The distribution and evolutionary dynamics of dopaminergic neurons in molluscs.

Dopamine is one of the most versatile neurotransmitters in invertebrates. It's distribution and plethora of functions is likely coupled to feeding ecology, especially in Euthyneura (the largest clade of molluscs), which presents the broadest spectrum of environmental adaptations. Still, the analyses of dopamine-mediated signaling were dominated by studies of grazers. Here, we characterize the distribution of dopaminergic neurons in representatives of two distinct ecological groups: the sea angel - obligate predatory pelagic mollusc Clione limacina (Pteropoda, Gymnosomata) and its prey - the sea devil Limacina helicina (Pteropoda, Thecosomata) as well as the plankton eater Melibe leonina (Nudipleura, Nudibranchia). By using tyrosine hydroxylase-immunoreactivity (TH-ir) as a reporter, we showed that the dopaminergic system is moderately conservative among euthyneurans. Across all studied species, small numbers of dopaminergic neurons in the central ganglia contrast to significant diversification of TH-ir neurons in the peripheral nervous system, primarily representing sensory-like cells, which predominantly concentrated in the chemotactic areas and projecting afferent axons to the central nervous system. Combined with α-tubulin immunoreactivity, this study illuminates the unprecedented complexity of peripheral neural systems in gastropod molluscs, with lineage-specific diversification of sensory and modulatory functions.

Illustrated Neuroanatomy of Ctenophores: Immunohistochemistry.

Ctenophores or comb jellies are representatives of an enigmatic lineage of early branching metazoans with complex tissue and organ organization. Their biology and even microanatomy are not well known for most of these fragile pelagic and deep-water species. Here, we present immunohistochemical protocols successfully tested on more than a dozen ctenophores. This chapter also illustrates neural organization in several reference species of the phylum (Pleurobrachia bachei, P. pileus, Mnemiopsis leidyi, Bolinopsis microptera, Beroe ovata, and B. abyssicola) as well as numerous ciliated structures in different functional systems. The applications of these protocols illuminate a very complex diversification of cell types comparable to many bilaterian lineages.

Scanning Electron Microscopy of Ctenophores: Illustrative Atlas.

Scanning electron microscopy (SEM) is a powerful tool for ultrastructural analyses of biological specimens at their surface. With comb jellies being very soft and full of water, many methodological difficulties limit their microanatomical studies via SEM. Here, we describe SEM protocols and approaches successfully tested on ctenophores Pleurobrachia bachei and Beroe abyssicola. Our SEM investigation revealed the astonishing diversity of ciliated structures in all major functional systems, different receptor types, and complex muscular architecture. These protocols can also be practical for various basal bilaterian lineages such as cnidarians.

Recording Cilia Activity in Ctenophores.

Pelagic ctenophores swim in the water with the help of eight rows of long fused cilia. Their entire behavioral repertoire is dependent to a large degree on coordinated cilia activity. Therefore, recording cilia beating is paramount to understanding and registering the behavioral responses and investigating its neural and hormonal control. Here, we present a simple protocol to monitor and quantify cilia activity in semi-intact ctenophore preparations (using Pleurobrachia and Bolinopsis as models), which includes a standard electrophysiological setup for intracellular recording.

A peripheral subepithelial network for chemotactile processing in the predatory sea slug Pleurobranchaea californica.

Many soft-bodied animals have extensive peripheral nervous systems (PNS) with significant sensory roles. One such, the sea slug Pleurobranchaea californica, uses PNS computations in its chemotactile oral veil (OV) in prey tracking, averaging olfactory stimuli across the OV to target likely source direction, or "stimulus place". This suggests a peripheral subepithelial network (SeN) interconnecting sensory sites to compute the directional average. We pursued anatomy and connectivity of previously described ciliated putative sensory cells on OV papillae. Scanning electron microscopy (SEM) confirmed paddle-shaped cilia in clusters. Anti-tubulin and phalloidin staining showed connections to branching nervelets and muscle fibers for contraction and expansion of papillae. Ciliary cell processes could not be traced into nerves, consistent with sensory transmission to CNS via secondary afferents. Anti-tyrosine hydroxylase-stained ciliated cells in clusters and revealed an at least partially dopaminergic subepithelial network interconnecting clusters near and distant, connections consistent with PNS averaging of multiple stimulated loci. Other, unidentified, SeN neurotransmitters are likely. Confirming chemotactile functions, perfusible suction electrodes recorded ciliary spiking excited by both mechanical and appetitive chemical stimuli. Stimuli induced sensory nerve spiking like that encoding stimulus place. Sensory nerve spikes and cilia cluster spikes were not identifiable as generated by the same neurons. Ciliary clusters likely drive the sensory nerve spikes via SeN, mediating appetitive and stimulus place codes to CNS. These observations may facilitate future analyses of the PNS in odor discrimination and memory, and also suggest such SeNs as potential evolutionary precursors of CNS place-coding circuitry in the segmented, skeletonized protostomes and deuterostomes.

A common modular design of nervous systems originating in soft-bodied invertebrates.

Nervous systems of vertebrates and invertebrates show a common modular theme in the flow of information for cost-benefit decisions. Sensory inputs are incentivized by integrating stimulus qualities with motivation and memory to affect appetitive state, a system of homeostatic drives, and labelled for directionality. Appetitive state determines action responses from a repertory of possibles and transmits the decision to a premotor system that frames the selected action in motor arousal and appropriate postural and locomotion commands. These commands are then sent to the primary motor pattern generators controlling the motorneurons, with feedback at each stage. In the vertebrates, these stages are mediated by forebrain pallial derivatives for incentive and directionality (olfactory bulb, cerebral cortex, pallial amygdala, etc.) interacting with hypothalamus (homeostasis, motivation, and reward) for action selection in the forebrain basal ganglia, the mid/hindbrain reticular formation as a premotor translator for posture, locomotion, and arousal state, and the spinal cord and cranial nuclei as primary motor pattern generators. Gastropods, like the predatory sea slug Pleurobranchaea californica, show a similar organization but with differences that suggest how complex brains evolved from an ancestral soft-bodied bilaterian along with segmentation, jointed skeletons, and complex exteroceptors. Their premotor feeding network combines functions of hypothalamus and basal ganglia for homeostasis, motivation, presumed reward, and action selection for stimulus approach or avoidance. In Pleurobranchaea, the premotor analogy to the vertebrate reticular formation is the bilateral "A-cluster" of cerebral ganglion neurons that controls posture, locomotion, and serotonergic motor arousal. The A-cluster transmits motor commands to the pedal ganglia analogs of the spinal cord, for primary patterned motor output. Apparent pallial precursors are not immediately evident in Pleurobranchaea's central nervous system, but a notable candidate is a subepithelial nerve net in the peripheral head region that integrates chemotactile stimuli for incentive and directionality. Evolutionary centralization of its computational functions may have led to the olfaction-derived pallial forebrain in the ancestor's vertebrate descendants and their analogs in arthropods and annelids.

Recording cilia activity in ctenophores: effects of nitric oxide and low molecular weight transmitters.

Cilia are the major effectors in Ctenophores, but very little is known about their transmitter control and integration. Here, we present a simple protocol to monitor and quantify cilia activity and provide evidence for polysynaptic control of cilia coordination in ctenophores. We also screened the effects of several classical bilaterian neurotransmitters (acetylcholine, dopamine, L-DOPA, serotonin, octopamine, histamine, gamma-aminobutyric acid (GABA), L-aspartate, L-glutamate, glycine), neuropeptide (FMRFamide), and nitric oxide (NO) on cilia beating in Pleurobrachia bachei and Bolinopsis infundibulum. NO and FMRFamide produced noticeable inhibitory effects on cilia activity, whereas other tested transmitters were ineffective. These findings further suggest that ctenophore-specific neuropeptides could be major candidates for signal molecules controlling cilia activity in representatives of this early-branching metazoan lineage.

Nitric oxide suppresses cilia activity in ctenophores.

Cilia are the major effectors in Ctenophores, but very little is known about their transmitter control and integration. Here, we present a simple protocol to monitor and quantify cilia activity in semi-intact preparations and provide evidence for polysynaptic control of cilia coordination in ctenophores. Next, we screen the effects of several classical bilaterian neurotransmitters (acetylcholine, dopamine, L-DOPA, serotonin, octopamine, histamine, gamma-aminobutyric acid (GABA), L-aspartate, L-glutamate, glycine), neuropeptides (FMRFamide), and nitric oxide (NO) on cilia beating in Pleurobrachia bachei and Bolinopsis infundibulum . Only NO inhibited cilia beating, whereas other tested transmitters were ineffective. These findings further suggest that ctenophore-specific neuropeptides could be major candidate signaling molecules controlling cilia activity in representatives of this early-branching metazoan lineage.

Development of the nervous system in the early hatching larvae of the ctenophore Mnemiopsis leidyi.

Ctenophores are descendants of an early branching basal metazoan lineage, which may have evolved neurons and muscles independently from other animals. Mnemiopsis is one of the important reference ctenophore species. However, little is known about its neuromuscular organization. Here, we mapped and tracked the development of the neural and muscular elements in the early hatching cydippid larvae, as well as adult Mnemiopsis leidyi. The overall development of the neuromuscular system in Mnemiopsis was very similar to Pleurobrachia bachei, although in Mnemiopsis the entire process occurred significantly faster. The subepithelial neural cells were observed immediately after hatching. This population consisted of a dozen of separated individual neurons with short neurites. In about 2 days, when their neurites grew significantly longer and connected to their neighbors, they began to form a canonical polygonal subepithelial network. Mesogleal neural elements prominent in all studied adult ctenophores were not detectable in Mnemiopsis larvae but were clearly labeled in closely related Lobata species Bolinopsis infundibulum. Hatched larvae also had putative mechanoreceptors with long stereocilia and approximately two dozen muscle cells. In adult Mnemiopsis, the feeding lobes and auricles contained two distinct populations of neurons and neural ensembles that were not observed in other ctenophore lineages and likely represented elaborate neuronal innovations characteristic for the clade Lobata and their lifestyles.

Structure and function of the nervous system in nectophores of the siphonophore Nanomia bijuga.

Although the bell-shaped nectophores of the siphonophore Nanomia bijuga are clearly specialized for locomotion, their complex neuroanatomy described here testifies to multiple subsidiary functions. These include secretion, by the extensively innervated 'flask cells' located around the bell margin, and protection, by the numerous nematocytes that line the nectophore's exposed ridges. The main nerve complex consists of a nerve ring at the base of the bell, an adjacent column-shaped matrix plus two associated nerve projections. At the top of the nectophore the upper nerve tract appears to have a sensory role; on the lower surface a second nerve tract provides a motor input connecting the nectophore with the rest of the colony via a cluster of nerve cells at the stem. N. bijuga is capable of both forward and backward jet-propelled swimming. During backwards swimming the water jet is redirected by the contraction of the Claus' muscle system, part of the muscular velum that fringes the bell aperture. Contractions can be elicited by electrical stimulation of the nectophore surface, even when both upper and lower nerve tracts have been destroyed. Epithelial impulses elicited there, generate slow potentials and action potentials in the velum musculature. Slow potentials arise at different sites around the bell margin and give rise to action potentials in contracting Claus' muscle fibres. A synaptic rather than an electrotonic model more readily accounts for the time course of the slow potentials. During backward swimming, isometrically contracting muscle fibres in the endoderm provide the Claus' fibres with an immobile base.

Comparative neuroanatomy of ctenophores: Neural and muscular systems in Euplokamis dunlapae and related species.

Ctenophora is an early-branching basal metazoan lineage, which may have evolved neurons and muscles independently from other animals. However, despite the profound diversity among ctenophores, basal neuroanatomical data are limited to representatives of two genera. Here, we describe the organization of neuromuscular systems in eight ctenophore species focusing on Euplokamis dunlapae-the representative of the lineage sister to all other ctenophores. Cydippids (Hormiphora hormiphora and Dryodora glandiformis) and lobates (Bolinopsis infundibulum and Mnemiopsis leidyi) were used as reference platforms to cover both morphological and ecological diversity within the phylum. We show that even with substantial environmental differences, the basal organization of neural systems is conserved among ctenophores. In all species, we detected two distributed neuronal subsystems: the subepithelial polygonal network and the mesogleal elements. Nevertheless, each species developed specific innovations in neural, muscular, and receptor systems. Most notable Euplokamis-specific features are the following: (a) Comb nerves with giant axons. These nerves directly coordinate the rapid escape response bypassing the central integrative structure known as the aboral sensory organ. (b) Neural processes in tentacles along the rows of "boxes" providing structural support and located under striated muscles. (c) Radial muscles that cross the mesoglea and connect the outer wall to the aboral canal. (d) Flat muscles, encircling each meridional canal. Also, we detected a structurally different rectangular neural network in the feeding lobes of Lobata (Mnemiopsis/Bolinopsis) but not in other species. The described lineage-specific innovations can be used for future single-cell atlases of ctenophores and analyses of neuronal evolution in basal metazoans.

Atlas of the neuromuscular system in the Trachymedusa Aglantha digitale: Insights from the advanced hydrozoan.

Cnidaria is the sister taxon to bilaterian animals, and therefore, represents a key reference lineage to understand early origins and evolution of the neural systems. The hydromedusa Aglantha digitale is arguably the best electrophysiologically studied jellyfish because of its system of giant axons and unique fast swimming/escape behaviors. Here, using a combination of scanning electron microscopy and immunohistochemistry together with phalloidin labeling, we systematically characterize both neural and muscular systems in Aglantha, summarizing and expanding further the previous knowledge on the microscopic neuroanatomy of this crucial reference species. We found that the majority, if not all (~2,500) neurons, that are labeled by FMRFamide antibody are different from those revealed by anti-α-tubulin immunostaining, making these two neuronal markers complementary to each other and, therefore, expanding the diversity of neural elements in Aglantha with two distinct neural subsystems. Our data uncovered the complex organization of neural networks forming a functional "annulus-type" central nervous system with three subsets of giant axons, dozen subtypes of neurons, muscles, and a variety of receptors fully integrated with epithelial conductive pathways supporting swimming, escape and feeding behaviors. The observed unique adaptations within the Aglantha lineage (including giant axons innervating striated muscles) strongly support an extensive and wide-spread parallel evolution of integrative and effector systems across Metazoa.

Organization of Buccal Cone Musculature in the Pteropod Mollusc Clione limacina.

The pteropod mollusc Clione limacina is a feeding specialist, preying on shelled pteropods of the genus Limacina. Specialized prey-capture structures, called buccal cones, are hydraulically everted from within the mouth to capture the prey. Once captured, the prey is manipulated so the shell opening is over the mouth of Clione. Analyses of high-speed cine sequences of prey capture suggest that the mouth is actively opened rather than passively forced open by buccal cone eversion. The inflated buccal cones are initially straight and form a wide angle (maximum, 113°) prior to prey contact. Individual buccal cones bend orally following prey contact, suggesting a sensory trigger. To determine the muscular basis of buccal cone movements, the musculature of the buccal cones is described. Three distinct muscle fiber types include circular smooth muscle, longitudinal smooth muscle, and longitudinal striated muscle. The organization, distribution, and innervation of the muscle types suggest that circular muscle is used during buccal cone eversion, longitudinal smooth muscle is used for buccal cone withdrawal, and longitudinal striated muscle is used for oral bending of the buccal cones after prey contact and for manipulation of the prey.

Neural system and receptor diversity in the ctenophore Beroe abyssicola.

Although, neurosensory systems might have evolved independently in ctenophores, very little is known about their organization and functions. Most ctenophores are pelagic and deep-water species and cannot be bred in the laboratory. Thus, it is not surprising that neuroanatomical data are available for only one genus within the group-Pleurobrachia. Here, using immunohistochemistry and scanning electron microscopy, we describe the organization of two distinct neural subsystems (subepithelial and mesogleal) and the structure of different receptor types in the comb jelly Beroe abyssicola-the voracious predator from North Pacific. A complex subepithelial neural network of Beroe, with five receptor types, covers the entire body surface and expands deep into the pharynx. Three types of mesogleal neurons are comparable to the cydippid Pleurobrachia. The predatory lifestyle of Beroe is supported by the extensive development of ciliated and muscular structures including the presence of giant muscles and feeding macrocilia. The obtained cell-type atlas illustrates different examples of lineage-specific innovations within these enigmatic marine animals and reveals the remarkable complexity of sensory and effector systems in this clade of basal Metazoa.

Neuromuscular organization of the Ctenophore Pleurobrachia bachei.

Ctenophores are descendants of one of the earliest branching metazoan lineage with enigmatic nervous systems. The lack of convenient neurogenic molecules and neurotransmitters suggests an extensive parallel evolution and independent origins of neurons and synapses. However, the field lags due to the lack of microanatomical data about the neuro-muscular systems in this group of animals. Here, using immunohistochemistry and scanning electron microscopy, we describe the organization of both muscular and nervous systems in the sea gooseberry, Pleurobrachia bachei, from North Pacific. The diffuse neural system of Pleurobrachia consists of two subsystems: the subepithelial neural network and the mesogleal net with about 5,000-7,000 neurons combined. Our data revealed the unexpected complexity of neuromuscular organization in this basal metazoan lineage. The anatomical diversity of cell types includes at least nine broad categories of neurons, five families of surface receptors and more than two dozen types of muscle cells as well as regional concentrations of neuronal elements to support ctenophore feeding, complex swimming, escape, and prey capture behaviors. In summary, we recognize more than 80 total morphological cell types. Thus, in terms of cell-type specification and diversity, ctenophores significantly exceed what we currently know about other prebilaterian groups (placozoan, sponges, and cnidarians), and some basal bilaterians.

Development of neuromuscular organization in the ctenophore Pleurobrachia bachei.

The phylogenetic position of the phylum Ctenophora and the nature of ctenphore nervous systems are highly debated topics in modern evolutionary biology. However, very little is known about the organization of ctenophore neural and muscular systems, and virtually nothing has been reported about their embryogenesis. Here we have characterized the neural and muscular development of the sea gooseberry, Pleurobrachia bachei, starting from the cleavage stages to posthatching larvae. Scanning electron microscopy and immunochemistry were used to describe the formation of the embryonic mouth, tentacles, combs, aboral organ, and putative sensory cells. The muscles started their specification at the end of the first day of Pleurobrachia development. In contrast, neurons appeared 2 days after myogenesis, just before the hatching of fully formed cydippid larvae. The first tubulin-immunoreactive neurons, a small group of four to six cells with neuronal processes, was initially recognized at the aboral pole during the third day of development. Surprisingly, this observed neurogenesis occurred after the emergence of distinct behavioral patterns in the embryos. Thus, the embryonic behavior associated with comb cilia beatings and initial muscle organization does not require morphologically defined neurons and their elongated neurites. This study provides the first description of neuromuscular development in the enigmatic ctenophores and establishes the foundation for future research using emerging genomic tools and resources.

The ctenophore genome and the evolutionary origins of neural systems.

The origins of neural systems remain unresolved. In contrast to other basal metazoans, ctenophores (comb jellies) have both complex nervous and mesoderm-derived muscular systems. These holoplanktonic predators also have sophisticated ciliated locomotion, behaviour and distinct development. Here we present the draft genome of Pleurobrachia bachei, Pacific sea gooseberry, together with ten other ctenophore transcriptomes, and show that they are remarkably distinct from other animal genomes in their content of neurogenic, immune and developmental genes. Our integrative analyses place Ctenophora as the earliest lineage within Metazoa. This hypothesis is supported by comparative analysis of multiple gene families, including the apparent absence of HOX genes, canonical microRNA machinery, and reduced immune complement in ctenophores. Although two distinct nervous systems are well recognized in ctenophores, many bilaterian neuron-specific genes and genes of 'classical' neurotransmitter pathways either are absent or, if present, are not expressed in neurons. Our metabolomic and physiological data are consistent with the hypothesis that ctenophore neural systems, and possibly muscle specification, evolved independently from those in other animals.

Neural control of heartbeat during two antagonistic behaviors: whole body withdrawal and escape swimming in the mollusk Clione limacina.

Two cardioexcitatory and one cardioinhibitory neural groups have been previously identified as the central cardioregulatory system in the pteropod mollusk Clione limacina. We describe in this study one additional element of the central cardioregulatory system, which consists of a large intestinal neuron named Z-cell with a novel effect on the heart activity. Intracellular stimulation of the Z-cell induced only auricle contractions with no effect on the ventricle activity. The Z-cell processes were traced down to the heart, and vigorous branching was found in the auricle tissue. Specific patterns of activity of the Z-cell as well as intestinal heart excitatory and inhibitory neurons were studied during initiation of two behaviors--whole body withdrawal and escape swimming. It was found that initiation of both behaviors was accompanied by activation of Z-cell and intestinal heart excitor neurons. The firing rate of neurons induced by sensory stimuli was sufficient to trigger auricle contractions in the semi-intact preparations. Video analysis of heart activity revealed that auricle indeed was activated during both active and passive avoidance reactions, though the intensity and delay of the activation were different. The possible physiological role of the auricle contractions during antagonistic forms of behavior is discussed.

Neural mechanisms underlying co-activation of functionally antagonistic motoneurons during a Clione feeding behavior.

The ability of some neural networks to produce multiple motor patterns required during different behaviors is a well-documented phenomenon. We describe here a dramatic transition from coordinated inhibition between two functionally antagonistic groups of motoneurons to their co-activation in the feeding neural network of the predatory mollusk Clione limacina. To seize its prey, Clione uses specialized oral appendages, called buccal cones, which are controlled by two groups of motoneurons: cerebral A (Cr-A) neurons controlling buccal cone protraction and cerebral B (Cr-B) neurons controlling buccal cone retraction. When Cr-A neurons are active, Cr-B neurons usually receive strong inhibitory inputs that terminate their firing, which leads to the full protraction and elongation of the buccal cones. We have found, however, that the Cr-A and Cr-B motoneurons sometimes burst simultaneously without any traces of inhibition in the Cr-B motoneurons. This transformation of the neural network activity from inhibitory interactions to co-activation presumably occurs during the late "extraction" period of the feeding behavior when buccal cones become partially retracted and rhythmically active. The transition from the inhibitory interaction to co-activation is controlled by the activity of a single pair of cerebral interneurons (Cr-Aint interneurons), which are electrically coupled to the Cr-A neurons and monosynaptically inhibit Cr-B neurons. Normally, the Cr-Aint interneurons are active along with Cr-A motoneurons and inhibit Cr-B motoneurons. During a period of co-activation, however, these interneurons do not produce spikes, thus allowing Cr-A motoneuron activation without inhibition of the Cr-B motoneurons.

Coordinated excitatory effect of GABAergic interneurons on three feeding motor programs in the mollusk Clione limacina.

Coordination between different motor centers is essential for the orderly production of all complex behaviors. Understanding the mechanisms of such coordination during feeding behavior in the carnivorous mollusk Clione limacina is the main goal of the current study. A bilaterally symmetrical interneuron identified in the cerebral ganglia and designated Cr-BM neuron produced coordinated activation of neural networks controlling three main feeding structures: prey capture appendages called buccal cones, chitinous hooks used for prey extraction from the shell, and the toothed radula. The Cr-BM neuron produced strong excitatory inputs to motoneurons controlling buccal cone protraction. It also induced a prominent activation of the neural networks controlling radula and hook rhythmic movements. In addition to the overall activation, Cr-BM neuron synaptic inputs to individual motoneurons coordinated their activity in a phase-dependent manner. The Cr-BM neuron produced depolarizing inputs to the radula protractor and hook retractor motoneurons, which are active in one phase, and hyperpolarizing inputs to the radula retractor and hook protractor motoneurons, which are active in the opposite phase. The Cr-BM neuron used GABA as its neurotransmitter. It was found to be GABA-immunoreactive in the double-labeling experiments. Exogenous GABA mimicked the effects produced by Cr-BM neuron on the postsynaptic neurons. The GABA antagonists bicuculline and picrotoxin blocked Cr-BM neuron-induced PSPs. The prominent coordinating effect produced by the Cr-BM neuron on the neural networks controlling three major elements of the feeding behavior in Clione suggests that this interneuron is an important part of the higher-order system for the feeding behavior.