Circulation of hemocoelic fluid during slow and fast swimming in the pteropod mollusc Clione limacina

In the pteropod mollusc Clione limacina Phipps 1774, individuals possess an open circulatory system that fills their body cavities and functions as a hydrostatic skeleton. Individuals of C. limacina demonstrate two distinct swimming behaviors, slow and fast swimming, and their wings are supported by their hydrostatic skeleton. We investigated the circulation of fluid within the body cavities of individuals of C. limacina by injecting dye into the hemocoelic compartments to visualize flow during both slow swimming and serotonin‐induced fast swimming. Hemocoelic fluid was observed to have a defined pattern of flow: rostrally from the heart into the wings and head, then following a dorsal pathway caudally into the body and tail before being taken up by the heart again. During patterned attack behavior, the neck constricted in width as the head's buccal cones were hydraulically inflated with hemocoelic fluid.     

Pleural-buccal interneurons in the pteropod mollusc Clione limacina

Paired, Phe-Met-Arg-Phe-NH₂-ergic pleural-to-buccal projecting neurons of the pleural ganglia were suggested to be responsible for feeding arrest associated with defensive withdrawal in freshwater and terrestrial pulmonate molluscs. In the present study, the pleural-to-buccal projecting cells were, for the first time, identified in a representative opisthobranch, the carnivorous marine pteropod Clione limacina. Two symmetric neurons of its pleural ganglia were found to be similar to the pulmonate pleural-to-buccal projecting neurons in the number of neurons, positions of their cell bodies in the central nervous system, a unique, indirect route of their axon, electrotonic coupling of the left and right cells, and expression of Phe-Met-Arg-Phe-NH₂-like immunoreactivity and inhibitory action on neurons participating in the motor program for feeding. In their turn, pleural-to-buccal projecting neurons receive excitatory inputs from the protractor interneurons involved in the feeding rhythm generation. Also, it was demonstrated that the pleural-to-buccal projecting cells activity positively correlates with spontaneous and induced acceleration of the locomotor rhythm. Accordingly, stimulation of the cerebral command neuron for locomotion, cell CPA1, excited pleural-to-buccal projecting neurons. We conclude that the neuronal network underlying feeding behavior in both pulmonate and opisthobranch molluscs is similarly linked to defensive behavior by pleural Phe-Met-Arg-Phe-NH₂-ergic neurons, thus indicating evolutionary conservation of these pleural-buccal projections. Accepted: 22 June 1999     

Organization of the dorsoventral musculature in the wings of the pteropod mollusc Clione limacina

The wings of the pteropod mollusc Clione limacina provide forward propulsive force through flapping movements in which the wings bend throughout their length in both dorsal and ventral directions. The musculature of the wings includes oblique, striated muscle bundles that generate the swimming movements of the wings, longitudinal and transverse (smooth) muscle bundles that collapse the wings and pull them into the body during a wing withdrawal response, and dorsoventral muscles that control the thickness of the wings. All muscles act against a hydrostatic skeleton that forms a central hemocoelic space within the wings. Of these muscle types, all have been thoroughly described and studied except the dorsoventral muscles. The fortuitous discovery that the dorsoventral musculature can be intensely labeled with an antibody against the vertebrate hyperpolarization‐activated cation channel (HCN2) provided the opportunity to describe the organization of the dorsoventral musculature in detail. In addition, electrical recordings and microelectrode dye injections supported the immunohistochemical data, and provided preliminary data on the activity of the muscle fibers. The organization and activity of the dorsoventral musculature suggests it may be involved in regulation of wing stiffness during the change from slow to fast swimming.     

Cerebral neurons underlying prey capture movements in the pteropod mollusc,Clione limacina

The pteropod mollusc Clione limacina is a highly specialized carnivore which feeds on shelled pteropods and uses, for their capture, three pairs of oral appendages, called buccal cones. Contact with the prey induces rapid eversion of buccal cones, which then become tentacle-like and grasp the shell of the prey. In the previous paper, a large group of electrically coupled, normally silent cells (A motoneurons) has been described in the cerebral ganglia of Clione. Activation of A neurons induces opening of oral skin folds and extrusion of the buccal cones. The present study continues the analysis of the electrical properties of A motoneurons.Brief intracellular stimulation of an A neuron can produce prolonged firing (afterdischarge), lasting up to 40 s, in the entire population of A neurons. Afterdischarge activity is based on an afterdepolarization evoked by an initial strong burst of A neuron spikes. The data suggest that this afterdepolarization represents excitatory synaptic input from unidentified neurons which in turn receive excitatory inputs from A neurons, thus organizing positive feedback. The main functional role of this positive feedback is the spread and synchronization of spike activity among all A neurons in the population. In addition, it serves to transform a brief excitatory input to A neurons into their prolonged and stable firing, which is required during certain phases of feeding behavior in Clione.     

Differential effects of serotonergic and peptidergic cardioexcitatory neurons on the heart activity in the pteropod mollusc, Clione limacina

A group of four cardioexcitatory neurons has been identified in the intestinal ganglia of the mollusc Clione limacina. Relatively weak stimulation of the intestinal neurons induced auricle contractions only, while strong stimulation produced initial auricle contractions followed by full-cycle auricle-ventricle contractions. Intestinal cardioexcitatory neurons probably utilized as their transmitter a peptide similar to Tritonia pedal peptide – they showed pedal peptide-like immunoreactivity, and their effects were mimicked by application of the exogenous pedal peptide. The pedal cardioexcitatory neuron was found to produce strong excitatory effects only on the ventricle contractions. Its stimulation induced ventricle contractions in the quiescent heart or significantly accelerated the rate of ventricle contractions in the rhythmically active heart. The pedal cardioexcitatory neuron apparently utilized serotonin as a neurotransmitter, based upon serotonin immunoreactivity, blocking effect of serotonin antagonists mianserin and methysergide, and the observation that exogenous serotonin mimicked its effect. A dense network of pedal peptide-like immunoreactivity was found both in the auricle and ventricle tissue. Serotonin immunoreactivity was densely present in the ventricle, while the auricle contained only a separate serotonin-immunoreactive unbranched axon. Thus, there are two separate groups of central cardioexcitatory neurons with different effects on heart activity, which together might provide a complex cardio-regulatory function in Clione. Accepted: 14 August 1999     

Morphology of wing mechanoreceptors involved in the wing retraction reflex in the pteropod mollusc Clione limacina

Tactile stimulation of the wings (parapodia) of actively swimming Clione limacina results in inhibition of swimming and retraction of the wings. Electrophysiological evidence suggests that wing mechanoreceptors have central cell bodies and wide innervation fields in the ipsilateral wing. Scanning electron microscopy of expanded wings reveals ciliary cone processes arranged in a pattern that is similar to the electrophysiologically‐determined innervation fields of wing mechanoreceptors. Transmission electron microscopy suggests that the ciliary cone structures are terminal processes of neuron‐like cells. Three‐dimensional reconstructions of serially‐sectioned terminal processes indicate that cell bodies are not found in the wing epithelium or immediately under the epithelium, further supporting the notion that the wing mechanoreceptors have central cell bodies.     

An identified cerebral interneuron initiates different elements of prey capture behavior in the pteropod mollusc,Clione limacina

The prey capture phase of feeding behavior in the pteropod molluscClione limacina consists of an explosive extrusion of buccal cones, specialized oral appendages which are used to catch the prey, and significant acceleration of swimming. Several groups of neurons which control different components of prey capture behavior inClione have been previously identified in the CNS. However, the question of their coordination in order to develop a normal behavioral reaction still remains open. We describe here a cerebral interneuron which has wide-spread excitatory and inhibitory effects on a number of neurons in the cerebral and pedal ganglia, directed toward the initiation of prey capture behavior inClione. This bilaterally symmetrical neuron, designated Cr-PC (Cerebral interneuron initiating Prey Capture), produced monosynaptic activation of Cr-A motoneurons, which control buccal cone extrusion, and inhibition of Cr-B and Cr-L motoneurons, whose spike activities maintain buccal cones in a withdrawn position inside the head in non-feeding animals. In addition, Cr-PC produced monosynaptic activation of a number of swim motoneurons and interneurons of the swim central pattern generator (CPG) in the pedal ganglia, pedal serotonergic Pd-SW neurons involved in a peripheral modulation of swimming and the serotonergic Heart Excitor neuron.     

Smooth muscle fiber types and a novel pattern of thick filaments in the wing of the pteropod mollusc Clione limacina

Summary Wing (parapodial) retraction in the pteropod mollusc Clione limacina is a reflex triggered by tactile stimulation. Light and transmission electron microscopy revealed three groups of smooth muscles in the wing hemocoel that participate in retraction movements: transverse, longitudinal, and dorsoventral. Among these, two subtypes of muscle cells were identified. The first (type A) appears in all three groups and forms a well-organized lattice-like structure. The second (type B) is the major component of transverse muscles and runs in one direction only. Quantitative ultrastructural comparisons of dimensions, abundance, and organization of dense bodies, thick and thin filaments, membrane invaginations, sarcoplasmic reticulum, and mitochondria suggest that type A cells are able to contract and relax more quickly with less endurance whereas type B cells are capable of generating stronger contractions with more endurance and slower relaxation speed. Furthermore, type A cells have a unique pattern of thick filament organization, here referred to as pseudosarcomeres. The roles played by the different cell types in wing retraction are discussed.     

Feeding and growth in Clione limacina (Phipps), a pteropod mollusc. II. Assimilation,metabolism, and growth efficiency

The gymnosomatous pteropod Clione limacina (Phipps) assimilated carbon from its prey Spiratella retroversa (Fleming) with greater than 90 % efficiency and with nearly 100 % efficiency for nitrogen.The respiratory rate of Clione was found to vary with locality and season, but the weight exponent did not differ significantly nor was it affected by temperature. Starving animals lost weight about twice as rapidly as would be predicted from the respiratory rate, but the rate of loss was unaffected by temperature over the normal range (2–17 °C) for the animal. A T-line of Paloheimo and Dickie calculated from the rate of weight loss had approximately the same slope as that obtained from direct respirometry, but a significantly greater slope (weight exponent) than those obtained for T-lines calculated from growth experiments. Feeding also greatly increased the level of metabolism, suggesting that a fed animal might be twenty times more metabolically active than a starved one.First order growth efficiency K₁ for Clione was often in excess of 50 % lending further support to the hypothesis that the prey is virtually all “physiologically useful ration”. K-lines of Paloheimo and Dickie calculated for individual Clione grown over a period of weeks showed no significant slope whether log K was plotted against size of predator (Clione), amount of prey ingested (Spiratella) or mean size of the prey. Multiple regression and correlation analyses suggest that a negative slope for the K-line would result if an animal were grown with a constant size of food. Increasing the ration markedly, by increasing both temperature and size of prey as the predator grew, tended to maintain the slope of the K-line near to zero.     

Prey capture phase of feeding behavior in the pteropod mollusc,clione limacina: neuronal mechanisms

The prey capture phase of feeding behavior in the pteropod mollusc Clione limacina consists of an explosive extrusion of buccal cones, specialized structures which are used to catch the prey, and acceleration of swimming with frequent turning and looping produced by tail bend. A system of neurons which control different components of prey capture behavior in Clione has been identified in the cerebral ganglia. Cerebral B and L neurons produce retraction of buccal cones and tightening of the lips over them — their spontaneous spike activities maintain buccal cones in the withdrawn position. Cerebral A neurons inhibit B and L cells and produce opening of the lips and extrusion of buccal cones. A pair of cerebral interneurons C-BM activates cerebral A neurons and synchronously initiates the feeding motor program in the buccal ganglia. Cerebral T neurons initiate acceleration of swimming and produce tail bending which underlies turning and looping during the prey capture. Both tactile and chemical inputs from the prey produce activation of cerebral A and T neurons. This reaction appears to be specific, since objects other than alive Limacina or Limacina juice do not initiate activities of A and T neurons.     

The contribution of the pleural type 12 interneuron to swim acceleration in Clione limacina

The pteropod mollusc, Clione limacina, swims by alternate dorsal–ventral flapping movements of its wing-like parapodia. The basic swim rhythm is produced by a network of pedal swim interneurons that comprise a swim central pattern generator (CPG). Serotonergic modulation of both intrinsic cellular properties of the swim interneurons and network properties contribute to swim acceleration, the latter including recruitment of type 12 interneurons into the CPG. Here we address the role of the type 12 interneurons in swim acceleration. A single type 12 interneuron is found in each of the pleural ganglia, which contributes to fast swimming by exciting the dorsal swim interneurons while simultaneously inhibiting the ventral swim interneurons. Each type 12 interneuron sends a single process through the pleural–pedal connective that branches in both ipsilateral and contralateral pedal ganglia. This anatomical arrangement allowed us to manipulate the influence of the type 12 interneurons on the swim circuitry by cutting the pleural–pedal connective followed by a “culture” period of 48 h. The mean swim frequency of cut preparations was reduced by 19% when compared to the swim frequency of uncut preparations when stimulated with 10⁻⁶ M serotonin; however, this decrease was not statistically significant. Additional evidence suggests that the type 12 interneurons may produce a short-term, immediate effect on swim acceleration while slower, modulatory inputs are taking shape.     

Effects of elevated ammonia concentrations on survival, metabolic rates, and glutamine synthetase activity in the Antarctic pteropod mollusk Clione limacina antarctica

Information on the effects of elevated ammonia on invertebrates in general, and polar Mollusks in particular, is scant. Questions of ammonia sensitivity are interesting for several reasons, particularly since predicted global change scenarios include increasing anthropogenic nitrogen and toxic ammonia. Furthermore, polar zooplankton species are often lipid-rich, and authors have speculated that there is a linkage between elevated levels of lipids/trimethylamine oxide and enhanced ammonia tolerance. In the present study, we sought to examine ammonia tolerance and effects of elevated exogenous ammonia on several key aspects of the physiology and biochemistry of the pteropod mollusk, Clione limacina antarctica. We determined that the 96-h LC₅₀ value for this species is 7.465?mM total ammonia (Upper 95% CL?=?8.498?mM and Lower 95% CL?=?6.557?mM) or 0.51?mg/L as unionized ammonia (NH₃) (at a pH of 7.756). While comparative data for mollusks are limited, this value is at the lower end of reported values for other species. When the effects of lower ammonia concentrations (0.07?mM total ammonia) on oxygen consumption and ammonia excretion rates were examined, no effects were noted. However, total ammonia levels as low as 0.1?mM (or 0.007?mg/l NH₃) elevated the activity of the ammonia detoxification enzyme glutamine synthetase by approximately 1.5-fold. The values for LC₅₀ and observable effects on biochemistry for this one species are very close to permissible marine ammonia concentrations, indicating a need to more broadly determine the sensitivity of zooplankton to potential elevated ammonia levels in polar regions.     

The Calculation of Frequency-Shift Functions for Chains of Coupled Oscillators, with Application to a Network Model of the Lamprey Locomotor Pattern Generator

Chains of coupled limit-cycle oscillators are considered, in which the coupling is assumed to be weak and only between adjacent oscillators. For such a system the change in frequency of an oscillator due to the coupling can be expressed, up to first order in thecoupling strength, by functions that depend only on the phase difference between the coupled oscillators. In this article a numerical algorithm is developed for the evaluation of these functions (the H-functions) in terms of a single oscillator and the interactions between coupled oscillators. The technique is applied to a connectionist model for the locomotor pattern generator in the lamprey spinal cord.An H-function so derived is compared to a function derived empirically(the C-function) from simulations of the same system. The phase lagsthat develop between adjacent oscillators in a simulated chain are compared with those predicted theoretically, and it is shown that coupling thatis functionally strong is nonetheless weak enough to behave as predicted.     

Regeneration of central and peripheral synaptic connections in the locomotor system of the pteropod molluscClione limacina

The neural network underlying rhythmic wing movements in the molluscClione limacina is well-studied. Two different groups of motoneurons innervate two distinct groups of wing muscles. The locomotor rhythm generated in the left and right pedal ganglia is synchronized by interneurons. When the axons of the locomotor motoneurons are crushed, numerous fine neurites sprout towards the denervated muscles and reach them in 8–15 days. At this stage motoneurons project to and synapse on not only correct but equally incorrect muscle targets. After 2 weeks of regeneration the number of incorrect neurites and synaptic connections begins to decrease and following 1.5–2 months all incorrect connections are eliminated, incorrect axons are withdrawn and the behavioral deficit is compensated. In this study the regeneration of interneurons and the growth profiles of inter- and motoneurons were also studiedin vitro. Two individually isolated pedal ganglia were co-cultured in three different configurations: a) the wing nerve stump from one ganglion was fixed against the commissural stump from another ganglion; b) the wing nerve stumps were fixed against each other; c) the commissural stumps were fixed against each other. Under the above experimental conditions we found that the interneurons were able to cross only the contact between two commissural stumps, and in this case found their original targets, restored correct connections and synchronized the rhythm in two pedal ganglia. In contrast, motoneurons were able to cross all types of contacts.     

Shining light into the black box of spinal locomotor networks

Rhythmic activity is responsible for numerous essential motor functions including locomotion, breathing and chewing. In the case of locomotion, it has been realized for some time that the spinal cord contains sufficient circuitry to produce a sophisticated stepping pattern. However, the central pattern generator for locomotion in mammals has remained a ‘black box’ where inputs to the network were manipulated and the outputs interpreted. Over the last decade, new genetic approaches and techniques have been developed that provide ways to identify and manipulate the activity of classes of interneurons. The use of these techniques will be critically discussed and related to current models of network function.     

Anatomical study of serotonergic innervation and 5-HT1A receptor in the human spinal cord

Serotonergic innervation of the spinal cord in mammals has multiple roles in the control of motor, sensory and visceral functions. In rats, functional consequences of spinal cord injury at thoracic level can be improved by a substitutive transplantation of serotonin (5-HT) neurons or regeneration under the trophic influence of grafted stem cells. Translation to either pharmacological and/or cellular therapies in humans requires the mapping of the spinal cord 5-HT innervation and its receptors to determine their involvement in specific functions. Here, we have performed a preliminary mapping of serotonergic processes and serotonin-lA (5-HT_1A) receptors in thoracic and lumbar segments of the human spinal cord. As in rodents and non-human primates, 5-HT profiles in human spinal cord are present in the ventral horn, surrounding motoneurons, and also contact their presumptive dendrites at lumbar level. 5-HT_1A receptors are present in the same area, but are more densely expressed at lumbar level. 5-HT profiles are also present in the intermediolateral region, where 5-HT_1A receptors are absent. Finally, we observed numerous serotonergic profiles in the superficial part (equivalent of Rexed lamina II) of the dorsal horn, which also displayed high levels of 5-HT_1A receptors. These findings pave the way for local specific therapies involving cellular and/or pharmacological tools targeting the serotonergic system.     

Modulation of the feeding system by a radular mechanosensory neuron in the terrestrial slug,Incilaria fruhstorferi

We investigated the modulatory role of a radular mechanoreceptor (RM) in the feeding system of Incilaria. RM spiking induced by current injection evoked several cycles of rhythmic buccal motor activity in quiescent preparations, and this effect was also observed in preparations lacking the cerebral ganglia. The evoked rhythmic activity included sequential activation of the inframedian radular tensor, the supramedian radular tensor, and the buccal sphincter muscles in that order.In addition to the generation of rhythmic motor activity, RM spiking enhanced tonic activities in buccal nerve 1 as well as in the cerebrobuccal connective, showing a wide excitatory effect on buccal neurons. The excitatory effect was further examined in the supramedian radular tensor motoneuron. RM spiking evoked biphasic depolarization in the tensor motoneuron consisting of fast excitatory postsynaptic potentials and prolonged depolarization lasting after termination of RM spiking. These depolarizations also occurred in high divalent cation saline, suggesting that they were both monosynaptic.When RM spiking was evoked in the fictive rasp phase during food-induced buccal motor rhythm, the activity of the supramedian radular tensor muscle showed the greatest enhancement of the three muscles tested, while the rate of ongoing rhythmic motor activity showed no increase.Abbreviations CPG central pattern generator - EPSP excitatory postsynaptic potential - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

Hexamethonium sensitivity of the swim musculature of the pteropod mollusc, <Emphasis Type="Italic">Clione limacina</Emphasis>

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.