Why do cubomedusae have only four swim pacemakers?

The classic view of swimming control in scyphozoan and cubozoan jellyfish involves a diffuse motor nerve net activated by multiple pacemaker sites that interact in a simple resetting hierarchy. Earlier modeling studies of jellyfish swimming, utilizing resetting linkages of multiple pacemakers, indicated that increases in pacemaker number were correlated with increases in the rate and regularity of network activity. We conducted a similar study using the cubozoan jellyfish Carybdea marsupialis, concentrating not only on the adaptive features of multiple pacemaker networks but also on the mechanism of pacemaker interaction. The best fit for our experimental data is a model in which pacemakers express a degree of independence. Thus, our results challenge the idea that pacemaker interactions in scyphozoan and cubozoan medusae are based on a strict resetting hierarchy. Furthermore, our data suggest that the combination of semi-independent linkage of pacemakers with the small pacemaker number characteristic of cubomedusae is important in (i) maintaining a biphasic modulatory capability in the swimming system, and (ii) allowing behaviorally appropriate directional responses to asymmetrical sensory inputs in the radially arranged jellyfish nervous system.

Distribution of NADPH-diaphorase reactivity and effects of nitric oxide on feeding and locomotory circuitry in the pteropod mollusc, Clione limacina.

The action of nitric oxide (NO) and the distribution of putative nitric oxide synthase-containing cells in the pelagic pteropod mollusc Clione limacina were studied using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and conventional microelectrode techniques in the isolated central nervous system and in semi-intact preparations. The majority of NADPH-d-reactive neuronal somata were restricted to the cerebral ganglia. The labeled cells were small in diameter (20-30 microm) and were located in the medial areas of the ganglia. A pair of symmetrical neurons was found in the peripheral "olfactory organ." NADPH-d-reactive non-neuronal cells were detected in the periphery and were mainly associated with secretorylike cells and organs of the renopericardial system. The NO donor, diethylamine NO complex sodium salt (10-100 microM), activated neurons from both feeding and locomotory circuits. The cGMP analog, 8-Br-cGMP, mimicked the effects of NO on neurons. We suggest that NO is an endogenous neuromodulator involved in the control of some aspects of feeding and locomotor behavior of Clione.

Serotonin-induced spike narrowing in a locomotor pattern generator permits increases in cycle frequency during accelerations.

During serotonin-induced swim acceleration in the pteropod mollusk Clione limacina, interneurons of the central pattern generator (CPG) exhibit significant action potential narrowing. Spike narrowing is apparently necessary for increases in cycle frequency during swim acceleration because, in the absence of narrowing, the combined duration of the spike and the inhibitory postsynaptic potential (IPSP) of a single cycle is greater than the available cycle duration. Spike narrowing could negatively influence synaptic efficacy in all interneuron connections, including reciprocal inhibitory connections between the two groups of antagonistic CPG interneurons as well as the interneuron-to-motoneuron connections. Thus compensatory mechanisms must exist to produce the overall excitatory behavioral change of swim acceleration. Such mechanisms include 1) a baseline depolarization of interneurons, which brings them closer to spike threshold, 2) enhancement of their postinhibitory rebound, and 3) direct modulation of swim motoneurons and muscles, all through inputs from serotonergic modulatory neurons.

Distribution of myomodulin-like and buccalin-like immunoreactivities in the central nervous system and peripheral tissues of the mollusc, Clione limacina.

The distribution of the myomodulin-like and buccalin-like immunoreactivities in the central nervous system and peripheral tissues associated with feeding was examined in the pteropod mollusc Clione limacina by using wholemount immunohistochemical techniques. Immunoreactive neurons and cell clusters were located in all central ganglia except the pleural ganglia, with approximately 50 central neurons reactive to myomodulin antiserum and 60 central neurons reactive to buccalin antiserum. All central ganglia contained a dense network of myomodulin- and buccalin-immunoreactive processes in their neuropil regions and connectives. In the periphery, the primary attention was focused on the tissues associated with feeding, especially feeding structures unique to Clione, such as hook sacs and buccal cones, which are used for prey capture and acquisition. All of these feeding structures contained myomodulin-immunoreactive and buccalin-immunoreactive fibers, with each peptide family showing specific innervation fields that were common in buccal cones and were totally different in the hook sacs. The specific central and peripheral distribution of myomodulin-like and buccalin-like immunoreactivities as well as specific effects of the exogenous peptides on identified neurons involved in the control of feeding behavior and swimming suggest that neuropeptides from myomodulin and buccalin families act as neurotransmitters or neuromodulators in a variety of central circuits and in the peripheral neuromuscular systems associated with feeding in Clione limacina.

Startle phase of escape swimming is controlled by pedal motoneurons in the pteropod mollusk Clione limacina.

Escape swimming in the pteropod mollusk Clione limacina includes an initial startle response in which one or two powerful wing beats propel the animal up to 18 body lengths per second, followed by a variable period of fast swimming with a maximal speed of 6 body lengths per second. The initial startle response is the focus of this report. Two pairs of large pedal neurons (50-60 microns) initiate wing contractions that are several times stronger than those produced during slow or fast swimming. These "startle" neurons are silent, with very low resting potentials and high activation thresholds. Each startle neuron has widespread innervation fields in the ipsilateral wing, with one pair of neurons innervating the dorsal musculature and producing dorsal flexion of the wing (d-phase) and the other innervating the ventral musculature and producing a ventral flexion of the wing (v-phase). Startle neurons are motoneurons, because they produce junctional potentials or spike-like responses in both slow-twitch and fast-twitch muscle cells with 1:1 ratios of spikes to excitatory postsynaptic potentials. Muscle activation persists in high-divalent saline, suggesting monosynaptic connections. The musculature innervated by startle neurons is the same used during normal slow and fast swimming. However, startle neuron activity is independent of normal swimming activity: startle neurons do not influence the activity of swim pattern generator interneurons or motoneurons, nor do swim neurons alter the activity of startle neurons. The startle response shows significant response depression with repetitive mechanical stimulation of the tail or wings. A major focus for this depression is at the neuromuscular junction. In reduced preparations, repetitive direct stimulation of a startle neuron does not result in a significant decrease in spike number or frequency, but does produce a decrease in force generation (decrease to 20% of original value after 5 stimuli delivered at 3-s intervals). Inputs that activate the wing retraction reflex as well as swim inhibition inhibit startle neurons. The inhibition appears to originate in the retraction interneurons, because direct connections from retraction sensory cells or retraction motoneurons are not found. Mechanical stimulation of a wing or the tail, which usually initiates startle response in intact animals, produces spikes or large EPSPs in startle neurons. The startle neurons appear to be likely candidates for direct control of the swim musculature during the startle phase of escape swimming in Clione.

Cholinergic activation of startle motoneurons by a pair of cerebral interneurons in the pteropod mollusk Clione limacina.

The holoplanktonic pteropod mollusk Clione limacina exhibits an active escape behavior that is characterized by fast swimming away from the source of potentially harmful stimuli. The initial phase of escape behavior is a startle response that is controlled by pedal motoneurons whose activity is independent of the normal swim pattern generator. In this study, a pair of cerebral interneurons is described that produces strong activation of the d-phase startle motoneurons, which control dorsal flexion of the wings. These interneurons were designated cerebral startle (Cr-St) interneurons. Each Cr-St neuron has a small cell body on the dorsal surface of the cerebral ganglia and one large axon that runs into the ipsilateral cerebral-pedal connective and the neuropile of the ipsilateral pedal ganglion. Each spike in a Cr-St neuron produces a fast, high-amplitude (up to 50 mV) excitatory postsynaptic potential (EPSP) in the d-phase startle motoneurons. This 1:1 ratio of spikes to EPSPs and the stable short synaptic latencies (2 ms) persist in high-Mg2+, high-Ca2+ seawater, suggesting monosynaptic connections. Synaptic transmission between Cr-St neurons and startle motoneurons exhibits a very slow synaptic depression, because a number of spikes in Cr-St neurons is required to achieve a noticeable decrease in EPSP amplitude. Synaptic transmission between Cr-St interneurons and startle motoneurons appears to be cholinergic. In startle neurons, 20 microM atropine and 50 microM d-tubocurarine reversibly block EPSPs produced by spike activity in Cr-St interneurons. Hexamethonium only partially blocks EPSPs in startle neurons, and much higher concentrations are required. Exogenous acetylcholine (1 microM) produces a dramatic depolarization of startle motoneurons in high-Mg2+ seawater, and this depolarization is reversibly blocked by atropine. Nicotine also has a depolarizing effect on startle motoneurons, although higher concentrations are required. Cr-St interneurons and startle motoneurons are also electrically coupled; however, the coupling is weak. Stimuli that are known to initiate escape responses in intact animals, such as tactile stimulation of the tail or wings, produce excitatory inputs to Cr-St interneurons. In addition, tactile stimulation of the lips and buccal cones, which is known to trigger prey capture reactions in Clione, also produces excitatory inputs to Cr-St interneurons and startle motoneurons, suggesting involvement of the startle neuronal system in prey capture behavior of Clione.

Modulation of swimming speed in the pteropod mollusc, Clione limacina: role of a compartmental serotonergic system.

In locomotory systems, the central pattern generator and motoneuron output must be modulated in order to achieve variability in locomotory speed, particularly when speed changes are important components of different behavior acts. The swimming system of the pteropod mollusc Clione limacina is an excellent model system for investigating such modulation. In particular, a system of central serotonergic neurons has been shown to be intimately involved in regulating output of the locomotory pattern generator and motor system of Clione. There are approximately 27 pairs of serotonin-immunoreactive neurons in the central nervous system of Clione, with about 75% of these identified. The majority of these identified immunoreactive neurons are involved in various aspects of locomotory speed modulation. A symmetrical cluster of pedal serotonergic neurons serves to increase wing contractility without affecting wing-beat frequency or motoneuron activity. Two clusters of cerebral cells produce widespread responses that lead to an increase in pattern generator cycle frequency, recruitment of swim motoneurons, activation of the pedal serotonergic neurons and excitation of the heart excitor neuron. A pair of ventral cerebral neurons provides weak excitatory inputs to the swimming system, and strongly inhibits neurons of the competing whole-body withdrawal network. Overall, the serotonergic system in Clione is compartmentalized so that each subsystem (usually neuron cluster) can act independently or in concert to produce variability in locomotory speed.

Whole body withdrawal circuit and its involvement in the behavioral hierarchy of the mollusk Clione limacina.

1. The behavioral repertoire of the holoplanktonic pteropod mollusk Clione limacina includes a few well-defined behaviors organized in a priority sequence. Whole body withdrawal takes precedence over slow swimming behavior, whereas feeding behavior is dominant over withdrawal. In this study a group of neurons is described in the pleural ganglia, which controls whole body withdrawal behavior in Clione. Each pleural withdrawal (Pl-W) neuron has a high threshold for spike generation and is capable of inducing whole body withdrawal in a semi-intact preparation: retraction of the body-tail, wings, and head. Each Pl-W neuron projects axons into the main central nerves and innervates all major regions of the body. 2. Stimulation of Pl-W neurons produces inhibitory inputs to swim motor neurons that terminate swimming activity in the preparation. In turn, Pl-W neurons receive inhibitory inputs from the cerebral neurons involved in the control of feeding behavior in Clione, neurons underlying extrusion of specialized prey capture appendages. Thus it appears that specific inhibitory connections between motor centers can explain the dominance of withdrawal behavior over slow swimming and feeding over withdrawal in Clione.

Cerebral serotonergic neurons reciprocally modulate swim and withdrawal neural networks in the mollusk Clione limacina.

1. A pair of serotonin-immunoreactive neurons has been identified in the cerebral ganglia of the pteropod mollusk Clione limacina, which produce coordinated, excitatory/inhibitory effects on neurons controlling two incompatible behaviors, swimming and whole body withdrawal. These cells were designated cerebral serotonergic ventral (Cr-SV) neurons. 2. Activation of Cr-SV neurons produces a prominent inhibition of the pleural withdrawal neurons, which have been previously shown to induce whole body withdrawal in Clione. In addition, the cerebral neurons produce weak excitatory inputs to swim motor neurons, pedal serotonergic neurons involved in the peripheral modulation of swimming, and to the serotonergic heart excitor neuron. 3. Inhibitory and excitatory effects appear to be produced by serotonin because they are mimicked by exogenous serotonin and are blocked by the serotonin antagonist mianserin. 4. All serotonergic neurons identified thus far in the CNS of Clione appear to function in a coordinated manner, altering a variety of neural centers all directed toward the activation of swimming behavior.

Serotonergic modulation of swimming speed in the pteropod mollusc Clione limacina. I. Serotonin immunoreactivity in the central nervous system and wings.

Serotonin-immunoreactive somata in the pteropod mollusc Clione limacina were restricted to the cerebral and pedal ganglia. 10-14 pairs of cells were consistently found in the cerebral ganglia, including one large pair that had soma positions and axon branching patterns reminiscent of those of the metacerebral cells of other molluscs. Two clusters of somata were found on the midline near the cerebral commissure, one on the anterior-lateral margin and one posterior-laterally. A distinct paired cluster of up to nine somata was found on the dorso-lateral margin of the pedal ganglia, near the emergence of the pedal commissure. Up to five of these cells innervated the ipsilateral wing via the wing nerve. Dye-fills of these cells showed that they branch repeatedly in the ipsilateral wing and innervate the swim musculature. Double-labelling experiments indicated that the filled neurons were also serotonin-immunoreactive. Neurobiotin fills that were processed for electron microscopy revealed two types of terminals associated with the swim musculature: direct contacts and reactive terminals adjacent to non-labelled presynaptic terminals. Additional immunoreactive neurons in the pedal ganglia included the asymmetrical heart excitor neuron of the left pedal ganglion and up to nine ventral somata.

Serotonergic modulation of swimming speed in the pteropod mollusc Clione limacina. II. Peripheral modulatory neurons.

A symmetrical cluster of serotonin-immunoreactive neurons in the pedal ganglia of Clione limacina has been described morphologically and physiologically. At least five of the cluster neurons send axons to the ipsilateral wing that branch throughout the entire wing area. Activation of these cells did not produce a motor effect in non-swimming preparations, but did enhance contractility in swimming preparations. Activity in the pedal neurons did not produce detectable central effects as neither swim interneuron nor swim motor neuron activities were altered. Most notable was a lack of a change in swim frequency, a characteristic of swim acceleration. Activity in the pedal neurons did enhance the size of muscle junctional potentials and spike-like responses, but only in slow-twitch muscles. The peripheral modulatory effect was blocked by the serotonin antagonist mianserin.

Serotonergic modulation of swimming speed in the pteropod mollusc Clione limacina. III. Cerebral neurons.

Swim acceleration in Clione limacina can occur via central inputs to pattern generator interneurons and motor neurons and through peripheral inputs to the swim musculature. In the previous paper, peripheral modulation of the swim muscles was shown to increase wing contractility. In the present paper, central inputs are described that trigger an increase in swim frequency and an increase in motor neuron activity. In dissected preparations, spontaneous acceleration from slow to fast swimming included an increase in the cycle frequency, a baseline depolarization in the swim interneurons and an increase in the intensity of motoneuron firing. Similar effects could be elicited by bath application of 10(-5) mol l-1 serotonin. Two clusters of cerebral serotonin-immunoreactive interneurons were found to produce acceleration of swimming accompanied by changes in neuronal activity. Posterior cluster neurons triggered an increase in swim frequency, depolarization of the swim interneurons, an increase in general excitor motoneuron activity and activation of type 12 interneurons and pedal peripheral modulatory neurons. Cells from the anterior cerebral cluster also increased swim frequency, increased activity in the swim motoneurons and activated type 12 interneurons, pedal peripheral modulatory neurons and the heart excitor neuron. The time course of action of the anterior cluster neurons did not greatly outlast the duration of spike activity, while that of the posterior cluster neurons typically outlasted burst duration. It appears that the two discrete clusters of serotonin-immunoreactive neurons have similar, but not identical, effects on swim neurons, raising the possibility that the two serotonergic cell groups modulate the same target cells through different cellular mechanisms.

Small cardioactive peptide B increases the responsiveness of the neural system underlying prey capture reactions in the pteropod mollusc, Clione limacina.

Effects of small cardioactive peptide B (SCPB) on cerebral neurons which underlie prey capture in the carnivorous pteropod mollusc, Clione limacina, were investigated. SCPB in concentrations of 10 microM and higher produced direct activation of cerebral ganglion neurons underlying extrusion of buccal cones used in prey capture. SCPB in lower concentrations, between 1 and 5 microM, did not have a noticeable effect on the membrane potentials of these neurons; however, it significantly increased their responsiveness to sensory inputs from the tactile stimulation of the head, and their ability to generate afterdischarge activity. SCPB immunoreactivity was observed in cell bodies in buccal, cerebral, pedal, and intestinal ganglia, as well as in the anterior esophagus and in buccal cones where fibers stained intensely. These electrophysiological and immunohistochemical data suggest that SCPB may have a physiological role in feeding arousal in Clione.

Co-Activation of Antagonistic Motoneurons as a Mechanism of High-Speed Hydraulic Inflation of Prey Capture Appendages in the Pteropod Mollusk Clione limacina.

The predatory pteropod mollusk Clione limacina catches its prey by using specialized oral appendages called buccal cones. Eversion and elongation of buccal cones is a hydraulic phenomenon. In the cerebral ganglia, two groups of motoneurons have been identified that underlie functionally opposite movements of buccal cones: extrusion and retraction. We suggest that the remarkably rapid inflation of buccal cones (50 ms) is achieved through initial co-activation of antagonistic neurons, which presumably produces high pressure in the head hemocoel prior to buccal cone extrusion. The subsequent sudden inhibition of retractor motoneuron activity results in a very rapid and powerful inflation of the buccal cones. Cerebral interneurons that evoke co-activation are described.

FMRFamide and GABA Produce Functionally Opposite Effects on Prey-Capture Reactions in the Pteropod Mollusk Clione limacina.

The effects of FMRFamide and gamma-aminobutyric acid (GABA) on prey-capture reactions in Clione and on cerebral A and B neurons, which control opposite movements of prey capture appendages, have been studied. FMRFamide hyperpolarized A neurons and depolarized and increased spike activity in B neurons. FMRFamide thus had a reciprocal effect on A and B neurons, triggering buccal cone withdrawal. In addition, FMRFamide inhibited swimming, acceleration of which is a component of feeding arousal. Many neurons throughout the central nervous system showed FMRFamide immunoreactivity. Dense networks of immunoreactive fibers were localized in the head wall, buccal mass and in buccal cones, adjacent to striated longitudinal muscle cells. In wings, immunoreactive processes were found mainly in association with smooth retractor muscles. GABA depolarized and activated A neurons but hyperpolarized and inhibited B neurons. The overall effect of GABA thus resulted in extrusion of buccal cones. Both direct GABA responses and inhibitory postsynaptic potentials (IPSPs) induced in B neurons by A neuron activity were chloride-mediated. However, picrotoxin and bicuculline did not block IPSPs or direct GABA responses in B cells.

Neuromuscular organization in the swimming system of the pteropod mollusc Clione limacina.

Swim motor neurons of the pteropod mollusc Clione limacina were identified by a combination of electrophysiological and morphological characteristics. Two types of motor neurons were found, including small motor neurons which are active during both slow and fast swimming and which innervated restricted fields of the ipsilateral wing. General excitor motor neurons have large cell bodies, innervate widespread fields and are recruited into activity for fast swimming. Small motor neurons monosynaptically innervate slow-twitch muscle cells, whereas general excitors monosynaptically innervate both slow-twitch and fast-twitch muscle cells. Activity in general excitors can centrally enhance that in small motor neurons because the neurons are electrically coupled. Neuromuscular recordings and lesion experiments indicate that a peripheral nerve network does not appear to play an important role in the spread of excitation throughout the muscle fields.

Cerebral neurons underlying prey capture movements in the pteropod mollusc, Clione limacina. I. Physiology, morphology.

The pteropod mollusc Clione limacina feeds on shelled pteropods capturing them with 3 pairs of oral appendages, called buccal cones. A group of electrically-coupled putative motoneurons (A neurons) has been identified in the cerebral ganglia, whose activation induces opening of the oral skin folds and extrusion of the buccal cones. These cells are normally silent and have one or two axons in the ipsilateral head nerves. Electrical coupling between A neurons is relatively weak and normally does not produce 1:1 spike synchronization. Coupling coefficients ranged from 0.05 to 0.25. A second type of putative motoneurons (B neurons) controls retraction and withdrawal of buccal cones. B neurons show spontaneous spike activity which maintains the buccal cones in a continuous retracted state. All B neurons have one axon running into the head nerves. Ipsilateral B motoneurons are electrically coupled to each other. A neurons strongly inhibit B neurons, however, seven identified A motoneurons which were specifically tested do not form monosynaptic contacts with B motoneurons. Appropriate stimuli from the prey activate A motoneurons, which in turn inhibit B motoneurons and evoke extrusion of the buccal cones. One mechanism promoting the speed of this extremely rapid reaction is brief co-activation of antagonistic A and B neuron groups, which provides a notable increase in fluid pressure inside the head. Mechanical stimulation of buccal cones provides excitatory inputs to A motoneurons. Similar stimulation from captured prey would serve to prolong buccal cone protraction during the manipulatory phase of feeding.

Fast-Strike Feeding Behavior in a Pteropod Mollusk, Clione limacina Phipps.

High speed cinematography and video recordings were used to evaluate the fast-strike feeding response by which Clione limacina captures its prey, Limacina helicina. The acquisition phase of feeding involves rapid mouth opening and extrusion of three pairs of buccal cones. Mouth opening occurs in 10 to 20 ms, while hydrostatic inflation of the buccal cones takes 50 to 70 ms. Buccal cones are immediately retracted if prey are not contacted. Buccal cones surround the prey and release a viscous material that may be used as an adhesive attachment to the prey shell. Surface ultrastructure of the buccal cones reveals that they are studded with clusters of capitulate papillae, which appear to be the source of the viscous secretory material.

Neural Control of Speed Changes in an Opisthobranch Locomotory System.

Three forms of forward locomotion have been described in the pteropod mollusk Clione limacina, including slow, fast, and escape swimming. The neuromuscular organization of the swimming system suggests that a two-geared system operates for slow and fast swimming, while the escape response is superimposed on fast swimming. In addition to escape, changes in locomotory speed can occur through a dramatic "change-of-gears," or through a more subtle change of speed within gears. The former involves reconfiguration of the central pattern generator and recruitment of previously inactive motor units. The latter can be due to: changes in tonic inputs to the central neurons, central modulation that is not sufficient to "change gears," endogenous properties of muscle cells, and peripheral modulation of muscle contractility. The initial ballistic phase of escape swimming is believed to be triggered by activity in a newly identified pair of swim motor neurons that neither receive information from, nor provide input to, the central pattern generator. These neurons appear to produce a startle response. Evidence presented suggests that most, if not all, of these variables help produce locomotory plasticity in Clione.

Decreases in heterologous metabolic and dye coupling, but not in electrical coupling, accompany meiotic resumption in hamster oocyte-cumulus complexes.

The temporal relationship between resumption of meiosis and reduction in either heterologous intercellular coupling, or magnitude of oocyte or cumulus cell resting potential in hamster oocyte-cumulus complexes was investigated. Coupling was assessed qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites or electrical current after culture of complexes in various systems previously characterized either to maintain meiotic arrest or to permit meiotic resumption. In each of the three systems which permitted meiotic resumption, cumulus to oocyte metabolic and dye coupling and oocyte to cumulus dye coupling decreased progressively with time after release from meiotic arrest. In contrast, no similar temporal changes in metabolic or dye coupling were observed in any complex after culture in either of the two systems which maintained meiotic arrest. Analysis of the extent of heterologous ionic coupling revealed that in neither direction was a decrease in ionic uncoupling consistently associated with reinitiation of meiosis. Furthermore, while the resting potential of both the oocyte and cumulus cell underwent changes characteristic of each system employed, the level of neither cell membrane potential was specific to meiotic status. These results support the hypothesis that meiotic maturation in hamster oocytes is accompanied by disruption of the integrity of intercellular, non-ionic coupling between the oocyte and its adherent cumulus cells. The data show, however, that no specific alteration either in the extent of ionic coupling or in the oocyte or cumulus cell resting potential is prerequisite for meiotic resumption in this species.