Neural correlates of swimming behavior in Melibe leonina

The nudibranch Melibe leonina swims by rhythmically bending from side to side at a frequency of 1 cycle every 2-4 s. The objective of this study was to locate putative swim motoneurons (pSMNs) that drive these lateral flexions and determine if swimming in this species is produced by a swim central pattern generator (sCPG). In the first set of experiments, intracellular recordings were obtained from pSMNs in semi-intact, swimming animals. About 10-14 pSMNs were identified on the dorsal surface of each pedal ganglion and 4-7 on the ventral side. In general, the pSMNs in a given pedal ganglion fired synchronously and caused the animal to flex in that direction, whereas the pSMNs in the opposite pedal ganglion fired in anti-phase. When swimming stopped, so did rhythmic pSMN bursting; when swimming commenced, pSMNs resumed bursting. In the second series of experiments, intracellular recordings were obtained from pSMNs in isolated brains that spontaneously expressed the swim motor program. The pattern of activity recorded from pSMNs in isolated brains was very similar to the bursting pattern obtained from the same pSMNs in semi-intact animals, indicating that the sCPG can produce the swim rhythm in the absence of sensory feedback. Exposing the brain to light or cutting the pedal-pedal connectives inhibited fictive swimming in the isolated brain. The pSMNs do not appear to participate in the sCPG. Rather, they received rhythmic excitatory and inhibitory synaptic input from interneurons that probably comprise the sCPG circuit.     

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.     

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.

     

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.     

Mechanisms of Locomotory Speed Change: The Pteropod Solution

Three primary factors contribute to locomotory speed changesin the pteropod mollusk Clione limacina. (1) An increase incycle frequency of locomotory appendages is associated witha baseline depolarization and enhancement of postinhibitoryrebound in central pattern generator (CPG) interneurons, anda reorganization of the CPG through recruitment of additionalinterneurons. (2) An increase in the force of appendage movementsis generated through enhancement of activity of active motoneurons,recruitment of additional motoneurons and peripheral modulationof swim musculature. (3) Changes in biomechanical aspects ofappendage movements are presumably achieved, at least in part,through changes in the activity of motoneurons and swim muscle.All changes associated with non-startle swim acceleration areproduced by a serotonergic arousal system that acts at all threelevels of the swimming system: CPG interneurons, motoneuronsand swim musculature.     

Neurochemical and neuroanatomical identification of central pattern generator neuron homologues in Nudipleura molluscs

Certain invertebrate neurons can be identified by their behavioral functions. However, evolutionary divergence can cause some species to not display particular behaviors, thereby making it impossible to use physiological characteristics related to those behaviors for identifying homologous neurons across species. Therefore, to understand the neural basis of species-specific behavior, it is necessary to identify homologues using characteristics that are independent of physiology. In the Nudipleura mollusc Tritonia diomedea, Cerebral Neuron 2 (C2) was first described as being a member of the swim central pattern generator (CPG). Here we demonstrate that neurochemical markers, in conjunction with previously known neuroanatomical characteristics, allow C2 to be uniquely identified without the aid of electrophysiological measures. Specifically, C2 had three characteristics that, taken together, identified the neuron: 1) a white cell on the dorsal surface of the cerebral ganglion, 2) an axon that projected to the contralateral pedal ganglion and through the pedal commissure, and 3) immunoreactivity for the peptides FMRFamide and Small Cardioactive Peptide B. These same anatomical and neurochemical characteristics also uniquely identified the C2 homologue in Pleurobranchaea californica (called A1), which was previously identified by its analogous role in the Pleurobranchaea swim CPG. Furthermore, these characteristics were used to identify C2 homologues in Melibe leonina, Hermissenda crassicornis, and Flabellina iodinea, species that are phylogenetically closer to Tritonia than Pleurobranchaea, but do not display the same swimming behavior as Tritonia or Pleurobranchaea. These identifications will allow future studies comparing and contrasting the physiological properties of C2 across species that can and cannot produce the type of swimming behavior exhibited by Tritonia.     

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.     

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.     

Evolutionary divergence in developmental strategies and neuromodulatory control systems of two amphibian locomotor networks

Attempts to understand the neural mechanisms which produce behaviourmust consider both prevailing sensory cues and the central cellularand synaptic changes they direct. At each level, neuromodulationcan additionally shape the final output. We have investigatedneuromodulation in the developing spinal motor networks in hatchlingtadpoles of two closely related amphibians, Xenopus laevis andRana temporaria to examine the subtle differences in their behavioursthat could be attributed to their evolutionary divergence. At the point of hatching, both species can swim in responseto a mechanosensory stimulus, however Rana embryos often displaya more forceful, non-locomotory coiling behaviour. Whilst thesynaptic drive that underlies these behaviours appears similar,subtle inter-specific differences in neuronal properties shapemotor outputs in different ways. For example, Rana neurons expressN-methyl-D-aspartate (NMDA)/serotonin (5-HT)-dependent oscillations,not present in hatchling Xenopus and many also exhibit a prominentslow spike after-hyperpolarisation. Such properties may endowthe spinal circuitry of Rana with the ability to produce a moreflexible range of outputs. Finally, we compare the roles of the neuromodulators 5-HT, noradrenaline(NA) and nitric oxide (NO) in shaping motor outputs. 5-HT increasesburst durations during swimming in both Xenopus and Rana, but5-HT dramatically slows the cycle period in Rana with littleeffect in Xenopus. Three distinct, but presumably homologousNO-containing brainstem clusters of neurons have been described,yet the effects of NO differ between species. In Xenopus, NOslows and shortens swimming in a manner similar to NA, yet inRana NO and NA elicit the non-rhythmic coiling pattern.     

两种软体动物神经系统一氧化氮合酶的组织化学定位

运用一氧化氮合酶(NOS)组织化学方法研究了软体动物门双壳纲种类中国蛤蜊和腹足纲种类嫁Qi神经系统中NOS阳性细胞以及阳性纤维的分布。结果表明：在蛤蜊脑神经节腹内侧,每侧约有10-15个细胞呈强NOS阳性反应,其突起也呈强阳性反应,并经脑足神经节进入足神经节的中央纤维网中;足神经节内只有2个细胞呈弱阳性反应,其突起较短,进入足神经节中央纤维网中,但足神经节中,来自脑神经节阳性细胞和外周神经系统的纤维大多呈NOS阳性反应;脏神经节的前内侧部和后外侧部各有一个阳性细胞团,其突起分别进入后闭壳肌水管后外套膜神经和脑脏神经索。脏神经节背侧小细胞层以及联系两侧小细胞层的纤维也呈NOS阳性反应。嫁Qi中枢神经系统各神经节中没有发现NOS阳性胞体存在;脑神经节、足神经节、侧神经节以及脑—侧、脑—足、侧—脏连索中均有反应程度不同的NOS阳性纤维,这些纤维均源于外周神经。与已研究的软体动物比较,嫁Qi和前鳃亚纲其它种类一样,神经系统中NO作为信息分子可能主要存在于感觉神经。而中国蛤蜊的神经系统中一氧化氮作为信息分子则可能参与更广泛的神经调节过程。     

Regulation of Drosophila Visual System Development by Nitric Oxide and Cyclic GMP

Photoreceptors undergoing target selection in the optic lobeof Drosophila express a nitric oxide sensitive soluble guanylatecyclase (sGC). At the same time, cells in the target regionof the optic lobe express nitric oxide synthase (NOS). Pharmacologicalinhibition of NOS, NO or sGC leads to disruption of the retinalprojection pattern in vitro, and the extension of individualretinal axons beyond their appropriate targets. The disruptiveeffects of NOS inhibition in vitro are prevented by adding acGMP analog. Mutations in the sGC alpha subunit gene, Gc1, reducesGC expression and attenuate NO-sensitive retinal cGMP productionin the visual system. Although the retinal projection patternis undisturbed in Gc1 mutants, they lack positive phototaxisas adults, suggesting inappropriate connections exist betweenthe photoreceptors and optic lobe interneurons in these flies.Preliminary results show that heat-shock expression of wild-typeGc1 during metamorphosis can restore positive phototaxis insevere Gc1 mutants. These in vivo results support the in vitrofindings that NOS and sGC activity are required to promote theappropriate retinal innervation of the optic lobe.     

The role of postinhibitory rebound in the locomotor central-pattern generator of Clione limacina

In animals, networks of central neurons, called central-patterngenerators (CPGs), produce a variety of locomotory behaviorsincluding walking, swimming, and flying. CPGs from diverse animalsshare many common characteristics that function at the systemlevel, circuit level, and cellular level. However, the relativeroles of common CPG characteristics are variable among differentanimal species, in ways that suit different forms of locomotionin different environmental contexts. Here, we examine some ofthese common features within the locomotor CPG in a model systemused to investigate changes in locomotory speed—the swimsystem of the pteropod mollusk, Clione limacina. In particular,we discuss the role of one cellular characteristic that is essentialfor locomotor pattern generation in Clione, postinhibitory rebound.     

Involvement of GnRH neuron in the spermatogonial proliferation of the scallop, Patinopecten yessoensiss

The aim of this study was to quantitatively analyze a pattern of proliferation of gonial cells and to demonstrate neural involvement in spermatogonial proliferation of the scallop by the in vitro experiment. Immunocytochemistry for incorporated BrdU was used to identify mitotically active gonial cells. The pattern of proliferation of gonial cells was divided into two phases: phase I; oogonia and spermatogonia slowly proliferate through the growing stage: phase II; oogonia develop into oocytes and spermatogonia start to proliferate rapidly from the mature stage through the spawning stage. The neurons detected with anti-mammalian (m)GnRH antibody were distributed sparsely in the pedal ganglion and predominantly in the cerebral ganglion of both sexes at the growing stage. The extracts from the cerebral and pedal ganglion (CPG) of both sexes collected at the growing stage promoted proliferation of spermatogonia in the in vitro culture of the testicular tissue as well as mGnRH. However, CPG extract had no effect on oogonial proliferation. The increased mitotic activity induced by CPG and mGnRH was abolished by the addition of mGnRH antagonists and anti-mGnRH antibody, suggesting that the spermatogonial proliferation is regulated by GnRH-like peptide in CPG of the scallop. The same mitotic activity as CPG extract and mGnRH was observed in the hemocyte lysate, but not in the serum. These findings suggest that the spermatogonial proliferation at phase II in the scallop may be under the neuroendocrine control by GnRH neuron in CPG.     

Cellular Mechanisms Underlying Swim Acceleration in the Pteropod Mollusk Clione limacina

The pteropod mollusk Clione limacina swims by dorsal-ventralflapping movements of its wing-like parapodia. Two basic swimspeeds are observed—slow and fast. Serotonin enhancesswimming speed by increasing the frequency of wing movements.It does this by modulating intrinsic properties of swim interneuronscomprising the swim central pattern generator (CPG). Here weexamine some of the ionic currents that mediate changes in theintrinsic properties of swim interneurons to increase swimmingspeed in Clione. Serotonin influences three intrinsic propertiesof swim interneurons during the transition from slow to fastswimming: baseline depolarization, postinhibitory rebound (PIR),and spike narrowing. Current clamp experiments suggest thatneither I_h nor I_A exclusively accounts for the serotonin-inducedbaseline depolarization. However, I_h and I_A both have a stronginfluence on the timing of PIR—blocking I_h increases thelatency to PIR while blocking I_A decreases the latency to PIR.Finally, apamin a blocker of I_K(Ca) reverses serotonin-inducedspike narrowing. These results suggest that serotonin may simultaneouslyenhance I_h and I_K(Ca) and suppress I_A to contribute to increasesin locomotor speed.     

Specialized brain regions and sensory inputs that control locomotion in leeches

Locomotor systems are often controlled by specialized cephalic neurons and undergo modulation by sensory inputs. In many species, dedicated brain regions initiate and maintain behavior and set the duration and frequency of the locomotor episode. In the leech, removing the entire head brain enhances swimming, but the individual roles of its components, the supra- and subesophageal ganglia, in the control of locomotion are unknown. Here we describe the influence of these two structures and that of the tail brain on rhythmic swimming in isolated nerve cord preparations and in nearly intact leeches suspended in an aqueous, “swim-enhancing” environment. We found that, in isolated preparations, swim episode duration and swim burst frequency are greatly increased when the supraesophageal ganglion is removed, but the subesophageal ganglion is intact. The prolonged swim durations observed with the anterior-most ganglion removed were abolished by removal of the tail ganglion. Experiments on the nearly intact leeches show that, in these preparations, the subesophageal ganglion acts to decrease cycle period but, unexpectedly, also decreases swim duration. These results suggest that the supraesophageal ganglion is the primary structure that constrains leech swimming; however, the control of swim duration in the leech is complex, especially in the intact animal.     

Nitric Oxide as an Orthograde Cotransmitter at Central Synapses of Aplysia: Responses of Isolated Neurons in Culture

Nitric oxide serves as an orthograde synaptic cotransmitterbetween identified neurons in the cerebral ganglion of Aplysia.Nitric oxide synthase, the enzyme that produces nitric oxide,is localized in a few specific neurons in the ganglia, includingneuron C2. Guanylyl cyclase the target enzyme of nitric oxide,is found in neurons C4 and MCC, which are synaptic followersof C2. Stimulation of C2 causes a vsEPSP in these neurons thatis reduced to 50% of its amplitude by nitric oxide synthaseinhibitors and guanylyl cyclase inhibitors. The remaining portionof the vsEPSP is mediated by histamine. Thus, nitric oxide andhistamine act as orthograde cotransmitters in producing thevsEPSP. Both cotransmitters cause closure of a background potassiumchannel, which depolarizes the neuron and enhances its responseto synaptic inputs. Exogenous nitric oxide (released by nitricoxide donor molecules) and histamine mimic the vsEPSP's depolarizationand decreased membrane conductance. When neurons C4 or MCC areisolated in cell culture they respond just as they do in theganglion, i.e., the nitric oxide response but not the histamineresponse is blocked by guanylyl cyclase inhibitors, and themembrane conductance is decreased by both histamine and nitricoxide. Aplysia hemolymph partially suppresses the response tonitric oxide, due to nitric oxide scavenging by hemocyanin,which contains copper and is the equivalent of hemoglobin. NeuronC2 followers that are hyperpolarized by histamine are insensitiveto nitric oxide. Thus, only select follower neurons respondto both transmitters.     

Nitric oxide decreases lung liquid production in fetal lambs

Cummings, James J. Nitric oxide decreases lung liquidproduction in fetal lambs. J. Appl.Physiol. 83(5): 1538-1544, 1997.To examine theeffect of nitric oxide on fetal lung liquid production, I measured lungliquid production in fetal sheep at 130 ± 5 days gestation (range122-137 days) before and after intrapulmonary instillation ofnitric oxide. Thirty-one studies were done in which net lung luminalliquid production (Jv) was measured by plotting the change in lung luminal liquid concentration ofradiolabeled albumin, an impermeant tracer that was mixed into the lungliquid at the start of each study. To see whether changes inJvmight be associated with changes in pulmonary hemodynamics, pulmonary and systemic pressures were measured and left pulmonary arterial flowwas measured by an ultrasonic Doppler flow probe. Variables weremeasured during a 1- to 2-h control period and for 4 h after a smallbolus of isotonic saline saturated with nitric oxide gas (10 or 100%)was instilled into the lung liquid. Control (saline) instillations(n = 6) caused no change in anyvariable over 6 h. Nitric oxide instillation significantly decreasedJv and increased pulmonary blood flow;these effects were sustained for 1-2 h. There was also asignificant but transient decrease in pulmonary arterial pressure. Thusintrapulmonary nitric oxide causes a significant decrease in lungliquid and is associated with a decrease in pulmonary vascularresistance. In a separate series of experiments either amiloride orbenzamil, which blocks Na⁺transport, was mixed into the lung liquid before nitric oxide instillation; still, there was a similar reduction in lung liquid production. Thus the reduction in lung liquid secretion caused bynitric oxide does not appear to depend on apicalNa⁺ efflux.

     

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in theregulation of shikonin formation in Onosma paniculatum cells.NO generated after cells were inoculated into shikonin productionmedium reached the highest level after 2 d of culture, whichwas 16 times that at the beginning of the experiment, and maintaineda high level for 6 d. A nitric oxide synthase (NOS) inhibitor,N-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor,sodium azide (SoA), consistent with their inhibition of NO biosynthesis,decreased shikonin formation significantly. This reduction couldbe alleviated or even abolished by exogenous NO supplied bysodium nitroprusside (SNP), suggesting that the inhibition ofNO biosynthesis resulted in decreased shikonin formation. However,when endogenous NO biosynthesis was up-regulated by the elicitorfrom Rhizoctonia cerealis, shikonin production was enhancedfurther, showing a dependence on the elicitor-induced NO burst.Real-time PCR analysis showed that NO could significantly up-regulatethe expression of PAL, PGT and HMGR, which encode key enzymesinvolved in shikonin biosynthesis. These results demonstratedthat NO plays a critical role in shikonin formation in O. paniculatumcells.