Muscle function in animal movement: passive mechanical properties of leech muscle

We investigated passive properties of leech body wall as part of a larger project to understand better mechanisms that control locomotion and to establish mathematical models that predict such dynamical behavior. In tests of length-tension relationships in 2-segment-long preparations of body wall through step-stretch manipulations (step size = 1 mm), we discovered that these relationships are nonlinear, with significant hysteresis, even for the relatively small changes in length that occur during swimming. We developed a mathematical model comprising three nonlinear springs, two in series with nonlinear dashpots that describe well the tension statics and dynamics for step-stretch experiments. This model suggested that body wall dynamics are slow enough to be neglected when predicting the tension generated by imposed sinusoidal length changes (about ±10% of nominal) at 1–3 Hz, mimicking swimming. We derived a static model, comprising one nonlinear spring, which predicts sinusoidal data accurately, even when preparations were exposed to serotonin (0.1–10 μM). Preparations bathed in saline-serotonin had significantly reduced steady-state and peak tensions, without alterations in tension dynamics. Anesthetizing preparations (8% ethanol) reduced body wall tension by 77%, indicating that passive tension in the obliquely striated longitudinal muscles of leeches results primarily from a resting tonus.     

Development of swimming in the medicinal leech,the gradual acquisition of a behavior

Observing the development of behavior provides an assay for the developmental state of an embryo’s nervous system. We have previously described the development of behaviors that were largely confined to one or a few segments. We now extend the work to a kinematic analysis of the development of swimming, a behavior that requires coordination of the entire body. When leech embryos first begin to swim they make little forward progress, but within several days they swim as effectively as adults. This increase in efficacy depends on changes in body shape and on improved intersegmental coordination of the swim central pattern generator. These kinematic details suggest how the swim central pattern generating circuit is assembled during embryogenesis.     

Modulation of swimming behavior in the medicinal leech

The effects of serotonin on the electrical properties of swim-gating neurons (cell 204) were examined in leech (Hirudo medicinalis) nerve cords. Exposure to serotonin decreased the threshold current required to elicit swim episodes by prolonged depolarization of an individual cell 204 in isolated nerve cords. This effect was correlated with a more rapid depolarization and an increased impulse frequency of cell 204 in the first second of stimulation. In normal leech saline, brief depolarizing current pulses (1 s) injected into cell 204 failed to elicit swim episodes. Following exposure to serotonin, however, identical pulses consistently evoked swim episodes. Thus, serotonin appears to transform cell 204 from a gating to a trigger cell.Serotonin had little effect on the steady-state currentvoltage relation of cell 204. However, serotonin altered the membrane potential trajectories in response to injected current pulses and increased the amplitude of rebound responses occurring at the offset of current pulses. These changes suggest that serotonin modulates one or more voltage dependent conductances in cell 204, resulting in a more rapid depolarization and greater firing rate in response to injected currents. Thus, modulation of intrinsic ionic conductances in cell 204 may account in part for the increased probability of swimming behavior induced by serotonin in intact leeches.Abbreviations AHP afterhyperpolarizing potential - DCC discontinuous current clamp - DP dorsal posterior nerve - G2 segmental ganglion 2 - PIR postinhibitory rebound - RMP resting membrane potential     

Modulation of swimming behavior in the medicinal leech

Expression of swimming in the medicinal leech (Hirudo medicinalis) is modulated by serotonin, a naturally occurring neurohormone. Exogenous application of serotonin engenders spontaneous swimming activity in nerve-cord preparations. We examined whether this activity is due to enhanced participation of swim motor neurons (MNs) in generating the swimming rhythm. We found that depolarizing current injections into MNs during fictive swimming are more effective in shifting cycle phase in nerve cords following serotonin exposure. In such preparations, the dynamics of membrane potential excursions following current injection into neuronal somata are substantially altered. We observed: 1) a delayed outward rectification (relaxation) during depolarizing current injection, most marked in inhibitory MNs; and 2) in excitor MNs, an enhancement of postinhibitory rebound (PIR) and afterhyperpolarizing potentials (AHPs) following hyperpolarizing and depolarizing current pulses, respectively. In contrast, we found little alteration in MN properties in leech nerve cords depleted of amines. We propose that enhanced expression of swimming activity in leeches exposed to elevated serotonin is due, partly, to enhancement of relaxation, PIR and AHP in MNs. We believe that as a consequence of alterations in cellular properties and synaptic interactions (subsequent paper) by serotonin, MNs are reconfigured to more effectively participate in generating and expressing the leech swimming rhythm.Abbreviations AHP Afterhyperpolarizing potential - DCC Discontinuous current clamp - DE Dorsal excitor motor neuron - DI Dorsal inhibitor motor neuron - IPSP Inhibitory postsynaptic potential - MN Motor neuron - PIR Postinhibitory rebound - VE Ventral excitor motor neuron - VI Ventral inhibitor motor neuron     

Celebrity with a neglected taxonomy: molecular systematics of the medicinal leech (genus Hirudo)

The medicinal leech is the most famous representative of the Hirudinea. It is one of few invertebrates widely used in medicine and as a scientific model object. It has recently been given considerable conservation effort. Despite all attention there is confusion regarding the taxonomic status of different morphological forms, with many different species described in the past, but only two generally accepted at present. The results of the phylogenetic analysis of a nuclear (ITS2+5.8S rRNA) and two mitochondrial gene sequences (12S rRNA, COI) suggest that the genus Hirudo is monophyletic. It consists, apart form the type Hirudo medicinalis and the East Asian Hirudo nipponia, of three other, neglected species. All of them have already been described either as species or morphological variety, and can readily be identified by their coloration pattern. The type species is in weakly supported sister relation with Hirudo sp. n. (described as variety orientalis) from Transcaucasia and Iran. Sister to them stands Hirudo verbana from southeastern Europe and Turkey, which is nowadays predominantly bred in leech farms and used as 'medicinal leech.' The North African Hirudo troctina is the sister taxon to this group of Western Eurasian species, whereas the basal split is between H. nipponia and the Western Palaearctic clade.     

Multivariable harmonic balance analysis of the neuronal oscillator for leech swimming

Biological systems, and particularly neuronal circuits, embody a very high level of complexity. Mathematical modeling is therefore essential for understanding how large sets of neurons with complex multiple interconnections work as a functional system. With the increase in computing power, it is now possible to numerically integrate a model with many variables to simulate behavior. However, such analysis can be time-consuming and may not reveal the mechanisms underlying the observed phenomena. An alternative, complementary approach is mathematical analysis, which can demonstrate direct and explicit relationships between a property of interest and system parameters. This paper introduces a mathematical tool for analyzing neuronal oscillator circuits based on multivariable harmonic balance (MHB). The tool is applied to a model of the central pattern generator (CPG) for leech swimming, which comprises a chain of weakly coupled segmental oscillators. The results demonstrate the effectiveness of the MHB method and provide analytical explanations for some CPG properties. In particular, the intersegmental phase lag is estimated to be the sum of a nominal value and a perturbation, where the former depends on the structure and span of the neuronal connections and the latter is roughly proportional to the period gradient, communication delay, and the reciprocal of the intersegmental coupling strength.

Abolition of magnesium chelatase activity by the gun5 mutation and reversal by Gun4

The chlorophyll-deficient gun5-1 and cch Arabidopsis mutants carry single point mutations in the CHLH subunit of the magnesium chelatase enzyme, which catalyses the first committed step of chlorophyll biosynthesis. Recombinant Synechocystis ChlH subunits carrying the gun5-1 or cch mutations are inactive in Mg-chelatase assays, despite being able to bind both substrate and product, and retaining a capacity to form a ChlH–ChlI–ChlD Mg-chelatase complex. These mutant subunits act as inhibitors of ChlH, showing that the ChlH-porphyrin complex associates reversibly with the ChlI and D subunits during the catalytic cycle. This inhibition is reversed upon addition of Gun4.     

Control of leech swimming activity by the cephalic ganglia

We investigated the role played by the cephalic nervous system in the control of swimming activity in the leech, Hirudo medicinalis, by comparing swimming activity in isolated leech nerve cords that included the head ganglia (supra- and subesophageal ganglia) with swimming activity in nerve cords from which these ganglia were removed. We found that the presence of these cephalic ganglia had an inhibitory influence on the reliability with which stimulation of peripheral (DP) nerves and intracellular stimulation of swim-initiating neurons initiated and maintained swimming activity. In addition, swimming activity recorded from both oscillator and motor neurons in preparations that included head ganglia frequently exhibited irregular bursting patterns consisting of missed, weak, or sustained bursts. Removal of the two head ganglia as well as the first segmental ganglion eliminated this irregular activity pattern. We also identified a pair of rhythmically active interneurons, SRN1, in the subesophageal ganglion that, when depolarized, could reset the swimming rhythm. Thus the cephalic ganglia and first segmental ganglion of the leech nerve cord are capable of exerting a tonic inhibitory influence as well as a modulatory effect on swimming activity in the segmental nerve cord.     

The activity of abdominal stretch receptors during non-giant swimming in the crayfish<Emphasis Type="Italic"> Cherax destructor</Emphasis> and their role in hydrodynamic efficiency

Recordings were made from the nerve innervating the stretch receptors of the abdominal muscle receptor organs and slow extensor muscles of tethered crayfish, Cherax destructor, during so-called non-giant swimming. The stretch receptors were active during the flexor phase of swimming but the duration and pattern of activity varied from cycle to cycle. Their pattern of firing was modified by the activity of the large accessory neurons which make direct inhibitory synapses upon them. Neither the stretch receptors nor the accessory neurons were active during the extensor phase of the cycle. The timing and extent of tailfan movements during the period of stretch receptor activity were measured from video records before and after the stretch receptor nerves were cut in the second to fifth segments. The promotion of the tailfan during flexion was significantly delayed and the minimum angle to which the uropods were remoted at the end of flexion significantly larger in denervated animals. We propose that afferent information from the stretch receptors coordinates the timing and extent of tailfan movements according to variations in the positioning and movement of the abdominal segments such that the hydrodynamic efficiency of the tailfan is enhanced on a cycle by cycle basis during non-giant swimming.Abbreviations A# abdominal segment number - Acc accessory neuron - LUU large unidentified unit - MRO muscle receptor organ - NGS non-giant swimming - SEMN slow extensor motor neuron - SR stretch receptor neuron     

Conserved origin of the ventral pancreas in chicken

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.     

Irregular helicoids in leech cocoon membranes

Helicoids in the cocoon membrane of leeches Theromyzon tessulatum and Erpobdella punctata comprise a twisted superposition of layers, each containing a variable number of planes formed by unidirectional fibrils. Straight fibrils intersecting at different angles were displayed in tangential sections through the cocoon wall of each species. When the sectioning angle was below a certain value (i.e., the critical angle), bow-shaped lines apparent in oblique sections were replaced by a succession of layers containing straight fibrils, permitting a direct measurement of step-angle change between successive layers in a helicoid. By this methodology, we determined that no regularities exist in the succession of step-angles or in layer thicknesses within the cocoon membranes, but that the distribution of step-angles between layers was unique for each cocoon type.     

Divergent actions of serotonin receptor activation during fictive swimming in frog embryos

We have investigated the pharmacology underlying locomotor system responses to serotonin (5-HT) in embryos of the frog, Rana temporaria, to provide a comparison to studies in embryos of its close relative, Xenopus laevis. Our findings suggest that two divergent mechanisms underlie the modulation of locomotion by 5-HT in Rana. Bath-applied 5-HT or 5-carboxamidotyptamine, a 5-HT_1,5A,7 receptor agonist, can modulate fictive swimming in a dose-dependent manner, increasing burst durations and cycle periods. However, activation of 5-HT_1,7 receptors with R8-OHDPAT or 8-OHDPAT fails to mimic 5-HT, and in some cases exerts exactly the opposite response; decreasing burst durations and cycle periods. Elevating endogenous 5-HT levels by blocking re-uptake with clomipramine transiently increases burst durations. The receptors involved in this endogenous response include 5-HT_1A receptors, as in Xenopus, but also 5-HT₇ receptors. However, like the 8-OHDPAT enantiomers, prolonged re-uptake inhibition can result in a motor response in the opposite direction to exogenous 5-HT. This effect is not reversed by 5-HT_1A and/or 5-HT₇ receptor antagonism, implicating 5-HT_1B/1D receptors. Remarkably, antagonism of these receptors using methiothepin unmasks a dose-dependent response to clomipramine, reminiscent of exogenous 5-HT. Our data suggest that 5-HT_1A,7 and 5-HT_1B/1D receptors act as gain-setters of burst durations, whilst 5-HT_5A receptors are involved in the effects of bath-applied 5-HT on locomotion.     

Theoretical study on the body form and swimming pattern of Anomalocaris based on hydrodynamic simulation

Anomalocarid arthropod is the largest known predatory animal of middle Cambrian. Studies on Anomalocaris have been piled up in the past two decades since the first reasonable reconstruction had achieved in 1980s. Recent finding of legs beneath lobes on Parapeytoia Yunnanensis shows arthropod affinities, however, many researchers believe that it must be a powerful swimmer by the use of developed lobes. In this work, we investigate swimming behaviour of Anomalocaris in water by performing hydrodynamical calculation. As a result of simulation using moving particle method possible swimming motion of Anomalocaris is obtained. In the computer we can change the morphology from known bauplan of Anomalocaris found as fossil record. It makes us possible to discuss on the variants of Anomalocaris at the intermediate state of evolution process. Such new methodology using computer reveals how and from where Anomalocaris evolved.     

Opposing interactions between homothorax and Lobe define the ventral eye margin of Drosophila eye

Patterning in multi-cellular organisms involves progressive restriction of cell fates by generation of boundaries to divide an organ primordium into smaller fields. We have employed the Drosophila eye model to understand the genetic circuitry responsible for defining the boundary between the eye and the head cuticle on the ventral margin. The default state of the early eye is ventral and depends on the function of Lobe (L) and the Notch ligand Serrate (Ser). We identified homothorax (hth) as a strong enhancer of the L mutant phenotype of loss of ventral eye. Hth is a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD). Hth is known to bind Extradenticle (Exd) via its MH domain for its nuclear translocation. Loss-of-function of hth, a negative regulator of eye, results in ectopic ventral eye enlargements. This phenotype is complementary to the L mutant phenotype of loss-of-ventral eye. However, if L and hth interact during ventral eye development remains unknown. Here we show that (i) L acts antagonistically to hth, (ii) Hth is upregulated in the L mutant background, and (iii) MH domain of Hth is required for its genetic interaction with L, while its homeodomain is not, (iv) in L mutant background ventral eye suppression function of Hth involves novel MH domain-dependent factor(s), and (v) nuclear localization of Exd is not sufficient to mediate the Hth function in the L mutant background. Further, Exd is not a critical rate-limiting factor for the Hth function. Thus, optimum levels of L and Hth are required to define the boundary between the developing eye and head cuticle on the ventral margin.     

spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

The swimming and feeding behavior of Mesocyclops

The swimming and feeding behaviors of Mesocyclops are described from a review of the literature and personal observations.Mesocyclops exhibits considerable behavioral flexibility in response to environmental stimuli. Mesocyclops edax exhibits an increase in horizontal looping behavior at high prey densities, and performs a tight vertical looping behavior in response to the loss of captured prey. Ingestion rates by Mesocyclops are a complex function of prey density, morphology, and behavior in addition to prey size. Vertebrate predators induce a rapid escape response in Mesocyclops and may be responsible at least in part for their extensive diel vertical migrations. The complex behavioral patterns of Mesocyclops suggest that its distribution and abundance in nature will be distinctly nonrandom and influenced as much by its own behavioral responses as by other external physical factors such as water circulation patterns.     

Activation of two forms of locomotion by a previously identified trigger interneuron for swimming in the medicinal leech

Higher-order projection interneurons that function in more than one behavior have been identified in a number of preparations. In this study, we document that stimulation of cell Tr1, a previously identified trigger interneuron for swimming in the medicinal leech, can also elicit the motor program for crawling in isolated nerve cords. We also show that motor choice is independent of the firing frequency of Tr1 and amount of spiking activity recorded extracellularly at three locations along the ventral nerve cord prior to Tr1 stimulation. On the other hand, during Tr1 stimulation there is a significant difference in the amount of activity elicited in the ventral nerve cord that correlates with the motor program activated. On average, Tr1 stimulation trials that lead to crawling elicit greater amounts of activity than in trials that lead to swimming.