laterofrontal tentacle nerves. The discovery of the inner nerve ring in adult phoronids makes the architecture of the lophophore nervous system similar in all lophophorates and thereby supports the monophyly of this group. The presence of intertentacular nerves, perikarya, and groups of perikarya is a typical feature of the nervous system in lophophorate presumably coordinating movements of the tentacles and thereby increasing the efficiency of lophophore functioning.

Neuroanatomy of Hyalinella punctata: Common patterns and new characters in phylactolaemate bryozoans.

Studies on the bryozoan adult nervous system employing immunocytochemical techniques and confocal laser scanning microscopy are scarce. To gain a better view into the structure and evolution of the nervous system of the Phylactolaemata, the earliest extant branch and sister taxon to the remaining Bryozoa, this work aims to characterize the nervous system of Hyalinella punctata with immunocytochemical techniques and confocal laser scanning microscopy. The cerebral ganglion is located between the anus and the pharynx and contains a lumen. Two ganglionic horns and a circum-oral nerve ring emanate from the cerebral ganglion. The pharynx is innervated by a diffuse neural plexus with two prominent neurite bundles. The caecum is innervated by longitudinal neurite bundles and a peripheral plexus. The intestine is characterized by longitudinal and circular neurite bundles, mostly near the anus. Novel putative sensory cells were found in the foregut and intestine. The tentacle sheath is innervated by a diffuse neural plexus, which emanates from several neurite bundles from the cerebral ganglion, but also parts of the pharyngeal plexus. There are six tentacle neurite bundles of intertentacular origin. The retractor muscles are innervated by two thin neurite bundles. Several characters are described herein for the first time in Phylactolaemata: Longitudinal neurite bundles and a peripheral plexus of the caecum, putative sensory structures of the gut, retractor muscle innervation, specific duplicature band neurite bundles. The tentacle innervation differs from previous descriptions of phylactolaemates regarding the origin of the three abfrontal neurite bundles. In general, most organ systems are innervated by a diffuse plexus in phylactolaemates as opposed to gymnolaemates. In contrast to the Gymnolaemata, representatives of Phylactolaemata show a higher number of tentacle nerves. Although the plesiomorphic condition for zooidal features among bryozoans remains unclear, having a diffuse nerve plexus may represent an ancestral feature for freshwater bryozoans.

The serotonin-lir nervous system of the Bryozoa (Lophotrochozoa): a general pattern in the Gymnolaemata and implications for lophophore evolution of the phylum.

BackgroundSerotonin represents an evolutionary ancient neurotransmitter that is ubiquitously found among animals including the lophotrochozoan phylum Bryozoa, a group of colonial filter-feeders. Comparatively little is known on their nervous system, and data on their serotonin-lir nervous system currently are mostly limited to the basal phylactolaemates. Previous investigations indicated a common ground-pattern of the serotonin-lir nervous system in these animals, but in order to assess this on a larger scale, 21 gymnolaemate species from 21 genera were comparatively analysed herein.MethodsTwenty-one species from 21 gymnolaemate genera were analysed by immunocytochemical stainings and confocal laser scanning microscopy.ResultsIn all species the serotonin-lir signal is concentrated in the cerebral ganglion from where a nerve tract emanates laterally and traverses orally to engulf the foregut. Serotonin-lir perikarya are situated at the base of the tentacles that almost always correspond to the number of tentacles minus two. The oral side in almost all species shows three serotonin-lir perikarya followed by a ‘serotonergic gap’ that to our knowledge is not reflected in the morphology of the nervous system. Some species show additional serotonin-lir signal in tentacle nerves, visceral innervation and pore complexes. Paludicella articulata is exceptional as it shows signal in the latero-visceral nerves with serotonin-lir perikarya in the esophagus, parts of the tentacle sheath nerves as well as the frontal body wall around the parietal muscle bundles.ConclusionsIn general, the serotonin-lir nervous system in the Bryozoa shows a consistent pattern among its different clades with few deviations. Preliminary data on phylactolaemates suggest the presence of a ‘serotonergic gap’ similar to gymnolaemates. Both show a subset of oral tentacles and the remaining tentacles in gymnolaemates which correspond to the lateral tentacles of phylactolaemates. The lophophoral concavity lacks serotonin-lir perikarya indicating that due to their larger sizes and increased tentacle number, the horse-shoe shaped arrangement could represent an apomorphy of phylactolaemates.

Comparative morphology of the nervous system in three phylactolaemate bryozoans.

BackgroundThough some elements of the bryozoan nervous system were discovered 180 years ago, few studies of their neuromorphology have been undertaken since that time. As a result the general picture of the bryozoan nervous system structure is incomplete in respect of details and fragmentary in respect of taxonomic coverage.ResultsThe nervous system of three common European freshwater bryozoans – Cristatella mucedo, Plumatella repens (both with a horseshoe-shaped lophophore) and Fredericella sultana (with a circular lophophore) had numerous differences in the details of the structure but the general neuroarchitecture is similar. The nervous system of the zooid consists of the cerebral ganglion, a circumpharyngeal ring and lophophoral nerve tracts (horns), both sending numerous nerves to the tentacles, and the nerve plexuses of the body wall and of the gut. A number of the important details (distal branching of the additional radial nerve, pattern of distribution of nerve cells and neurites in the ganglion, etc.) were described for the first time. The number and position of the tentacle nerves in Cristatella mucedo was ascertained and suggestions about their function were made. The revealed distribution of various neuromediators in the nervous system allowed us to suggest functional affinities of some major nerves.ConclusionsDespite the basic similarity, both the ganglion and the lophophore nervous system in Phylactolaemata have a more complex structure than in marine bryozoans (classes Gymnolaemata and Stenolaemata). First of all, their neuronal network has a denser and more complex branching pattern: most phylactolaemates have two large nerve tracts associated with lophophore arms, they have more nerves in the tentacles, additional and basal branches emitting from the main radial nerves, etc. This, in part, can be explained by the horseshoe shape of the lophophore and a larger size of the polypide in freshwater species. The structure of the nervous system in Fredericella sultana suggests that it underwent a secondary simplification following the reduction of the lophophore arms. Colony locomotion in Cristatella mucedo is based on co-ordinated activity of two perpendicular muscle layers of the sole and the plexus of motor neurons sandwiched between them. The trigger of this activity and the co-ordination mechanism remain enigmatic.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-015-0112-2) contains supplementary material, which is available to authorized users.

Nitric Oxide-Mediated Modulation of Central Network Dynamics during Olfactory Perception.

Nitric oxide (NO) modulates the dynamics of central olfactory networks and has been implicated in olfactory processing including learning. Land mollusks have a specialized olfactory lobe in the brain called the procerebral (PC) lobe. The PC lobe produces ongoing local field potential (LFP) oscillation, which is modulated by olfactory stimulation. We hypothesized that NO should be released in the PC lobe in response to olfactory stimulation, and to prove this, we applied an NO electrode to the PC lobe of the land slug Limax in an isolated tentacle-brain preparation. Olfactory stimulation applied to the olfactory epithelium transiently increased the NO concentration in the PC lobe, and this was blocked by the NO synthase inhibitor L-NAME at 3.7 mM. L-NAME at this concentration did not block the ongoing LFP oscillation, but did block the frequency increase during olfactory stimulation. Olfactory stimulation also enhanced spatial synchronicity of activity, and this response was also blocked by L-NAME. Single electrical stimulation of the superior tentacle nerve (STN) mimicked the effects of olfactory stimulation on LFP frequency and synchronicity, and both of these effects were blocked by L-NAME. L-NAME did not block synaptic transmission from the STN to the nonbursting (NB)-type PC lobe neurons, which presumably produce NO in an activity-dependent manner. Previous behavioral experiments have revealed impairment of olfactory discrimination after L-NAME injection. The recording conditions in the present work likely reproduce the in vivo brain state in those behavioral experiments. We speculate that the dynamical effects of NO released during olfactory perception underlie precise odor representation and memory formation in the brain, presumably through regulation of NB neuron activity.

The nervous system of Paludicella articulata - first evidence of a neuroepithelium in a ctenostome ectoproct.

IntroductionComparatively few data are available concerning the structure of the adult nervous system in the Ectoprocta or Bryozoa. In contrast to all other ectoprocts, the cerebral ganglion of phylactolaemates contains a central fluid-filled lumen surrounded by a neuroepithelium. Preliminary observations have shown a small lumen within the cerebral ganglion of the ctenostome Paludicella articulata. Ctenostome-grade ectoprocts are of phylogenetic relevance since they are considered to have retained ancestral ectoproct features. Therefore, the ctenostome Paludicella articulata was analyzed in order to contribute to the basal neural bauplan of ctenostomes and the Ectoprocta in general.ResultsThe presence of a lumen and a neuroepithelial organization of the nerve cells within the cerebral ganglion are confirmed. Four tentacle nerves project from the cerebral ganglion into each tentacle. Three of the tentacle nerves (one abfrontal and two latero-frontal nerves) have an intertentacular origin, whereas the medio-frontal nerve arises from the cerebral ganglion. Six to eight visceral nerves and four tentacle sheath nerves are found to emanate from the cerebral ganglion and innervate the digestive tract and the tentacle sheath, respectively.ConclusionsThe situation in P. articulata corresponds to the situation found in other ctenostomes and supports the notion that four tentacle nerves are the ancestral configuration in Ectoprocta and not six as proposed earlier. The presence of a lumen in the ganglion represents the ancestral state in Ectoprocta which disappears during ontogeny in all except in adult Phylactolaemata and P. articulata. It appears likely that it has been overlooked in earlier studies owing to its small size.

The central and peripheral nervous system of Cephalodiscus gracilis (Pterobranchia, Deuterostomia)

Nervous systems are important in assessing interphyletic phylogenies because they are conservative and complex. Regarding nervous system evolution within deuterostomes, two contrasting hypotheses are currently discussed. One that argues in favor of a concentrated, structured, central nervous system in the last common ancestor of deuterostomes (LCAD); the other reconstructing a decentralized nerve net as the nervous system of the LCAD. Here, we present a morphological analysis of the nervous system of the pterobranch deuterostome Cephalodiscus gracilis Harmer, 1905 based on transmission electron microscopy, confocal laser scanning microscopy, immunohistochemistry, and computer-assisted 3D reconstructions based on complete serial histological sections. The entire nervous system constitutes a basiepidermal plexus. The prominent dorsal brain at the base of the mesosomal tentacles contains an anterior concentration of serotonergic neurons and a posterior net of neurites. Predominant neurite directions differ between brain regions and synapses are present, indicating that the brain constitutes a centralized portion of the nervous system. Main structures of the peripheral nervous system are the paired branchial nerves, tentacle nerves, and the ventral stalk nerve. Serotonergic neurites are scattered throughout the epidermis and are present as concentrations along the anterior border of the branchial nerves. Serotonergic neurons line each tentacle and project into the brain. We argue that the presence of a centralized brain in C. gracilis supports the hypothesis that a nerve center was present in the LCAD. Moreover, based on positional and structural similarity, we suggest that the branchial nerves in C. gracilis could be homologous to branchial nerves in craniates, a hypothesis that should be further investigated.

Putative lateral inhibition in sensory processing for directional turns

Computing targeted responses is a general problem in goal-directed behaviors. We sought the sensory template for directional turning in the predatory sea slug Pleurobranchaea californica, which calculates precise turn angles by averaging multiple stimulus sites on its chemotactile oral veil (Yafremava LS, Anthony CW, Lane L, Campbell JK, Gillette R. J Exp Biol 210: 561-569, 2007). Spiking responses to appetitive chemotactile stimulation were recorded in the two bilateral pairs of oral veil nerves, the large oral veil nerve (LOVN) and the tentacle nerve (TN). The integrative abilities of the peripheral nervous system were significant. Nerve spiking responses to punctate, one-site stimulation of the oral veil followed sigmoid relations as stimuli moved between lateral tentacle and the midline. Receptive fields of LOVN and TN were unilateral, overlapping, and oppositely weighted for responsiveness across the length of oral veil. Simultaneous two-site stimulation caused responses of amplitudes markedly smaller than the sum of corresponding one-site responses. Plots of two-site nerve responses against the summed approximate distances from midline of each site were markedly linear. Thus the sensory paths in the peripheral nervous system show reciprocal occlusion similar to lateral inhibition. This outcome suggests a novel neural function for lateral inhibitory mechanisms, distinct from simple contrast enhancement, in computation of both sensory maps and targeted motor actions.

Organogenesis during budding and lophophoral morphology of Hislopia malayensis Annandale, 1916 (Bryozoa, Ctenostomata)

BackgroundBryozoans represent a large lophotrochozoan phylum with controversially discussed phylogenetic position and in group relationships. Developmental processes during the budding of bryozoans are in need for revision. Just recently a study on a phylactolaemate bryozoan gave a comprehensive basis for further comparisons among bryozoans. The aim of this study is to gain more insight into developmental patterns during polypide formation in the budding process of bryozoans. Particular focus is laid upon the lophophore, also its condition in adults. For this purpose we studied organogenesis during budding and lophophoral morphology of the ctenostome bryozoan Hislopia malayensis.ResultsPolypide buds develop on the frontal side of the developing cystid as proliferation of the epidermal and peritoneal layer. Early buds develop a lumen bordered by the inner budding layer resulting in the shape of a two-layered sac or vesicle. The hind- and midgut anlagen are first to develop as outpocketing of the prospective anal area. These grow towards the prospective mouth area where a comparatively small invagination marks the formation of the foregut. In between the prospective mouth and anus the ganglion develops as an invagination protruding in between the developing gut loop. Lophophore development starts with two lateral ridges which form tentacles very early. At the lophophoral base, intertentacular pits, previously unknown for ctenostomes, develop. The ganglion develops a circum-oral nerve ring from which the tentacle nerves branch off in adult zooids. Tentacles are innervated by medio-frontal nerves arising directly from the nerve ring, and medio-frontal and abfrontal nerves which originate both from an intertentacular fork.ConclusionsWe are able to show distinct similarities among bryozoans in the formation of the different organ systems: a two-layered vesicle-like early bud, the ganglion forming as an invagination of the epidermal layer in between the prospective mouth and anal area, the digestive tract mainly forming as an outpocketing of the prospective anal area, and the lophophore forming from two lateral anlagen that first fuse on the oral and afterwards on the anal side. Future studies will concentrate on cyclostome budding to complement our knowledge on developmental patterns of bryozoans.

Two morphological sub-systems within the olfactory organs of a terrestrial snail

In the present work, we have re-visited the problem of the olfactory neural system organization in the terrestrial snail. By staining the tentacle's nerves and their intrinsic tracts in different points of the cerebral ganglia–tentacles system we have found that the relatively small part of the primary sensory neurons from the sensory pad (7–8%) send their axons directly to the cerebral ganglia. The axons terminated in the metacerebral neuropil which suggests these receptors being not chemosensory but rather mechanosensory neurons. Majority of the primary sensory neurons are synaptically switching in the areas outside the cerebral ganglia, i.e. digits, glomeruli, tentacular ganglion. No primary sensory neurons of the olfactory pad were projecting directly to the procerebrum — the putative centre of olfactory information processing. The afferent tract innervating the procerebrum neuropil originated from the interneurons located in the tentacle ganglion and its digits. Our results suggest the presence of two different sub-systems within the snail nose – mechanosensory and chemosensory – with two different projection targets.

Specific spatio-temporal activities in the cerebral ganglion of Incilaria fruhstorferi in response to superior and inferior tentacle nerve stimulation

In terrestrial gastropod mollusks (slugs and snails), olfaction is the dominant sensory modality guiding various kinds of behavior. Anatomical studies indicate that olfactory information is processed in the brain (the cerebral ganglion) in two lobes in particular: the procerebrum (PC) and the metacerebrum (MtC). This implies that olfactory functions emerge from simultaneous and cooperative processing in the PC and the MtC. However, no previous physiological study has investigated the activity in these two lobes simultaneously. In the present study, the activity evoked by electrical stimulation of the olfactory nerves, the superior and inferior tentacle nerves, was recorded optically from the whole cerebral ganglion of the terrestrial slug, Incilaria fruhstorferi. The results indicated that the evoked activity in the PC and the MtC showed two specific spatio-temporal patterns. First, when either set of nerves was stimulated, the activity of the medial neuropilar region of the MtC (the mMtC) always preceded the activity in the PC. Second, stimulation of the superior tentacle nerves activated the medial and lateral halves of the mMtC almost evenly, whereas stimulation of the inferior tentacle nerves activated the lateral half of the mMtC more strongly than the medial half. These results suggest that the activated region of the mMtC plays an important role in olfactory processing, especially with respect to the functional differences between the superior and inferior tentacles.

Selective calcium imaging of olfactory interneurons in a land mollusk

The procerebrum (PC) of the land slug Limax is the olfactory center involved in olfactory discrimination and learning. The PC contains two types of neurons, bursting (B) and nonbursting (NB) neurons. B neurons are local interneurons and show spontaneous bursting while NB neurons directly receive input from olfactory nerve fibers, and are possibly involved in odor coding. In order to observe activity of NB neurons, selective calcium imaging was made by retrograde staining of NB neurons with the calcium sensitive dye, rhod-2 AM, from the internal mass (IM) of the PC. The stained cells, presumably NB neurons, appeared to be mostly silent, but showed transient calcium elevation in response to electrical stimulus to the tentacle nerve. Stimulation of the tentacle with an odorant also evoked calcium events in some of the stained cells. These included one or multiple calcium events, and these events were synchronized with each other. These results revealed characteristics of the activity of NB neurons.

Immunocytochemistry of the neuromuscular systems ofLoxosomella vivipara andL. parguerensis (Entoprocta: Loxosomatidae)

Little detailed information exists on the anatomy of the nervous system and the musculature of Entoprocta. Herein we describe the distribution of the neurotransmitters RFamide and serotonin as well as the myo-anatomy of adults and asexually produced budding stages of the solitary entoproct species Loxosomella vivipara and L. parguerensis using immunocytochemistry and epifluorescence as well as confocal microscopy. The development of the RFamidergic and serotonergic nervous system starts in early budding stages. In the adults, RFamide is present in the bilateral symmetric cerebral ganglion, a pair of oral nerves that innervate two pairs of nerve cell clusters in the heel of the foot, a pair of aboral nerves, the paired lateral nerves, the calyx nerves, the atrial ring nerve, the tentacle nerves, the stomach nerves, and the rectal nerves. Serotonin is only found in the cerebral ganglion, the oral nerves, and in the tentacle nerves. Some differences in the distribution of both neurotransmitters were found between L. vivipara and L. parguerensis and are most obvious in the differing number of large serotonergic perikarya associated with the oral nerves. Nerves arising from the cerebral ganglion and running in a ventral direction have not been described for Entoprocta before, and the homology of these to the ventral nerve cords of other Spiralia is considered possible. The body musculature of both Loxosomella species comprises longitudinal and diagonal muscles in the foot, the stalk, and the calyx. We found several circular muscles in the calyx. The stalk and parts of the foot and the calyx are surrounded by a fine outer layer of ring muscles. In addition to the congruent details regarding the myo-anatomy of both species, species-specific muscle structures could be revealed. The comparison of our data with recent findings of the myo-anatomy of two Loxosoma species indicates that longitudinal and diagonal body muscles, atrial ring muscles, tentacle muscles, esophageal and rectal ring muscles, as well as intestinal and anal sphincters are probably part of the ancestral entoproct muscle bauplan.

Ultrastructure of the tentacle nerve plexus and putative neural pathways in sea anemones

Abstract. Neurons of sea anemone tentacles receive stimuli via sensory cells and process and transmit information via a plexus of nerve fibers. The nerve plexus is best revealed by scanning electron microscopy of epidermal peels of the tentacles. The nerve plexus lies above the epidermal muscular layer where it appears as numerous parallel longitudinal and short interconnected nerve fibers in Calliactis parasitica. Bipolar and multipolar neurons are present and neurites form interneuronal and neuromuscular synaptic contacts. Transmission electron microscopy of cross sections of tentacles of small animals, both C. parasitica and Aiptasia pallida, reveals bundles of 50–100 nerve fibers lying above groups of longitudinal muscle fibers separated by intrusions of mesoglea. Smaller groups of 10–50 slender nerve fibers are oriented at right angles to the circular muscle formed by the bases of the digestive cells. The unmyelinated nerve fibers lack any glial wrapping, although some bundles of epidermal fibers are partially enveloped by cytoplasmic extensions of the muscle cells; small gastrodermal nerve bundles lie between digestive epithelial cells above their basal myonemes. A hypothetical model for sensory input and motor output in the epidermal and gastrodermal nerve plexuses of sea anemones is proposed.