Serotonergic Neural System not only Activates Swimming but also Inhibits Competing Neural Centers in a Pteropod Mollusc

Initiation of a particular behavior requires not only activationof the neural center directly involved in its control but alsoinhibition of the neural networks controlling competing behaviors.In the pteropod mollusc, Clione limacina, many identified serotonergicneurons activate or modulate different elements of the swimmingsystem resulting in the initiation or acceleration of the swimmingbehavior. Cerebral serotonergic neurons are described here,which produce excitatory inputs to the swimming system as wellas inhibitory inputs to the neural centers that control competingbehaviors. Whole-body withdrawal behavior is incompatible withswimming activity in Clione. The main characteristic of whole-bodywithdrawal is complete inhibition of swimming. Cerebral serotonergicneurons were found to produce a prominent inhibition of thepleural neurons that control whole-body withdrawal behavior.By inhibiting pleural withdrawal cells, serotonergic neuronseliminate its inhibitory influence on the swimming system andthus favor increased swimming speed. Serotonergic neurons alsoproduce a prominent inhibition of the Pleural White Cell, whichis presumably involved in reproductive or egg-laying behavior.Thus the serotonergic system directly activates swimming systemand, at the same time, alters a variety of other neural systemspreventing simultaneous initiation of incompatible behaviors.     

Evidence that the Central Pattern Generator for Swimming in Tritonia Arose from a Non-Rhythmic Neuromodulatory Arousal System: Implications for the Evolution of Specialized Behavior

Comparisons of the nervous systems of closely related invertebratespecies show that identified neurons tend to be highly conservedeven though the behaviors in which they participate vary. Allopisthobranch molluscs examined have a similar set of serotonin-immunoreactiveneurons located medially in the cerebral ganglion. In a smallnumber of species, these neurons have been physiologically andmorphologically identified. In the nudibranch, Tritonia diomedea,three of the neurons (the dorsal swim interneurons, DSIs) havebeen shown to be members of the central pattern generator (CPG)underlying dorsal/ventral swimming. The DSIs act as intrinsicneuromodulators, altering cellular and synaptic properties withinthe swim CPG circuit. Putative homologues of the DSIs have beenidentified in a number of other opisthobranchs. In the notaspid,Pleurobranchaea californica, the apparent DSI homologues (As1–3)play a similar role in the escape swim and they also have widespreadactions on other systems such as feeding and ciliary locomotion.In the gymnosomatid, Clione limacina, the presumed homologousneurons (Cr-SP) are not part of the swimming pattern generator,which is located in the pedal ganglia, but act as extrinsicmodulators, responding to noxious stimuli and increasing thefrequency of the swim motor program. Putative homologous neuronsare also present in non-swimming species such as the anaspid,Aplysia californica, where at least one of the cerebral serotonergicneurons, CC3 (CB-1), evokes neuromodulatory actions in responseto noxious stimuli. Thus, the CPG circuit in Tritonia appearsto have evolved from the interconnections of neurons that arecommon to other opisthobranchs where they participate in arousalto noxious stimuli but are not rhythmically active.     

Homologues of serotonergic central pattern generator neurons in related nudibranch molluscs with divergent behaviors

Homologues of a neuron that contributes to a species-specific behavior were identified and characterized in species lacking that behavior. The nudibranch Tritonia diomedea swims by flexing its body dorsally and ventrally. The dorsal swim interneurons (DSIs) are components of the central pattern generator (CPG) underlying this rhythmic motor pattern and also activate crawling. Homologues of the DSIs were identified in six nudibranchs that do not exhibit dorsal–ventral swimming: Tochuina tetraquetra, Melibe leonina, Dendronotus iris, D. frondosus, Armina californica, and Triopha catalinae. Homology was based upon shared features that distinguish the DSIs from all other neurons: (1) serotonin immunoreactivity, (2) location in the Cerebral serotonergic posterior (CeSP) cluster, and (3) axon projection to the contralateral pedal ganglion. The DSI homologues, named CeSP-A neurons, share additional features with the DSIs: irregular basal firing, synchronous inputs, electrical coupling, and reciprocal inhibition. Unlike the DSIs, the CeSP-A neurons were not rhythmically active in response to nerve stimulation. The CeSP-A neurons in Tochuina and Triopha also excited homologues of the Tritonia Pd5 neuron, a crawling efferent. Thus, the CeSP-A neurons and the DSIs may be part of a conserved network related to crawling that may have been co-opted into a rhythmic swim CPG in Tritonia. This material is based upon work supported by the National Science Foundation, under Grant No. 0445768, and a GSU Research Program Enhancement grant to PSK.     

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 molluscClione 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 ofClione. There are approximately 27 pairs of serotonin-immunoreactive neurons in the central nervous system ofClione, 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 inClione is compartmentalized so that each subsystem (usually neuron cluster) can act independently or in concert to produce variability in locomotory speed.     

The Role of the Escape Swim Motor Network in the Organization of Behavioral Hierarchy and Arousal in Pleurobranchaea

The escape swimming pattern generator of the notaspid opisthobranchPleurobranchaea drives a high threshold, override behavior.The pattern generator is integrated with neural networks ofother behaviors so as to coordinate unitary behavioral expressionand to promote general behavioral arousal. These functions areseparately produced by different swim network elements. Oneset of swim premotor neurons, the A1/A10 ensemble, A3 and I_VS,generate the swim pattern and, through corollary activity, suppresspotentially conflicting feeding behavior by exerting broad inhibitionat major feeding network interneurons. A second set of swimneurons, the serotonergic As1–4 neurons, provides intrinsicneuromodulatory excitation to the swim pattern generator thatsustains the escape swim episode through multiple cycles. TheAs1–4 also provide neuromodulatory excitation to importantmodulatory, serotonergic cells in the feeding motor networkand locomotor network, and may have a general regulatory rolein the distributed serotonergic arousal network of the mollusk.The As1–4 appear to be also necessary to both avoidanceand orienting turning, and are therefore likely to be critical,multi-functional components upon which much of the organizationof the animal's behavior rests.     

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.     

Modulation of swimming in Tritonia: excitatory and inhibitory effects of serotonin

1. In the mollusc Tritonia escape swimming is produced by a network of central pattern generator (CPG) neurons. The purpose of this study was to determine which neurotransmitters might be involved in the swim system.
2. Injection of serotonin (5HT) into whole animals elicited swimming followed by a long-lasting inhibition of swimming. In isolated brain preparations, bath-applied 5HT elicited a swim pattern at short latency and also caused a long-lasting inhibition of the swim pattern. The activation of swimming by 5HT was associated with a tonic depolarization of cerebral cell 2 (C2) and the dorsal swim interneurons (DSI) which form part of the swim CPG network.
3. In isolated brain preparations, bath applied glycine, histamine, proctolin, and FMFRamide had no effect on the swim motor pattern elicited by electrical stimulation of a peripheral nerve. Aspartate, carbacol, dopamine, glutamate, octopamine, pilocarpine, and small cardioactive peptide-B (SCP_B) inhibited the activation of swimming by nerve stimulation.
4. The 5HT antagonists cyproheptidine, tryptamine, and 7-methyltryptamine had no effect on swimming, but methysergide and fenfluramine inhibited swimming to both normal sensory stimuli and exogenously applied 5HT.
5. Staining with a polyclonal antibody indicated that one class of CPG neurons, the dorsal swim interneurons (DSI), was immunoreactive for 5HT.
6. Taken together, the data suggest that pattern generator interneurons, particularly the DSIs, use 5HT as a neurotransmitter.

     

Neuropeptide TPep action on salivary duct ciliary beating rate in the nudibranch molluscTritonia diomedea

Recently, in the marine molluscTritonia, a family of three peptides (TPep-NLS,-PLS,-PAR) from identified pedal ganglion neurons has been characterized and shown to regulate ciliary beat frequency in epithelia and isolated cells of the molluscan foot. In this study, using an antiserum raised against TPep-NLS, immunofluorescent labelling was observed in specific nerve cell bodies and axons in the buccal ganglia ofTritonia, as well as in axons leading to and innervating the salivary ducts, salivary glands, oesophagus and foregut. This pattern of innervation suggests that buccal ganglion neurons containing TPep control the beating rate of ciliated cells in feeding organs. Accordingly, TPeps were introduced to isolated ciliated salivary ducts. It was found that TPeps and serotonin increased the ciliary beat frequency of cells of the salivary duct similarly; other peptides (such as APep fromAplysia) had no such effect. Threshold sensitivity both for TPeps and serotonin was approximately 10⁻⁸ M, with maximal response occurring above 10⁻⁵ M. Taken together, these structural and physiological results suggest that TPep-like peptides are present in the salivary and other feeding organs ofTritonia and are involved in the regulation of salivary transport.     

Neural mechanisms generating the leech swimming rhythm: Swim-initiator neurons excite the network of swim oscillator neurons

Summary This paper describes newly identified excitatory connections linking the segmentally iterated swim-initiator interneurons with the network of oscillator neurons that generates the leech swimming rhythm. Apparently monosynaptic excitatory chemical connections are made from one class of swim-initiator neurons (cells 204/205) to several members of the swim oscillator network, including cells 28, 115 and, as described by Weeks (1982c), cell 208. A second class of swim-initiator neurons, cells 21 and 61, also excites this subset of the oscillator neurons.The unpaired swim oscillator neuron, cell 208, also chemically excites cells 28 and 115, apparently directly. Thus, in addition to its role as a member of the swim oscillator, the excitatory output from cell 208 to the swim oscillator adds to that provided by the swim-initiator neurons.The results of this paper enlarge the subset of identified swim oscillator neurons synaptically excited by the swim-initiator neurons. These newly described targets of the swim-initiators strengthen the hypotheses that: 1) the swim-initiator neurons supply much of the tonic excitatory drive responsible for activation and maintenance of the swim central motor program, and 2) the two classes of swim-initiators, cells 204/205 and cells 21/61, act synergistically to initiate and maintain swimming.Abbreviations EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential - CNS central nervous system     

Immunochemical and electrophysiological analyses of magnetically responsive neurons in the mollusc Tritonia diomedea

Tritonia diomedea uses the Earth’s magnetic field as an orientation cue, but little is known about the neural mechanisms that underlie magnetic orientation behavior in this or other animals. Six large, individually identifiable neurons in the brain of Tritonia (left and right Pd5, Pd6, Pd7) are known to respond with altered electrical activity to changes in earth-strength magnetic fields. In this study we used immunochemical, electrophysiological, and neuroanatomical techniques to investigate the function of the Pd5 neurons, the largest magnetically responsive cells. Immunocytochemical studies localized TPeps, neuropeptides isolated from Pd5, to dense-cored vesicles within the Pd5 somata and within neurites adjacent to ciliated foot epithelial cells. Anatomical analyses revealed that neurites from Pd5 are located within nerves innervating the ipsilateral foot and body wall. These results imply that Pd5 project to the foot and regulate ciliary beating through paracrine release. Electrophysiological recordings indicated that, although both LPd5 and RPd5 responded to the same magnetic stimuli, the pattern of spiking in the two cells differed. Given that TPeps increase ciliary beating and Tritonia locomotes using pedal cilia, our results are consistent with the hypothesis that Pd5 neurons control or modulate the ciliary activity involved in crawling during orientation behavior.     

Modulatory Mechanisms at a Primitive Neuromuscular Synapse: Membrane Currents, Transmitter Release and Modulation by Transmitters in a Cnidarian Motor Neuron

The phylum Cnidaria arose early in metazoan evolution and, assumingmonophyly, is regarded as being close to the ancestral metazoan.The simplicity of structure in the cnidariannervous system isnot reflected in the physiology of neurons. The motor neuronsthat control swimming in the jellyfish Polyorchis penicillatusepitomise this operational complexity. Synchrony in the contractionof the swimming muscle sheets is achieved by compensatingforthe conduction time of motor APs propagating to distant partsof the motor network. This depends on motor APs continuouslydecreasing in duration as they propagate through the networkwhich in turn leads to a decrease in the delay of muscle actionpotential initiation. Two membrane currents are critical forthis mechanism, a fast, transient K⁺ current (I_K-fast). anda transient Ca⁺⁺ current. A PCR-based screen of genomicDNA producedclones having considerable sequence identity with the Shaker,Shal, Shab and Shaw subfamilies. One full-length clone, jShaklwhen expressed in Xenopus oocytes reveals an A-like Shaker currentwhich activates at very positive voltages. Motor neuron activitycan be modulated by two endogenous transmitters, dopamine andFMRFamide-related peptides which are found endogenously. Dopaminecauses a long lasting hyperpolarization by activating a potassiumcurrent that is regulated by D₂ receptors. In addition dopaminereduces action potential duration. Pol-RFamides, on the otherhand have an excitatory effect by blocking the slowly inactivatingcurrent, I_K-slow     

The swimming behavior of the freshwater calanoid copepods Limnocalanus macrurus Sars, Senecella calanoides Juday and Epischura lacustris Forbes

The swimming behavior of the freshwater calanoid copepods Epischuralacustris, Senecella calanoides, and Limnocalanus macrurus wasexamined using a videotape analysis technique. E. lacustrisand S. calanoides alternate between periods of slow glidingand passive sinking. L. macrurus does not exhibit sinking behavior;its swimming activity consists of slow and steady gliding interruptedoccasionally by jumps. All three copepods exhibit behavioralresponses when exposed to prey organisms, as expressed by anincrease in the duration of sinking for E. lacustris and S.calanoides, a decrease in sinking speed for S. calanoides, anda less circuitous swimming path for L. macrurus. These behavioralchanges may allow the copepods to allocate more time for preydetection, to be less detectable by potential prey and to reducethe frequency of path re-crossing. The results demonstrate therelationship between the swimming and feeding behavior of calanoidcopepods and are consistent with previous findings that copepodshave the ability to modify their foraging behavior in responseto changes in food conditions.     

Assessing the roles of glutamatergic and cholinergic synaptic drive in the control of fictive swimming frequency in young Xenopus tadpoles

This paper investigates the proposal that the frequency of the swimming central pattern generator in young Xenopus tadpoles is partly determined by the population of glutamatergic premotor interneurons active on each cycle. During fictive swimming spinal neurons also receive cholinergic and electrotonic excitation from motoneurons. As frequency changes during swimming we make two predictions: first, since most motoneurons fire very reliably at all frequencies, the electrotonic and nicotinic drive from motoneurons should remain constant, and second, when swimming frequency decreases, the glutamatergic drive should decrease as the number of active premotor excitatory interneurons decreases. We have tested these predictions by measuring the excitatory synaptic drive to motoneurons as frequency changes during fictive swimming. The components of synaptic drive were revealed by the local microperfusion of strychnine together with different excitatory antagonists. After blocking the nicotinic acetylcholine receptor, the mainly glutmatergic excitatory synaptic drive still changed with frequency. However, when glutamate receptors or all chemical transmission was blocked, excitation did not change with frequency. Our predictions are confirmed, suggesting that premotor excitatory interneurons are a major factor in frequency control in the tadpole central pattern generator and that motoneurons provide a stable background excitation. Accepted: 14 August 1998     

Responses to magnetic stimuli recorded in peripheral nerves in the marine nudibranch mollusk <Emphasis Type="Italic">Tritonia diomedea</Emphasis>

Prior behavioral and neurophysiological studies provide evidence that the nudibranch mollusk Tritonia orients to the earth’s magnetic field. Earlier studies of electrophysiological responses in certain neurons of the brain to changing ambient magnetic fields suggest that although certain identified brain cells fire impulses when the ambient field is changed, these neuron somata and their central dentritic and axonal processes are themselves not primary magnetic receptors. Here, using semi-intact animal preparations from which the brain was removed, we recorded from peripheral nerve trunks. Using techniques to count spikes in individual nerves and separately also to identify, then count individual axonal spikes in extracellular records, we found both excitatory and inhibitory axonal responses elicited by changes in the direction of ambient earth strength magnetic fields. We found responses in nerves from many locations throughout the body and in axons innervating the body wall and rhinophores. Our results indicate that primary receptors for geomagnetism in Tritonia are not focally concentrated in any particular organ, but appear to be widely dispersed in the peripheral body tissues.     

Positive feedback loops sustain repeating bursts in neuronal circuits

Voluntary movements in animals are often episodic, with abrupt onset and termination. Elevated neuronal excitation is required to drive the neuronal circuits underlying such movements; however, the mechanisms that sustain this increased excitation are largely unknown. In the medicinal leech, an identified cascade of excitation has been traced from mechanosensory neurons to the swim oscillator circuit. Although this cascade explains the initiation of excitatory drive (and hence swim initiation), it cannot account for the prolonged excitation (10–100 s) that underlies swim episodes. We present results of physiological and theoretical investigations into the mechanisms that maintain swimming activity in the leech. Although intrasegmental mechanisms can prolong stimulus-evoked excitation for more than one second, maintained excitation and sustained swimming activity requires chains of several ganglia. Experimental and modeling studies suggest that mutually excitatory intersegmental interactions can drive bouts of swimming activity in leeches. Our model neuronal circuits, which incorporated mutually excitatory neurons whose activity was limited by impulse adaptation, also replicated the following major experimental findings: (1) swimming can be initiated and terminated by a single neuron, (2) swim duration decreases with experimental reduction in nerve cord length, and (3) swim duration decreases as the interval between swim episodes is reduced.     

The swimming behavior of the copepod Metridia pacfica

The swimming behavior of the copepod Metridia pacifica was studied.Animals exposed to algae showed lower average swimming speedand fewer high-speed bursts. Animals exposed to Artemia naupliialone exhibited no change in swimming behavior.     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

The aim of this study was to identify neurons in the subesophageal ganglion of the medicinal leech which initiate swimming activity and to determine their output connections. We found two bilaterally symmetrical pairs of interneurons, Tr1 and Tr2, located in the first division of the subesophageal ganglion which initiate swimming activity in the isolated nervous system when depolarized with brief (1-3 s) current pulses. Tr1 and Tr2 are considered trigger neurons because elicited swimming episodes outlast the stimulus duration, and because the length of elicited swim episodes is nearly independent of the intensity with which Tr1 and Tr2 are stimulated. Tr1 and Tr2 have similar morphologies. The neurites of both cells cross contralaterally in the subesophageal ganglion, project posteriorly, and exit the subesophageal ganglion in the contralateral connective. The axons of Tr1 and Tr2 extend as far posterior as segmental ganglion 18 of the ventral nerve cord. Tr1 provides direct excitatory drive to three groups of segmental neurons which are capable of initiating swimming: swim-initiating interneurons (cells 204 and 205), serotonin-containing interneurons (cells 61 and 21), and the serotonergic Retzius cells. In addition, all Retzius cells in the subesophageal ganglion are excited directly by Tr1. These three groups of neurons are excited even if Tr1 stimulation is subthreshold for swim initiation. In contrast to Tr1, Tr2 stimulation evokes transient inhibition in swim-initiating and serotonin-containing interneurons, and has little immediate effect on Retzius cells. In addition, Tr2 indirectly inhibits several oscillator neurons, including cells 208, 33, and 60. When Tr1 is stimulated during a swimming episode the swim period decreases for several cycles, while stimulation of Tr2 during swimming episodes reliably resets the ongoing swimming rhythm. Our findings indicate that Tr1 and Tr2 are trigger neurons which initiate swimming activity by different pathways. These neurons also have functional interactions with the swim oscillator network since either Tr1 or Tr2 stimulation during swimming can modulate the ongoing swimming rhythm.     

Advances in the neural bases of orientation and navigation

The ability to locomote in one direction (oriented movement),and the ability to navigate toward a distant goal are relatedbehaviors that are phylogenetically widespread. Orientationbehaviors include finding the source of an odor or acousticsignal, using a sun-compass for guidance, and moving relativeto fluid-dynamic cues. Such abilities might require little morethan directionally selective sensors coupled to a turning mechanism.This type of behavior, therefore, can be implemented by relativelysimple circuits. In contrast, navigation involves both the abilityto detect direction, as well as a map-sense that provides position.Navigation is less common and arguably requires greater braincomputation than does simple orientation, but may be presentin arthropods as well as in vertebrates. Great progress hasbeen made in exploring the biophysical and sensory bases forthese behaviors, and in recent years the locations and the identityof the cellular transducers of the sensory stimuli (for example,geomagnetic fields) have been narrowed in some taxa. Similarly,neurons within the central nervous that most likely functionin higher order computational processes have been identified.For example, direction-selective and position-responsive braincells have been located in the brains of mammals and birds,and these cells might contribute to a cognitive map that canenable navigation. One model organism in which orientation andnavigation has been extensively studied is the sea slug Tritoniadiomedea. This animal orients its crawling to chemical, hydrodynamic,and geomagnetic cues. The brain of Tritonia has 7000 relativelylarge neurons that facilitate circuit analysis. Recent workhas characterized both peripheral and central neural correlatesof orientation signals, as well as the control of thrust andturning, and studies of their field behavior have suggestedhow these disparate orientation systems may be integrated. Thesefindings provide the basis for future studies to determine howthe nervous system combines multiple sensory cues into a complexhierarchy of signals that can direct motor output and thereforeguide navigational tasks.     

The Site of Action of General Anaesthetics in Insect Olfactory Receptor Neurons

The effect of volatile anaesthetics such as N₂O, Xe, short-chainalkanes and cyclopropane, at pharmacologically relevant concentrations,on olfactory receptor neurons of insects was tested in electrophysiologicalrecordings. CO₂-receptor neurons in moths and files respondwith increased action potential activity, whereas in adherenceto the Meyer-Overton rule; alkanes of a chain length of 5 andabove are less effective or evoke suppression of action potentials.In olfactiory receptor neurons sensitive to benzoic acid infemale moths of Bombyx mori and in pheromone receptor neuronsof male moths of Antheraea polyphemus, anaesthetics are ineffectiveif applied alone; if superimposed on an excitatory olfactorystimulus, an inhibitory effect occurs, Local stimulation ofonly part of a sensory dendrite reveals that the anaestheticsreversibly block the reception of pheromone or its effect onthe conductance of the receptor cell memebrane. The observedinteractions are consistent with the hypothesis that the anaestheticsdo not interact with the primary transduction process, but ratheraffect a later stage such as the activation of ion channels. ^*Dedicated to H-J. Bestman, on the occasion of his 70th birthday.