Crustacean Neuromuscular Mechanisms

Properties of crustacean muscle fibers and neuromuscular synapsesare discussed, with particular reference to the problems offast and slow contraction, synaptic diversity, and peripheralinhibition. Electrical and mechanical responses of crustacean muscle fibersare variable, and govern to a large extent the muscle's performance.Fast and slow contractions are often mediated by distinct "phasic"and "tonic" muscle fibers, as in abdominal muscles, in whichsuch fibers are segregated into two parallel sets of muscles.In leg muscles the fibers are often heterogeneous in propertiesand innervation. In doubly-motor-innervated muscles of crabsthe axons producing fast and slow contractions preferentiallyinnervate rapidly and slowly contracting fibers, respectively. Crustacean neuromuscular synapses vary greatly in electricalbehavior and in ultrastructural characteristics. Some motoraxons possess both facilitating and nonfacilitating synapses.The proportion of the different types of synapse associatedwith a motor axon probably determines in large measure the propertiesof the postsynaptic potentials evoked by that axon. Pre-synaptic and post-synaptic inhibition both occur, sometimesin the same muscle. The latter type is more common. Pre-synapticinhibition is thought to be mediated by the action of an inhibitorytransmitter-substance on receptors of the motor nerve terminals.     

Degeneration and Regeneration in Crustacean Neuromuscular Systems

Crayfish motor neurons seem to repair damage to peripheral axonsby selective fusion of outgrowing proximal stumps with severeddistal processes that can survive morphologically and physiologicallyintact for over 200 days. Survival of isolated motor and CNSgiant axons is associated with much hypertrophy of their glialsheath. The severed stumps of peripheral sensory neurons oftendegenerate within 21 days and their glial sheath does not hypertrophy.Denervation and immobilization produce relatively little changein the morphology and physiology of the opener muscle, whereastenotomy produces much atrophy within 30-60 days. Crayfish motor and CNS giant neurons show no capability forregenerating ablated cell bodies, whereas peripheral sensorysomata regenerate after limb autotomy. An entire opener musclecan be replaced after limb autotomy but the organism shows littleor no ability to redifferentiate an entire muscle in the absenceof body part regeneration. However, a few opener muscle fiberscan be regenerated if the bulk of the muscle mass remains intact.The significance of all these findings are interpreted withrespect to the developmental capabilities and environmentaladaptations of the crayfish together with the evolution of regenerativeabilities in anthropods and vertebrates.     

Functional and Structural Parallels in Crustacean and Drosophila Neuromuscular Systems

Comparison of morphological and physiological phenotypes ofrepresentative crustacean motor neurons, and selected motorneurons of Drosophila larval abdominal muscles, shows severalfeatures in common. Crustacean motor nerve terminals, and thoseof Drosophila, possess numerous small synapses with well-definedactive zones. In crustaceans, neurons that are more tonicallyactive have markedly varicose terminals; synapses and mitochondriaare selectively localized in the varicosities. Phasic motoraxons have filiform terminals, sometimes with small varicosities;mitochondrial content is less than for tonic axons, and synapsesare distributed along the terminals. Tonic axons generate smallexcitatory potentials which facilitate strongly at higher frequencies,and which are resistant to depression. Thephasic neurons generatelarge excitatory potentials which exhibit relatively littlefrequency facilitation, and depress rapidly. In Drosophila,counterparts of crustacean phasic and tonic motor neurons havebeen found, but the differentiation is less pronounced. It isinferredthat cellular factors regulating the number of participatingsynapses and the probability of quantal release are similarin crustaceans and Drosophila, and that advantage can be takenof this in future to develop experiments addressing the regulationof synaptic plasticity.     

The Integration of Ventilation and Locomotion in Archosaurs

Movements of the pelvis have recently been found to contributeto ventilation in both crocodilians and birds. Alligators havea kinetic pelvis in which the ischiopubic and ischiotruncusmuscles rotate the pubic bones ventrally to increase abdominalvolume and thereby facilitate inspiration. In birds, the entirepelvis rocks on the axial skeleton to produce ventilation. Althoughthe mechanisms of pelvic aspiration are very different in crocodiliansand birds, it is unusual among vertebrates for the pelvic musculoskeletalsystem to play an active role in inspiration. This observationraises the possibility that the pelvic musculoskeletal systemmay have played an important role in the ventilation of basalarchosaurs. Based on the mechanism of pelvic aspiration in crocodiliansand the structure of gastralia in basal archosaurs, we suggestthat an ischiotruncus muscle pulled the medial aspect of thegastralia caudally, and thereby helped to produce inspirationby increasing the volume of the cuirassal basket. The proposedmechanism of cuirassal breathing in non-avian theropods leadsus to suggest that the phase relationship of the ventilatoryand locomotor cycles in running theropods was the opposite ofthat observed in running birds. Furthermore, we suggest thatthe ventilatory cycle of flying pterosaurs was entrained tothe locomotor cycle with a phase relationship that was the oppositeof that observed during flight in modern birds.     

The Distribution of Pre- and Postsynaptic Inhibition at Crustacean Neuromuscular Junctions

The relative contribution of pre- and postsynaptic mechanisms to peripheral inhibition has been analyzed in the abdominal slow flexor muscles of crayfish and lobsters. The conductance of the muscle fiber membrane may be increased to five or more times its resting value by repetitive stimulation of the peripheral inhibitory axon, and this effect accounts for all of the attenuation exerted by the inhibitor against excitatory junctional potentials. No "critical interval" has been found at which an inhibitory nerve impulse produces anomalously large reduction of a following depolarizing junctional potential; electrotonic depolarizations and junctional potentials are identically affected under all phase conditions. The presynaptic inhibitory mechanism is, therefore, absent in this system. In the dactyl opener muscle, on the contrary, most of the attenuation of excitatory junctional potentials is achieved presynaptically, though equally large postjunctional conductance changes are also seen (Dudel and Kuffler, 1961). The difference is correlated with a difference in the reflex operation of the two muscles. Reflex inhibition in the abdominal slow flexors is primarily central, whereas in the dactyl opener, inhibition is brought about by an increase in inhibitory nerve discharge frequency without central suppression of the single excitatory axon. The function of peripheral inhibition in the abdominal flexors is presumably to terminate residual depolarization by reducing the long time-constant of the muscle fibers.     

Locomotion Controls Spatial Integration in Mouse Visual Cortex

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Crustacean Neuromuscular Mechanisms: Functional Morphology of Nerve Terminals and the Mechanism of Facilitation

Two aspects of crustacean neuromuscular physiology are discussed:(1) the ultrastructural identification of the excitatory andinhibitory nerve terminals, and (2) the characteristics of,and the possible mechanisms for, facilitation. The first problem was studied in crayfish opener muscle whichhas one excitatory and one inhibitory axon. One of the nerveswas stimulated in the presence of DNP until synaptic transmissionfailed; the preparations were then fixed for electron microscopy.Whenever the excitatory nerve was stimulated, the terminalswith round synaptic vesicles were depleted while nearby terminalswith smaller elongate vesicles were normal. When the inhibitorynerve was stimulated, the converse was true. The possible reasons for the diversity in crustacean neuromuscularproperties are discussed. Large EPSP's with a high quantal content(m), appear to be produced by terminals which are invaded bya propagated spike. Small EPSP's (small m) appear to be producedby terminals which don't spike and which are depolarized bya decrementally conducted potential. There is an inverse relationshipbetween m and the amount of facilitation. The physiologicalbasis for facilitation is discussed; previous hypotheses arefound wanting and a new one is proposed, that of slow depolarization.     

Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs

The water flea, Daphnia, has been the subject of study in ecology, evolution, and environmental sciences for decades. Over the last few years, expressed sequence tags and a genome sequence have been determined. In addition, functional approaches of overexpression and gene silencing based on microinjection of RNAs into eggs have been established. However, the transient nature of these approaches prevents us from analyzing gene functions in later stages of development. To overcome this limitation, transgenesis would become a key tool. Here we report establishment of a transgenic line using microinjection of plasmid into Daphnia magna eggs. The green fluorescent protein (GFP) gene fused with the D. magna histone H2B gene under the control of a promoter/enhancer region of the elongation factor 1α-1 (EF1α-1) gene, EF1α-1::H2B-GFP, was used as a reporter providing high resolution visualization of active chromatin. Transgenic lines were obtained from 0.67% of the total fertile adults that survived the injections. One of the transgenic animals, which exhibited fluorescence in the nuclei of cells during embryogenesis and oogenesis, had two copies of EF1α-1::H2B-GFP in a head-to-tail array. This is the first report of a transgenesis technique in Daphnia and, together with emerging genome sequences, will be useful for advancing knowledge of the molecular biology of Daphnia.     

Glycogen in Crustacean Fast and Slow Muscles

Ultrastructural examination of crayfish superficial (tonic)and deep (phasic) abdominal extensor muscles reveals a distributionand quantitative difference in glycogen between these muscles.Both superficial and deep fibers have a dense accumulation ofglycogen in the interfibrillar sarcoplasm. In addition, thesuperficial extensors, but not the deep extensors, contain glycogenin the I band region. The glycogen granules are of the ßtype and can be removed enzymatically. The superficial medialand lateral fibers contain more glycogen than the medial andlateral deep fibers. A possible functional role for this differenceis suggested.     

Body Form and Locomotion in Sharks

A revised interpretation of the mode of action of the heterocercaltail in sharks shows that the upturned tail axis tends to producea thrust directed downwards behind the centre of balance ofthe fish and thus gives a moment turning the head upwards. Thisis countered in two ways—by the rotation of the tail alongits longitudinal axis during each lateral beat, and throughthe action of the ventral hypochordal lobe. The shape of thetail and the mode of action of the tail in all sharks so farconsidered reflects a balance between these three factors, inall of them the net effect being the production of a forwardthrust from the tail that passes directly through the centreof balance of the fiish. There is normally therefore no tendencyfor the fish to turn around the centre of balance in a sagittalplane but there is a net sinking effect that is countered bythe planning effect of the pectoral fins and the ventral surfaceof the head. A study of 56 species of sharks shows that the tail is constructedaccording to a remarkably consistent common plan, the extremesbeing the high angled rather symmetrical tail of pelagic sharkssuch as hums, Lamna and Rhincodon and the straight tails ofbenthic sharks such as Ginglymostoma in which a ventral hypochordallobe is absent. When the general body shape of sharks, includingthe position of insertion of the median and paired fins andthe pattern of growth of fin surface areas is considered, theuniformity of the shark body plan and locomolor function isfurther emphasised. Four patterns of body form in sharks are recognised: 1) Thefast swimming pelagic sharks and the whale sharks have a tailwith a high aspect ratio, a conical head, a lateral fluke onthe caudal peduncle. 2) The generalised sharks typified by theCarcharhinidae, have lower heterocercal angles, a flattenedventral surface on the head and lack the caudal fluke. 3) Thedemersal sharks typified by the catsharks (Scyliorhinidae) havea very low, almost straight tail. The ventral hypochordal lobeis absent and the first dorsal fin is posterior in position.4) The squalomorph sharks are distinct in the absence of theanal fin, presence of a marked epicaudal lobe in the tail andoften an elevated insertion of the pectorals. The anal and second dorsal fins are always the smallest finsand the pectorals grow at the fastest rate. In general thereis an inverse relationship between size and rale of growth ofall fins and the ventral surface of the head. In hammerheadsthe growth data confirms that the head has a significant planingaction in swimming. The pectoral, second dorsal and anal finsshow an extreme constancy of position of insertion in all sharksstudied. The locomotor mechanism of sharks is adapted for anefficient cruising swimming but at the same time, the potentialinstability in the sagittal plan allows for the production ofturning moments that are used in attack and feeding.     

Locomotion of Spirilla

The hydromechanics of spirilla locomotion is analyzed by considering the balance of both rectilinear and angular momenta of the surrounding viscous fluid which is otherwise at rest. The physical model of Spirillum adopted for the present analysis consists of a rigid helical body with flagella attached to both ends of the helix. The motion is supposed to be activated first by the polar flagella, both rotating in the same sense, thus causing the helical body to rotate in the opposite sense in angular recoil, which in turn pushes the body forward in response to the balance of linear momentum of the surrounding fluid. The sweeping back of the polar flagella during forward motion is ascribed to a certain bending flexibility of the flagella and of their conjunction with the body. Based on this model some quantitative results for Spirillum movement are predicted, and are found to be consistent with existing experimental data.     

Computerized Analysis of Crustacean Relationships

The strengths and weaknesses of phylogenetic analysis using computers are reviewed from the viewpoint of understanding crustacean evolution. Computerized methods require the explicit presentation of characters and character state homologies. New techniques allow investigators to design evolutionary models into a character data matrix, or to use evolutionary models that make minimal a priori assumptions. The computer analysis relieves the investigator from the highly repetitious testing of trees, allows the concentration on the character state data, and provides objective methods for comparing trees, primarily their length. These are regarded as the strengths of computerized methods. The weaknesses of these methods include the relatively inscrutable nature of the character data matrix compared with the overall ‘gestalt’ of resulting trees, the difficulties of defining discrete homologies within the Crustacea, especially for counts of segmentation, the lack of clear intermediate character states in some multistate segmental characters, and the inability to define evolutionary polarity. These difficulties may be overcome by analysing the data using the minimal assumption models of character evolution, and by a recognition that the trees are a result of the input data, and therefore the data should be criticized, rather than the trees themselves. A ‘consensus’ character data set, including most extant major groups of the Crustacea as well as several key fossils, was assembled and revised by the participants in the workshop. An artificial taxon, ‘ur-crustacean characters’, was introduced to root the tree. Three observations may be made from parsimony analyses using several weighting and tree rooting methods. (1) The currently accepted large scale phylogeny and classification of the Crustacea is not corroborated. (2) The number of supposed plesiomorphic traits possessed by a taxon is not a good index for early derivation in crustacean evolution. (3) The taxon Maxillopoda is not supported by the arrangement of any of the trees.     

Locomotion and posture in Lagothrix lagotricha

This long-term study of woolly monkey (Lagothrix) locomotor and postural behaviour employs methods identical to those used during a previous study of the locomotion and posture of two species of Ateles, allowing a detailed comparison between the two genera, which are strong competitors in extensive parts of the Amazon basin and northern Andes. As in Ateles, Lagothrix locomotion can be divided into five patterns, based on limb usage: quadrupedal walking and running, suspensory locomotion, climbing, bipedalism (very rare in wild woolly monkeys) and leaping. Lagothrix differs from Ateles primarily in its greater reliance on quadrupedal locomotion during both travel and feeding and on its de-emphasis of the use of suspensory locomotion as compared to Ateles, while the use of climbing and leaping is roughly equal in the two genera. Lagothrix exhibits more generalised (primitive) locomotive behaviour in accordance with its morphology, in comparison to the more specialised Ateles. The generic differences reflect differences in habitat use and particularly foraging ecology.     

Adaptive Staircase Measurement of Hand Proprioception

Clinicians and researchers often need to measure proprioception (position sense), for example to monitor the progress of disease, to identify the cause of movement or balance problems, or to ascertain the effects of an intervention. While researchers can use sophisticated equipment to estimate proprioceptive acuity with good precision, clinicians lack this option and must rely on the subjective and imprecise methods currently available in the clinic. Here we describe a novel technique that applies psychometric adaptive staircase procedures to hand proprioception with a simple tablet-style apparatus that could easily be adapted for the clinic. We report test-retest reliability, inter-rater reliability, and construct validity of the adaptive staircase method vs. two other methods that are commonly used in clinical settings: passive motion direction discrimination (PMDD) and matching. As a first step, we focus on healthy adults. Subjects ages 18–82 had their proprioception measured with each of the three techniques, at the metacarpophalangeal joint in the second finger of the right hand. A subset completed a second session in which the measures were repeated, to assess test-retest reliability. Another subset had the measurements done by two different testers to assess inter-rater reliability. Construct validity was assessed using stepwise regression on age and activity level, and correlations calculated across the three methods. Results suggest that of the three methods, the adaptive staircase method yields the best test-retest reliability, inter-rater reliability, and construct validity. The adaptive staircase method may prove to be a valuable clinical tool where more accurate assessment of proprioception is needed.     

Crustacean Neurosecretory Hormones and Physiological Specificity

Physiological effects of neurosecretory factors from crustaceansare examined for their hormonal specificity. Hyperglycemic hormone(HGH) from the eyestalk is a polypeptide, molecular weight about7,000; it may activate phosphorylase and inhibit glycogen synthetase. Four pigmentary effectors (distal retinal pigment, melanophores,erythrophores and leucophores) respond to a peptide hormone(DRPH) having a molecular weight of 2,000. An earlier suggestion(Kleinholz, 1970) that light-adaptation of the distal retinalpigment and dispersion of pigment granules in chromatophoresmay be regulated by the same hormonal molecule is confirmedby test with synthetic hormone. Concentrators oferythrophore(ECH) and leucophore (LCH) pigments are peptides with respectivemolecular weights of 1,000 and about 1,500 daltons. Three factors which affect succinate oxidation by mitochondriaappear in the three chromatographic zones eluted from G-25 Sephadexwhich contain DRPH, LCH and ECH, and therefore presumably areof about the same molecular size as the pigment hormones. Thesecond and third of these factors have been shown to be distinctlydifferent from the hormones with which they are initially eluted. Factors influencing hydromineral regulation, molt- and gonad-inhibition,cardiac activity, respiratory rates and lipid metabolism arereviewed.     

Crustacean peptide and peptide-like pheromones and kairomones

Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones are reviewed from a historical perspective. These crustacean information molecules are secondary functions of structural polymers. They are partial hydrolysis products, generated usually by the action of trypsin-like enzymes on proteins, and glycosidase enzymes on glycoproteins and proteoglycans. Structure-function studies based upon synthetic mimics of peptide information molecules show neutral amino acids with a basic carboxyl terminal are active in modifying physiological and or behavioral responses. Behaviorally active substituted amino sugar mimics are disaccharide hydrolysis products of heparin and chondroitin sulfate. Similar molecules are also used as information molecules by a variety of other marine organisms indicating they are a common biological theme.     

Crustacean phasic and tonic motor neurons

Crustacean motor neurons subserving locomotion are specializedfor the type of activity in which they normally participate.Neurons responsible for maintained activity (‘tonic’neurons) support moderate to high frequencies of nerve impulsesintermittently or continuously during locomotion, while thoserecruited for short-lasting rapid responses (‘phasic’neurons) generally fire a few impulses in a rapid burst duringrapid locomotion and are otherwise silent. The synaptic responsesof the two types, recorded at their respective neuromuscularjunctions, differ enormously: phasic neurons exhibit much higherquantal release per synapse and per muscle fibre, along withmore rapid synaptic depression and less short-term facilitation.We have analyzed the factors that are responsible for the largedifference in initial release of neurotransmitter. Several possibilities,including synapse and active zone size differences, entry ofcalcium at active zones, and immediately releasable vesiclepools, could not account for the large phasic-tonic differencein initial transmitter output. The most likely feature thatdifferentiates synaptic release is the sensitivity of the exocytoticmachinery to intracellular calcium. Molecular features of thephasic and tonic presynaptic nerve terminals are currently underinvestigation.