Cerebropleural Ganglia Research Articles

Neuropeptides regulate shell growth in the Mediterranean mussel (Mytilus galloprovincialis)

In bivalves, which are molluscs enclosed in a biomineralized shell, a diversity of neuropeptide precursors has been described but their involvement in shell growth has been largely neglected. Here, using a symmetric marine bivalve, the Mediterranean mussel (Mytilus galloprovincialis), we uncover a role for the neuroendocrine system and neuropeptides in shell production. We demonstrate that the mantle is rich in neuropeptide precursors and that a complex network of neuropeptide-secreting fibres innervates the mantle edge a region highly involved in shell growth. We show that shell damage and shell repair significantly modify neuropeptide gene expression in the mantle edge and the nervous ganglia (cerebropleural ganglia, CPG). When the CPG nerve commissure was severed, shell production was impaired after shell damage, and modified neuropeptide gene expression, the spatial organization of nerve fibres in the ganglia and mantle and biomineralization enzyme activity in the mantle edge. Injection of CALCIa and CALCIIa peptides rescued the impaired shell repair phenotype providing further support for their role in biomineralization. We propose that the regulatory mechanisms identified are likely to be conserved across bivalves and other shelled molluscs since they all share a similar nervous system, a common mantle biomineralization toolbox, and shell structure.

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina

The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomic data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of <10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 h. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm.

Qualitative and quantitative insights into the 3D microanatomy of the nervous ganglia of Scrobicularia plana (Bivalvia: Tellinoidea: Semelidae)

ABSTRACTThe nervous system of bivalves is bilaterally symmetrical and consists of interconnected cerebropleural, pedal and visceral ganglia, which may be partially to totally fused. We studied the microanatomy of the ganglia of Scrobicularia plana using three-dimensional (3D) reconstruction. We also examined whether intersex differences in the neural structure exist. Each type of ganglion had a characteristic 3D shape, and the cerebropleural ganglia shape was slightly asymmetrical. The visceral, pedal and cerebropleural ganglia are progressively smaller in volume, but only the pedal ganglion volume was positively correlated with the animal’s length, height or width; suggesting functional implications. As to total surface area, correlations were found for the cerebropleural and visceral ganglia, but it was the visceral that consistently showed strong positive correlations with each biometric parameter. The medulla may often penetrate the cortex and touch the capsule in areas that (contrary to what might be expected) are not connected with emerging nerves. Despite the differences in volume and surface area among ganglia, the volume ratio of cortex/medulla is fairly stable (c. 1.5), suggesting a functional optimum. Finally, we conclude that the ganglia of males and females do not show significant quantitative differences.

Taxonomic review of the genusPleurobranchaea(Gastropoda: Pleurobranchoidea) from Brazil, with description of a new species

Based on a literature review and anatomical analysis we recognize 17 valid species of the genus Pleurobranchaea worldwide and summarize their characters. Many of these species have been insufficiently described and many have numerous synonyms. Four species have been recorded from the western Atlantic: P. agassizii, P. obesa, P. tarda and P. inconspicua. Pleurobranchaea inconspicua was originally described based on only one specimen from northern Brazil. This study gives a detailed redescription of P. inconspicua, based on material from Brazil, and of the similar P. gela, based on specimens from West Africa, to clarify their taxonomic validity. A new species from the Brazilian coast, P. spiroporphyra n. sp., is described. The traditional characters used in characterization of Pleurobranchaea species are evaluated and novel characters proposed. Traditionally, anatomy of the male apparatus, particularly the shape of the cross section of the cuticular stylet, was considered the most useful character to distinguish species. We describe different portions of the cuticular stylet of P. inconspicua and show that shape and size vary along its length. These characters seem not to be species-specific if only one portion of the cuticular stylet is examined. Other features, such as the position of the aperture of the salivary duct, the position and presence or absence of extrinsic and intrinsic odontophoric muscles, the configuration of nerves of the cerebropleural and pedal ganglia and the shape of the seminal receptacle, can be used to distinguish species. Pleurobranchaea spiroporphyra can be distinguished from other species by having a bright orange labial cuticle in front of the jaws, by a welldeveloped odontophore muscle m2b, by a laterally flattened, purple cuticular stylet that coils 12–14 times inside the penial sac and shows a thin crest close to the gonopore.

3D- microanatomy of the semiterrestrial slug Gascoignella aprica Jensen, 1985—a basal plakobranchacean sacoglossan (Gastropoda, Panpulmonata)

The monophyly of the panpulmonate, usually marine benthic Sacoglossa—and its basal division into shelled Oxynoacea and shell-less Plakobranchacea—is undisputed, but family relationships are in doubt. Of particular interest is the potentially basal plakobranchacean family Platyhedylidae, comprising morphologically aberrant members lacking head tentacles or body appendages. Herein we re-describe the type species of the genus Gascoignella, G. aprica Jensen, 1985, from Hong Kong. Morphological data was generated by three-dimensional reconstruction from serial semi-thin sections using Amira software. Our microanatomical results largely confirm the original description. The anterior digestive system is sacoglossan-like but modified, e.g. the ascus is not demarcated externally and pharyngeal pouches are lacking. The digestive gland is bipartite, with two rami separated by a longitudinal, muscular, median septum, but fused in the rear end. The postpharyngeally situated nerve ring contains fused cerebropleural ganglia; the short visceral loop has three ganglia. Two major cerebral nerves were identified as rhinophoral and labiotentacular nerves, innervating sensory areas on the head velum. Gascoignella aprica is a hermaphrodite with a truly androdiaulic genital system of which some originally ambiguous characters are clarified. Bursa and prostate insert into a fertilization chamber proximal to a sac-like albumen gland and a tubular mucus gland. The cephalic copulatory apparatus contains a penis armed with a short and straight stylet and an accessory gland of unclear function; the presumed mode of sperm transfer is discussed. A well-developed heart and a large H-shaped kidney are present; the nephroduct opens into the intestine. Epidermal glands and body tissues are described for the first time. The presence of a unique longitudinal, median septum is considered diagnostic for Platyhedylidae, multiple further apomorphies are given. Morphological evidence supports the molecular phylogenetic hypothesis that the Platyhedylidae could be a basal non-shelled sacoglossan lineage.

Network interneurons underlying ciliary locomotion in Hermissenda.

In the nudibranch mollusk Hermissenda, ciliary locomotion contributes to the generation of two tactic behaviors. Light elicits a positive phototaxis, and graviceptive stimulation evokes a negative gravitaxis. Two classes of light-responsive premotor interneurons in the network contributing to ciliary locomotion have been recently identified in the cerebropleural ganglia. Aggregates of type I interneurons receive monosynaptic excitatory (I(e)) or inhibitory (I(i)) input from identified photoreceptors. Type II interneurons receive polysynaptic excitatory (II(e)) or inhibitory (II(i)) input from photoreceptors. The ciliary network also includes type III inhibitory (III(i)) interneurons, which form monosynaptic inhibitory connections with ciliary efferent neurons (CENs). Illumination of the eyes evokes a complex inhibitory postsynaptic potential, a decrease of I(i) spike activity, a complex excitatory postsynaptic potential, and an increase of I(e) spike activity. Here, we characterized the contribution of identified I, II, and III(i) interneurons to the neural network supporting visually guided locomotion. In dark-adapted preparations, light elicited an increase in the tonic spike activity of II(e) interneurons and a decrease in the tonic spike activity of II(i) interneurons. Fluorescent dye-labeled type II interneurons exhibited diverse projections within the circumesophageal nervous system. However, a subclass of type II interneurons, II(e(cp)) and II(i(cp)) interneurons, were shown to terminate within the ipsilateral cerebropleural ganglia and indirectly modulate the activity of CENs. Type II interneurons form monosynaptic or polysynaptic connections with previously identified components of the ciliary network. The identification of a monosynaptic connection between I(e) and III(i) interneurons shown here suggest that they provide a major role in the light-dependent modulation of CEN spike activity underlying ciliary locomotion.

Insemination by a kiss? Interactive 3D-microanatomy, biology and systematics of the mesopsammic cephalaspidean sea slug Pluscula cuica Marcus, 1953 from Brazil (Gastropoda: Euopisthobranchia: Philinoglossidae)

Increasing molecular evidence suggests that the phylogeny of euthyneuran gastropods differs greatly from centenary textbook concepts. The presence, homology and evolution of characters in major subgroups thus need to be reinvestigated. Traditionally basal opisthobranch Cephalaspidea (“head-shield snails and slugs”) were pruned to a new taxon concept, with benthic euopisthobranch and tentacle-bearing cephalaspidean lineages basal to burrowing, head-shield bearing philinoidean species. Among the latter, mesopsammic “microslug” lineages evolved at least twice. Herein we explore in 3D microanatomical detail the putatively basal philinoglossan Pluscula cuica (Marcus, Boletim da Faculdade de Filosofia, Ciencias e Letras. Universidade de Sao Paulo 164:165–203, 1953a) from its type locality in Brazil. The species possesses several “accessory” ganglia and a reduced posterior mantle cavity that retains some putative shell-building tissue and an osphradium. The hermaphroditic, monaulic genital system opens in a posterior position; it retains a bursa copulatrix but lacks a distinct receptaculum seminis. Autosperm is transferred to the cephalic copulatory organ via an external sperm groove, not through the hemocoel, as suggested in the original description. The penis opens through the oral tube, sperm is transferred by a “kiss”. A conspicuous yellow gland is discussed as a modified Blochmann’s gland. Retaining several putative symplesiomorphies with philinoids, Pluscula is discussed as the most basal offshoot in meiofaunal Philinoglossidae. However, the supposed “primitiveness” of the fused rather than separate cerebropleural ganglia and the triganglionate rather than pentaganglionate visceral nerve cord was based on misobservations. Higher categories such as Philinoglossacea for Philinoglossidae, and a separate family Plusculidae for P. cuica are no longer warranted. Inner cephalaspidean relationships and a scenario of more or less successive philinoglossid adaptation to meiofaunal environments should be investigated by molecular studies with more comprehensive taxon sampling.

Sacoglossa or Acochlidia? 3D reconstruction, molecular phylogeny and evolution of Aitengidae (Gastropoda: Heterobranchia)

The amphibious ‘bug-eating slug’ Aiteng ater Swennen & Buatip, 2009 shows a worm-like, compact body shape lacking any cephalic tentacles or body processes. Anatomically it has been described as showing an unusual mix of sacoglossan and acochlidian characters, thus the systematic affinities are uncertain. The species is redescribed here with an integrative microanatomical and molecular approach. All major organ systems were three-dimensionally reconstructed from serial histological sections using AMIRA software. Aiteng ater has a prepharyngeal nerve ring with separate cerebral and pleural ganglia rather than cerebro-pleural ganglia, and no sacoglossan-like ascus is detectable histologically. The radula is triseriate rather than uniseriate, showing one lateral tooth on each side of the rhachidian tooth. A well-developed two-chambered heart is present. The vas deferens in A. ater splits off distal to the female glands. The intestine is short and opens into a small mantle cavity. Long cavities in the connective tissue are remains of dissolved calcareous spicules. Only a few characters thus remain to support a closer relationship of A. ater to Sacoglossa, i.e. the Gascoignella-like body shape lacking cephalic tentacles, the presence of an elysiid-like system of dorsal vessels, and an albumen gland consisting of follicles. Additionally we describe in microanatomical detail an equally small and vermiform new aitengid species from Japan. Aiteng mysticus n. sp. differs from A. ater in habitat, body size and colour, central nervous system and presence of a kidney. Both aitengid species resemble acochlidians in the retractibility of the head, by possessing calcareous spicules, a prepharyngeal nerve ring with separated cerebral and pleural ganglia, a triseriate radula with an ascending and descending limb, but without sacoglossan-like ascus, and a special diaulic reproductive system. The prominent rhachidian tooth of Aitengidae, which is used to pierce insects and pupae in A. ater, and the large, laterally situated eyes closely resemble the anatomy of members of the limnic Acochlidiidae. The acochlidian nature of Aiteng is strongly indicated by our molecular analysis, in which it forms a basal hedylopsacean offshoot or the sister clade to limnic Acochlidiidae and brackish or marine Pseudunelidae within Hedylopsacea. Such a topology would, however, imply that Aitengidae have lost the most characteristic acochlidian apomorphy, the subdivision of the body into a headfoot complex and a free, elongated visceral hump. Also, the absence of cephalic tentacles gives the Aitengidae an appearance that is very different to other, strictly aquatic Acochlidia. Differences of the external morphology and the internal anatomy are discussed in the light of a habitat shift of Aitengidae within the Acochlidia.

Neactaeonina argentina, New Species, and Family Placement of the GenusNeactaeoninaThiele, 1912 (Mollusca: Gastropoda)

Neactaeonina argentina, a new species from the outer Argentine shelf off Buenos Aires province, is described. The species is characterized by the elongate, low-spired shell, with shouldered, nearly straight whorls, deep suture, and sculpture composed of numerous, broad, flat spiral cords. The radula is similar to that previously described for the type species, Neactaeonina cingulata Thiele, 1912, except in having a greater number of teeth. The present study provides the first information on the anatomy and histology of the genital system, and details of the radula anatomy of Neactaeonina, both unknown to date. The study of anatomical characters demonstrates that Neactaeonina has clear similarities with other Acteonidae, with a combination of features present in Acteon (such as the presence of a strongly calcified shell, with well-developed operculum; mantle cavity opening anteriorly; nervous system with only the cerebropleural ganglia fused; a single allosperm sac associated with the female system; radula without cardinal tooth; and oral cuticle with rod-like processes); and some characters present in acteonid genera other than Acteon (e.g., the number and morphology of teeth, and processes of the oral cuticle). However, Neactaeonina shows some characters thus far not reported for any other genus currently placed into the Acteonidae: salivary glands with laminar structure, male genital system with an open portion, a single allosperm sac opening distally, and a flat and triangular copulatory organ.

Localization of serotonin in neuronal ganglia of the freshwater pearl mussel, Hyriopsis (Hyriopsis) bialata

We investigated the histological features of neuronal ganglia and the localization of serotonin in the unionid mussel Hyriopsis (Hyriopsis) bialata in Thailand. The long-term goal of this study is to apply endocrine manipulation to enhance the synchronous release of larvae for in vitro culture of this freshwater mussel and to increase larval survival during settlement. Unlike the case in other bivalves, the cerebropleural ganglia in H. bialata were completely fused to form a single pear-shaped structure, and the pedal ganglia were situated within the visceral mass rather than in the foot. In all three ganglia of this mussel, the neuronal cell bodies were clustered at the periphery of the ganglia, and the nerve fibres were bundled in the central region. Of the three types of cells present in the ganglia, the large neurons were the only ones to stain with paraldehyde fuchsin, indicating a neurosecretory function. These same neurons were also intensely reactive with anti-serotonin, demonstrating their dual function. An intense staining of anti-serotonin was also observed in the nerve fibres and termini, particularly in the visceral ganglia. This suggests that serotonin may be conveyed from the large neurons within the visceral ganglion via an axonal route to reach the target reproductive organs, one of which is the demibranch, to stimulate larval release in this mussel.

5-HT and GABA Modulate Intrinsic Excitability of Type I Interneurons in Hermissenda

The sensory neurons (photoreceptors) in the visual system of Hermissenda are one site of plasticity produced by Pavlovian conditioning. A second site of plasticity produced by conditioning is the type I interneurons in the cerebropleural ganglia. Both photoreceptors and statocyst hair cells of the graviceptive system form monosynaptic connections with identified type I interneurons. Two proposed neurotransmitters in the graviceptive system, serotonin (5-HT) and gamma-aminobutyric acid (GABA), have been shown to modify synaptic strength and intrinsic neuronal excitability in identified photoreceptors. However, the potential role of 5-HT and GABA in plasticity of type I interneurons has not been investigated. Here we show that 5-HT increased the peak amplitude of light-evoked complex excitatory postsynaptic potentials (EPSPs), enhanced intrinsic excitability, and increased spike activity of identified type I(e(A)) interneurons. In contrast, 5-HT decreased spike activity and intrinsic excitability of type I(e(B)) interneurons. The classification of two categories of type I(e) interneurons was also supported by the observation that 5-HT produced opposite effects on whole cell steady-state outward currents in type I(e) interneurons. Serotonin produced a reduction in the amplitude of light-evoked complex inhibitory PSPs (IPSPs), increased spontaneous spike activity, decreased intrinsic excitability, and depolarized the resting membrane potential of identified type I(i) interneurons. In contrast to the effects of 5-HT, GABA produced inhibition in both types of I(e) interneurons and type I(i) interneurons. These results show that 5-HT and GABA can modulate the intrinsic excitability of type I interneurons independent of the presynaptic effects of the same transmitters on excitability and synaptic efficacy of photoreceptors.

Computer-based 3D anatomical reconstruction and systematic placement of the mesopsammic gastropod Platyhedyle denudata Salvini-Plawen, 1973 (Opisthobranchia, Sacoglossa)

The small marine interstitial gastropod species Platyhedyle denudata Salvini-Plawen, 1973 has a dorsoventrally flattened, worm-like body without a shell and head tentacles; its systematic position was enigmatic. Wawra's [1979. Zur systematischen Stellung von Platyhedyle denudata Salvini-Plawen, 1973 (Opisthobranchia, Gastropoda). Z. Zool. Syst. Evolutionsforsch. 17, 221–225] anatomical redescriptions based on paraffin-embedded histology preparations indicated the original description of P. denudata to be erroneous in many essential features. The present study uses newly collected specimens from the Mediterranean type locality, Secche della Meloria, Italy, for serial semi-thin histological sectioning. All major organ systems of P. denudata are reconstructed three-dimensionally with AMIRA software. Platyhedyle denudata is a simultaneous hermaphrodite with an androdiaulic, complex genital system lacking a receptaculum seminis. A heart could not be detected; the nephric duct opens into the intestine. The central nervous system is postpharyngeal and the cerebropleural ganglia are completely fused. The visceral loop is short, showing three distinct ganglia which connect to an additional, presumably osphradial ganglion, and to a posteriorly situated genital ganglion. Aggregations of precerebral accessory ganglia are present. The originally described jaws could not be detected. An ascus for storage of worn radula teeth clearly indicates that P. denudata is a sacoglossan. The digestive gland of the mesopsammic P. denudata resembles that of the depressed, tentacle-less semi-terrestrial sacoglossan Gascoignella aprica Jensen, 1985 by forming two long branches that are connected at the rear end. In G. aprica, the digestive-gland rami are separated by a unique median septum, which is described here, too, as part of the muscle system in P. denudata. The shared, most likely synapomorphic presence of this septum strongly supports the monophyly of a Platyhedyle+ Gascoignella clade in a sacoglossan family Platyhedylidae.

Development of Embryonic and Larval Cells Containing Serotonin, Catecholamines, and FMRFamide-Related Peptides in the Gastropod MolluscPhestilla sibogae

The present immunocytochemical study provides one of the first detailed descriptions of the development of cells containing a variety of neurotransmitters during much of the larval life of a nudibranch gastropod. Throughout much of early development, serotonergic cells were located only in the apical organ; as larvae approached metamorphosis, serotonergic cells were also detected in the cerebropleural and pedal ganglia. Cells exhibiting tyrosine hydroxylase immunoreactivity (indicative of catecholamine synthesis) were first located near the mouth but by late embryonic stages were also located in the apical organ and near the velum and eyes. By late larval stages, numerous catecholaminergic cells were found in the foot, with concentrations in the propodium. Finally, the first cells exhibiting FMRFamide immunoreactivity were detected posterior to the neuropil of the cerebropleural ganglia in the early embryo. Fibers that presumably originated from these cells subsequently invaded the cerebral and pedal ganglia and the apical organ. By early larval stages, a second pair of peptidergic neurons was located near the first pair, and additional peptidergic neurons were located in the apical organ and peripheral positions in the foot and medial and dorsal to the eyes. In addition to providing a unique phyletic perspective to our understanding of gastropod neural development, the present study also sets the stage for future studies into changes in the nervous system as this gastropod undergoes metamorphosis.

Serotonin-Immunoreactive CPT Interneurons inHermissenda: Identification of Sensory Input and Motor Projections

Serotonin immunoreactive (5-HT-IR) neurons identified as cerebropleural ganglion triplets (CPTs) in Hermissenda may be homologues of 5-HT-IR neurons identified in other opisthobranch molluscs. In studies of isolated nervous systems and semi-intact preparations we used a combination of immunohistochemical techniques and fluorescent labeling with Lucifer yellow to identify 5-HT-IR CPT neurons after investigating sensory inputs and motor neuron projections. Here we show that identified 5-HT-IR CPT interneurons receive sensory input from mechanoreceptors and photoreceptors. In semi-intact preparations with intact pedal nerves P1 and P2, cutaneous stimulation of the middle or tail regions of the foot with calibrated von Frey hairs elicited spikes recorded from identified CPT interneurons. Illumination of the eyes evoked a small complex excitatory postsynaptic potential (EPSP) and resulted in a modest increase in the spike discharge of CPT interneurons. Immunostaining of Lucifer yellow-labeled neurons revealed that CPT interneurons projected an axonal process to the contralateral pedal ganglion. Depolarization of CPT interneurons with extrinsic current evoked EPSPs and spikes recorded from identified VP2 pedal neurons, motor neurons previously shown to elicit movement of the anterior foot. Extrinsic current stimulation of CPT interneurons in semi-intact preparations evoked movement of the anterior foot but did not facilitate ciliary activity or evoke PSPs recorded in identified VP1 ciliary motor neurons. Our results show that CPT neurons are polysensory interneurons that contribute to reflexive foot contractions in Hermissenda.

Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of 125I-insulin and of insulin-related factor-1 (125I-IGF-1) by plasma membranes of the rat liver and brain. The IC50 value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (104 ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC50 value in the case of 125I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 104 ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.

Morphological characteristics and central projections of two types of interneurons in the visual pathway of Hermissenda.

The synaptic interactions between photoreceptors in the eye and second-order neurons in the optic ganglion of the nudibranch mollusk Hermissenda are well characterized. However, the higher-order neural circuitry of the visual system, consisting of cerebropleural interneurons that receive synaptic input from photoreceptors and project to pedal motor neurons that mediate visually guided behaviors, is only partially understood. In this report we have examined the central projections of two identified classes of cerebropleural interneurons that receive excitatory or inhibitory synaptic input from identified photoreceptors. The classification of the interneurons was based on both morphological and electrophysiological criteria. Type I interneurons received monosynaptic excitatory or inhibitory synaptic input from identified photoreceptors and projected to postsynaptic targets within the cerebropleural ganglion. Type II interneurons, characterized here for the first time, received polysynaptic excitatory or inhibitory synaptic input from identified photoreceptors and projected to postsynaptic targets in either the ipsilateral pedal ganglion or the contralateral cerebropleural ganglion. Type I interneurons exhibited unique intraganglionic projections to different regions of the cerebropleural ganglion, depending on whether they received excitatory or inhibitory synaptic input from identified photoreceptors. Type I interneurons that received monosynaptic excitatory input from identified B photoreceptors terminated near the cerebropleural commissure and had multiple regions of varicosities located at branches that projected from the primary axon. Type I interneurons that received monosynaptic inhibitory input from identified B photoreceptors projected to the anterior cerebropleural ganglion and exhibited varicosities localized to the terminal region of the primary axonal process. Type II interneurons that received polysynaptic inhibitory input from identified photoreceptors projected to the contralateral cerebropleural ganglion. Most type II interneurons that projected to the pedal ganglia received polysynaptic excitatory input from identified photoreceptors. These results indicate that there is at least one additional interneuron in the higher-order visual circuit between type I interneurons and pedal motor neurons responsible for the generation of phototactic locomotion in Hermissenda.

Histochemical survey of transmitters in the central ganglia of the gastropod mollusc Phestilla sibogae.

The aeolid nudibranch Phestilla sibogae is well studied in terms of its larval nervous system and neuronal involvement in metamorphosis. Central neurones in the adult have also been identified anatomically and electrophysiologically. We describe the neurotransmitter contents of these neurones and provide details of neuritic projections and developmental changes during growth (3 to 18 mm body length). Central ganglia from specimens of all sizes contained 100-115 serotonin-immunoreactive neurones, some of which appeared to be homologues of cells identified in other gastropods. Tyrosine hydroxylase immunoreactivity and aldehyde-induced fluorescence marked a common set of 28-30 catecholaminergic neurones located anteriorly in the cerebropleural ganglia and laterally in the pedal ganglia. Ganglionic neuropile and nerve trunks also contained many catecholaminergic fibres. About 65-100 intensely labelled FMRFamide-immunoreactive neurones were located symmetrically throughout the central ganglia, although one population was located only in the right pedal ganglion. Another 40-45 FMRFamide-immunoreactive neurones were weakly or variably stained. Central ganglia also contained 27-29 intensely labelled pedalpeptide-immunoreactive neurones, including those that were apparently homologues of cells previously described in Tritonia diomedea, and 16-19 weakly labelled pedal-peptide-immunoreactive neurones, including giant cerebropleural neurones coexhibiting FMRFamide immunoreactivity. Little cell addition involving any transmitter phenotype occurred as animals grew in body length, body growth being accommodated by growth in the size of individual cells, consistent with an approximate doubling in the size of the ganglia themselves.

Neuroethology ofMelibe leoninaSwimming Behavior

SYNOPSIS. The nudibranch Melibe leonina swims by rhythmically flexing its body from side to side at a frequency of 1 cycle every 2‐5 sec. Melibe swim spontaneously, when they are dislodged from the substrate, or when they come in contact with predatory seastars, such as Pycnopodia helianthoides. Intracellular recordings obtained from semi-intact swimming Melibe reveal a population of ;15 swim motoneurons (SMNs) in each pedal ganglion. In general, SMNs in one pedal ganglion fire out-of-phase with SMNs in the opposite pedal ganglion, resulting in rhythmic side-to-side bending movements. In isolated brains, recordings from SMNs yield similar results, indicating the existence of a swim central pattern generator (CPG). There is no evidence for synaptic interactions between SMNs and either inhibiting or exciting SMNs has no impact on the swim pattern. The SMNs are driven by a CPG consisting of 4 interneurons; 2 in the cerebropleural ganglia and 1 in each pedal ganglion. Appropriate bursting activity in the swim interneurons is necessary for swimming to occur. Either hyperpolarization or depolarization of any of the 4 CPG interneurons disrupts the normal swim pattern. Swimming behavior, and the fictive swim motor program expressed by the isolated brain, are inhibited by light and nitric oxide donors. NADPH-diaphorase staining and nitric oxide synthase (NOS) immunocytochemistry of Melibe brains suggests the source of nitric oxide might be a pair of bilaterally symmetrical cells located in the cerebropleural ganglia.

Identifiable nitrergic neurons in the central nervous system of the nudibranch Melibe leonina localized with NADPH-diaphorase histochemistry and nitric oxide synthase immunoreactivity.

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase (NOS). In this study, we used two different techniques-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry-to demonstrate that NOS is present in a pair of identifiable cells in the central nervous system of the nudibranch Melibe leonina. In the Melibe brain, NADPH-d histochemistry revealed only a single pair of bilaterally symmetrical cells in the cerebropleural ganglia. NOS activity also was found in the neuropil of the cerebral, pedal, and buccal ganglia; in the tentacles of the oral hood; in the sensory end of the rhinophores; and in the epithelial tissue of the mouth, preputium, and glans penis. Immunocytochemistry using NOS antisera corroborated the results of the NADPH-d histochemistry by staining the same two cells in the cerebropleural ganglia. Each of these identifiable nitrergic neurons projects into the ipsilateral pedal ganglion. Because the pedal ganglia play a critical role in the control of locomotion, our results provide morphological evidence suggesting that NO may influence swimming or crawling in Melibe leonina.

POLYCHLORINATED BIPHENYLS ARE SELECTIVELY NEUROTOXIC IN THE DEVELOPING SPISULA SOLIDISSIMA EMBRYO

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that accumulate to toxic levels in the food chain. Using Spisula solidissima (surf clam) embryos as a developmental model, it was shown that Aroclor 1254 specifically targets two neuronal structures during embryonic development. Embryos were exposed to 1, 10, or 100 ppm Aroclor 1254 or an acetone vehicle control posthatching for 24, 48, and 72 h. Embryos labeled with a serotonin antibody or a neural antigen antibody and a rhodamine-conjugated secondary antibody were viewed by confocal microscopy. The cerebropleural ganglion showed a decrease both in serotonin production and in the size of the serotonin-synthesizing region upon exposure to 10 and 100 ppm Aroclor 1254. These decreases were detectable as early as 48 h postfertilization. When exposed to 100 ppm Aroclor 1254, the primitive neural plexus, which coordinates the movements of the mouth and velum, showed a delay in onset and cessation of expression of a molluscan-specific neural antigen. Exposure to Aroclor 1254 did not affect the overall growth and morphology of the embryos. In addition, analyses of total protein profiles and heat-shock protein 70 levels showed that exposure to Aroclor 1254 did not trigger protein degradation or cause a stress or shock response. These results show that exposure of Spisula embryos to Aroclor 1254 specifically targets neurogenesis while having no effect on the overall growth of the embryo.