Cyclic Guanosine Monophosphate Modulates Locomotor Acceleration Induced by Nitric Oxide but not Serotonin in Clione limacina Central Pattern Generator Swim Interneurons.

Both nitric oxide (NO) and serotonin (5HT) mediate swim acceleration in the marine mollusk, Clione  limacina. In this study, we examine the role that the second messenger, cyclic guanosine monophosphate (cGMP), plays in mediating NO and 5HT-induced swim acceleration. We observed that the application of an analog of cGMP or an activator of soluble guanylyl cyclase (sGC) increased fictive locomotor speed recorded from Pd-7 interneurons of the animal's locomotor central pattern generator. Moreover, inhibition of sGC decreased fictive locomotor speed. These results suggest that basal levels of cGMP are important for slow swimming and that increased production of cGMP mediates swim acceleration in Clione. Because NO has its effect through cGMP signaling and because we show herein that cGMP produces cellular changes in Clione swim interneurons that are consistent with cellular changes produced by 5HT application, we hypothesize that both NO and 5HT function via a common signal transduction pathway that involves cGMP. Our results show that cGMP mediates NO-induced but not 5HT-induced swim acceleration in Clione.

Pedal serotonergic neuron clusters of the pteropod mollusc, Clione limacina, contain two morphological subtypes with different innervation targets.

Each pedal ganglion of the pteropod mollusc Clione limacina contains a cluster of serotonin-immunoreactive neurons that have been shown to modulate contractions of the slow-twitch musculature of the wing-like parapodia, and contribute to swim accelerations. Each cluster has a variable number of neurons, between 5 and 9, but there is no significant difference between right and left ganglia. In experiments with electrophysiological recordings followed by dye-injection (carboxyfluorescein), the clusters were found to contain two subsets of neurons. The majority innervate the ipsilateral wing via nerve n4. Two of the neurons in each cluster send processes out of the pedal ganglion in nerves n3 and n8. The processes in nerve n3 innervate the body wall of the neck region, while those in nerve n8 innervate the body wall of the tail. The baseline electrophysiological activity of the two subsets of neurons was different as "wing" neurons had constant barrages of small synaptic activity, while the "body wall" neurons had few synaptic inputs. The potential roles of the Pd-SW cluster in swim acceleration (wing neurons) and control of fluid pressure in the body and wing hemocoelic compartments (body wall neurons) are discussed.

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.

Tentacle Musculature in the Cubozoan Jellyfish Carybdea marsupialis.

The diploblastic cnidarian body plan comprising the epidermis and gastrodermis has remained largely unchanged since it evolved roughly 600 Ma. The origin of muscle from the mesoderm in triploblastic lineages is a central evolutionary question in higher animals. Triploblasts have three embryonic germ layers: the endoderm, mesoderm, and ectoderm, which develop into organs, muscle, and skin, respectively. Diploblasts lack the mesoderm, the layer thought to give rise to the skeletomuscular system. However, phyla such as Cnidaria and Ctenophora, which are typically classified as diploblasts, possess striated musculature. Within phylum Cnidaria, class Cubozoa includes carnivorous box jellyfish, which are capable of extending and contracting their tentacles for predation and defense mechanisms, thus suggesting a well-organized system of muscles. Here, the tentacle musculature of the cubomedusae Carybdea marsupialis is investigated using transmission electron microscopy in conjunction with light microscopy to further understand the arrangement of musculature in these primitive animals. Cross sections of tentacles confirmed that the gastrodermis is separated from the epidermis by a collagenous mesogleal layer containing numerous longitudinal muscle cells arranged in fascicles. Longitudinal muscles permit the tentacle to retract toward the bell during fast tentacle shortening and crumpling behavioral responses. Circular muscle cells were found in the gastrodermis and epidermis, encircling the layer of longitudinal muscle. These circular muscles likely enable the elongation process that allows the tentacles to return to a resting state after contraction. The presence of a definitive muscle cell layer within the mesoglea suggests that C. marsupialis has an advanced muscle morphology that is similar to triploblastic animals.

Control of vortex rings for manoeuvrability.

Manoeuvrability is critical to the success of many species. Selective forces acting over millions of years have resulted in a range of capabilities currently unmatched by machines. Thus, understanding animal control of fluids for manoeuvring has both biological and engineering applications. Within inertial fluid regimes, propulsion involves the formation and interaction of vortices to generate thrust. We use both volumetric and planar imaging techniques to quantify how jellyfish (Aurelia aurita) modulate vortex rings during turning behaviour. Our results show that these animals distort individual vortex rings during turns to alter the force balance across the animal, primarily through kinematic modulation of the bell margin. We find that only a portion of the vortex ring separates from the body during turns, which may increase torque. Using a fluorescent actin staining method, we demonstrate the presence of radial muscle fibres lining the bell along the margin. The presence of radial muscles provides a mechanistic explanation for the ability of scyphomedusae to alter their bell kinematics to generate non-symmetric thrust for manoeuvring. These results illustrate the advantage of combining imaging methods and provide new insights into the modulation and control of vorticity for low-speed animal manoeuvring.

Cnidarian Nerve Nets and Neuromuscular Efficiency.

Cnidarians are considered "nerve net animals" even though their nervous systems include various forms of condensation and centralization. Yet, their broad, two-dimensional muscle sheets are innervated by diffuse nerve nets. Do the motor nerve nets represent a primitive organization of multicellular nervous systems, do they represent a consequence of radial symmetry, or do they offer an efficient way to innervate a broad, two-dimensional muscle sheet, in which excitation of the muscle sheet can come from multiple sites of initiation? Regarding the primitive nature of cnidarian nervous systems, distinct neuronal systems exhibit some adaptations that are well known in higher animals, such as the use of oversized neurons with increased speed of conduction, and condensation of neurites into nerve-like tracts. A comparison of neural control of two-dimensional muscle sheets in a mollusc and jellyfish suggests that a possible primitive feature of cnidarian neurons may be a lack of regional specialization into conducting and transmitting regions.

The search for ancestral nervous systems: an integrative and comparative approach.

Even the most basal multicellular nervous systems are capable of producing complex behavioral acts that involve the integration and combination of simple responses, and decision-making when presented with conflicting stimuli. This requires an understanding beyond that available from genomic investigations, and calls for a integrative and comparative approach, where the power of genomic/transcriptomic techniques is coupled with morphological, physiological and developmental experimentation to identify common and species-specific nervous system properties for the development and elaboration of phylogenomic reconstructions. With careful selection of genes and gene products, we can continue to make significant progress in our search for ancestral nervous system organizations.

Organization of the ectodermal nervous structures in jellyfish: scyphomedusae.

Antibodies to α- or ß-tubulin and to the bioactive peptide FMRFamide were used to investigate the organization of the ectodermal nervous structures in five species of scyphomedusae. Within the swim system, morphological evidence, including a developmental sequence, suggests that the tubulin-immunoreactive nerve net in the subumbrella is the Giant Fiber Nerve Net (Motor Nerve Net) that directly activates the swim musculature, and the FMRFamide-immunoreactive nerve net is the Diffuse Nerve Net that serves a sensory function and also enhances swim muscle activity. Similar dual labeling was found in other structures, including those involved in feeding and protective reactions (pedalia and tentacles, radial strips of smooth muscle), and in the exumbrella, where the networks were associated with batteries of nematocysts. In addition, FMRFamide immuno-staining in the rhopalia and rhopalial niches suggests that sensory components of these networks may aid in the gravitational sense of scyphomedusae.

Organization of the ectodermal nervous structures in medusae: cubomedusae.

At least two conducting systems are well documented in cubomedusae. A variably diffuse network of large neurons innervates the swim musculature and can be visualized immunohistochemically using antibodies against α- or ß-tubulin. Despite the non-specificity of these antibodies, multiple lines of evidence suggest that staining highlights the primary motor networks. These networks exhibit unique neurite distributions among the muscle sheets in that network density is greatest in the perradial frenula, where neurites are oriented in parallel with radial muscle fibers. This highly innervated, buttress-like muscle sheet may serve a critical role in the cubomedusan mechanism of turning. In scyphomedusae, a second subumbrellar network immunoreactive to antibodies against the neuropeptide FMRFamide innervates the swim musculature, but it is absent in cubomedusae. Immunoreactivity to FMRFamide in cubomedusae is mostly limited to a small network of neurons in the pacemaker region of the rhopalia, the pedalial apex at the nerve ring junction, and a few neuron tracts in the nerve ring. However, FMRFamide-immunoreactive networks, as well as tubulin-immunoreactive networks, are nearly ubiquitous outside of the swim muscle sheets in the perradial smooth muscle bands, manubrium, pedalia, and tentacles. Here we describe in detail the peripheral nerve nets of box jellyfish on the basis of immunoreactivity to the antibodies above. Our results offer insight into how the peripheral nerve nets are organized to produce the complex swimming, feeding, and defensive behaviors observed in cubomedusae.

Multiple conducting systems in the cubomedusa Carybdea marsupialis.

Acute responses to mechanical, electrical, and photic stimuli were used to describe neural conducting systems in the cubomedusan jellyfish Carybdea marsupialis underlying three behaviors: contractile responses of single tentacles, protective crumple responses, and alterations of swimming activity by the visual system. Responses of single tentacles consisted of tentacular shortening and inward pedalial bending, and were accompanied by bursts of extracellularly recorded spike activity that were restricted to the stimulated tentacle. With nociceptive stimuli delivered to the subumbrella or margin, all four tentacles produced similar responses in a crumple response. The spike bursts in all four tentacles showed coordinated firing as long as the nerve ring was intact. Crumples were still produced following cuts through the nerve ring, but the activity in individual tentacles was no longer coordinated. Responses to light-on stimulation of a rhopalium, as recorded from the pacemaker region, were weak and inconsistent, but when present, resulted in a stimulation of swimming activity. In comparison, light-off responses were robust and resulted in temporary inhibition of swimming activity. Light-off responses were conducted in the nerve ring to unstimulated rhopalia. In conclusion, three conducting systems have been described as components of the rhopalia-nerve ring centralized system in Carybdea: the swim motor system, the crumple coordination system, and the light-off response system.

Toward an organismal neurobiology: integrative neuroethology.

Overt behavior is generated in response to a palette of external and internal stimuli and internal drives. Rarely are these variables introduced in isolation. This creates challenges for the organism to sort inputs that frequently favor conflicting behaviors. Under these conditions, the nervous system relies on established and flexible hierarchies to produce appropriate behavioral changes. The pteropod mollusc Clione limacina is used as an example to illustrate a variety of behavioral interactions that alter a baseline behavioral activity: slow swimming. The alterations include acceleration within the slow swimming mode, acceleration from the slow to fast swimming modes, whole body withdrawal (and inhibition of swimming), food acquisition behavior (with a feeding motivational state), and a startle locomotory response. These examples highlight different types of interaction between the baseline behavior and the new behaviors that involve external stimuli and two types of internal drives: a modular arousal system and a motivational state. The investigation of hierarchical interactions between behavioral modules is a central theme of integrative neuroethology that focuses on an organismal level of understanding of the neural control of behavior.

Changes in wingstroke kinematics associated with a change in swimming speed in a pteropod mollusk, Clione limacina.

In pteropod mollusks, the gastropod foot has evolved into two broad, wing-like structures that are rhythmically waved through the water for propulsion. The flexibility of the wings lends a tremendous range of motion, an advantage that could be exploited when changing locomotory speed. Here, we investigated the kinematic changes that take place during an increase in swimming speed in the pteropod mollusk Clione limacina. Clione demonstrates two distinct swim speeds: a nearly constant slow swimming behavior and a fast swimming behavior used for escape and hunting. The neural control of Clione's swimming is well documented, as are the neuromuscular changes that bring about Clione's fast swimming. This study examined the kinematics of this swimming behavior at the two speeds. High speed filming was used to obtain 3D data from individuals during both slow and fast swimming. Clione's swimming operates at a low Reynolds number, typically under 200. Within a given swimming speed, we found that wing kinematics are highly consistent from wingbeat to wingbeat, but differ between speeds. The transition to fast swimming sees a significant increase in wing velocity and angle of attack, and range of motion increases as the wings bend more during fast swimming. Clione likely uses a combination of drag-based and unsteady mechanisms for force production at both speeds. The neuromuscular control of Clione's speed change points to a two-gaited swimming behavior, and we consider the kinematic evidence for Clione's swim speeds being discrete gaits.

Do jellyfish have central nervous systems?

The traditional view of the cnidarian nervous system is of a diffuse nerve net that functions as both a conducting and an integrating system; this is considered an indicator of a primitive condition. Yet, in medusoid members, varying degrees of nerve net compression and neuronal condensation into ganglion-like structures represent more centralized integrating centers. In some jellyfish, this relegates nerve nets to motor distribution systems. The neuronal condensation follows a precept of neuronal organization of higher animals with a relatively close association with the development and elaboration of sensory structures. Nerve nets still represent an efficient system for diffuse, non-directional activation of broad, two-dimensional effector sheets, as required by the radial, non-cephalized body construction. However, in most jellyfish, an argument can be made for the presence of centralized nervous systems that interact with the more diffuse nerve nets.

A hyperpolarization-activated inward current alters swim frequency of the pteropod mollusk Clione limacina.

The pteropod mollusk, Clione limacina, exhibits behaviorally relevant swim speed changes that occur within the context of the animal's ecology. Modulation of C. limacina swimming speed involves changes that occur at the network and cellular levels. Intracellular recordings from interneurons of the swim central pattern generator show the presence of a sag potential that is indicative of the hyperpolarization-activated inward current (I(h)). Here we provide evidence that I(h) in primary swim interneurons plays a role in C. limacina swimming speed control and may be a modulatory target. Recordings from central pattern generator swim interneurons show that hyperpolarizing current injection produces a sag potential that lasts for the duration of the hyperpolarization, a characteristic of cells possessing I(h). Following the hyperpolarizing current injection, swim interneurons also exhibit postinhibitory rebound (PIR). Serotonin enhances the sag potential of C. limacina swim interneurons while the I(h) blocker, ZD7288, reduces the sag potential. Furthermore, a negative correlation was found between the amplitude of the sag potential and latency to PIR. Because latency to PIR was previously shown to influence swimming speed, we hypothesize that I(h) has an effect on swimming speed. The I(h) blocker, ZD7288, suppresses swimming in C. limacina and inhibits serotonin-induced acceleration, evidence that supports our hypothesis.

Ultrastructure of the retinal synapses in cubozoans.

Cubomedusae (box jellyfish) are well known for strong directional swimming, rapid responses to visual stimuli, and complex lensed eyes comparable to those of more advanced multicellular animals. They possess a total of 24 eyes that are of four morphologically different types, yet little is known about the neural organization of their eyes. The eyes are located on ganglion-like structures called rhopalia. Each of the four rhopalia contains an upper and a lower lensed eye (with a cornea, lens, and retina), two pit ocelli, and two slit ocelli. Transmission electron microscopy was used to examine the synaptic morphology of the eyes and pacemaker region of four species of cubozoans (Tamoya haplonema, Carybdea marsupialis, Tripedalia cystophora, and Chiropsalmus quadrumanus). Invaginated synapses were found in all four species, but only in the upper and lower lensed eyes. Density measurements indicated that the invaginated synapses were located close to the basal region of photoreceptor cells, and size differences of invaginated synapses were observed between the upper and lower lensed eyes, as well as between species. Four additional types of chemical synapses-clear unidirectional, dense-core unidirectional, clear bidirectional, and clear and dense-core bidirectional-were also observed in the rhopalia. The invaginated synapses of the lensed eyes may be useful as markers to help sort out the neural circuitry in the retinal region of these complex cubomedusan eyes.

Control of swimming in the hydrozoan jellyfish Aequorea victoria: subumbrellar organization and local inhibition.

The subumbrella of the hydrozoan jellyfish Aequorea victoria (previously classified as Aequorea aequorea) is divided by numerous radial canals and attached gonads, so the subumbrellar musculature is partitioned into subumbrellar segments. The ectoderm of each segment includes two types of muscle: smooth muscle with a radial orientation, used for local (feeding and righting) and widespread (protective) radial responses, and striated muscle with a circular orientation which produces swim contractions. Two subumbrellar nerve nets were found, one of which stained with a commercial antibody produced against the bioactive peptide FMRFamide. Circular muscle cells produce a single, long-duration action potential with each swim, triggered by a single junctional potential. In addition, the circular cells are electrically coupled so full contractions require both electrotonic depolarization from adjacent cells and synaptic input from a subumbrellar nerve net. The radial cells, which form a layer superficial to the circular cells, are also activated by a subumbrellar nerve net, and produce short-duration action potentials. The radial muscle cells are electrically coupled to one another. No coupling exists between the two muscle layers. Spread of excitation between adjacent segments is decremental, and nerve net-activated junctional potentials disappear during local inhibition of swimming (such as with a radial response). Variable swim contractions are controlled by a combination of synaptic input from the motor network of the inner nerve ring, synaptic input from a subumbrellar nerve net, and electrotonic depolarization from adjacent, active muscle cells.

Hexamethonium sensitivity of the swim musculature of the pteropod mollusc, Clione limacina.

Swimming in reduced electrophysiological preparations of the pteropod mollusc, Clione limacina, was blocked by bath application of hexamethonium even though pattern generator activity continued with this treatment. Neuromuscular recordings indicated that hexamethonium blocked synaptic input from Pd-3 and Pd-4 motoneurons to slow-twitch muscle cells, while connections from Pd-1A and Pd-2A motoneurons to fast-twitch muscle cells were variable in their response to hexamethonium-synaptic inputs were suppressed in most cases and occasionally blocked, but the latter only with high concentrations and long incubations. Acutely dissociated wing muscle cells showed a concentration-dependency in the percentage of contracted cells with bath application of acetylcholine, and this contractile activity was blocked in preparations that were first bathed in hexamethonium. Intracellular recordings from dissociated slow-twitch muscle cells showed conductance-increase depolarizations of approximately 20 mV following 1 s pressure ejections of 10(-4) M acetylcholine from micropipettes placed immediately adjacent to the muscle cells. These responses were blocked when hexamethonium was bath applied prior to the pressure-applied acetylcholine. The results suggest the Pd-3/Pd-4 motoneuron to slow-twitch muscle cell junctions are cholinergic with nicotinic-like receptors, while the Pd-1A/Pd-2A to fast-twitch muscle cell connections are likely cholinergic, but with a different receptor type.

Neuromechanics: an integrative approach for understanding motor control.

Neuromechanics seeks to understand how muscles, sense organs, motor pattern generators, and brain interact to produce coordinated movement, not only in complex terrain but also when confronted with unexpected perturbations. Applications of neuromechanics include ameliorating human health problems (including prosthesis design and restoration of movement following brain or spinal cord injury), as well as the design, actuation and control of mobile robots. In animals, coordinated movement emerges from the interplay among descending output from the central nervous system, sensory input from body and environment, muscle dynamics, and the emergent dynamics of the whole animal. The inevitable coupling between neural information processing and the emergent mechanical behavior of animals is a central theme of neuromechanics. Fundamentally, motor control involves a series of transformations of information, from brain and spinal cord to muscles to body, and back to brain. The control problem revolves around the specific transfer functions that describe each transformation. The transfer functions depend on the rules of organization and operation that determine the dynamic behavior of each subsystem (i.e., central processing, force generation, emergent dynamics, and sensory processing). In this review, we (1) consider the contributions of muscles, (2) sensory processing, and (3) central networks to motor control, (4) provide examples to illustrate the interplay among brain, muscles, sense organs and the environment in the control of movement, and (5) describe advances in both robotics and neuromechanics that have emerged from application of biological principles in robotic design. Taken together, these studies demonstrate that (1) intrinsic properties of muscle contribute to dynamic stability and control of movement, particularly immediately after perturbations; (2) proprioceptive feedback reinforces these intrinsic self-stabilizing properties of muscle; (3) control systems must contend with inevitable time delays that can simplify or complicate control; and (4) like most animals under a variety of circumstances, some robots use a trial and error process to tune central feedforward control to emergent body dynamics.