Leech Muscle Research Articles

Preliminary study on pathogen of Whitmania pigra disease in high temperature environment

In this paper, on the contrast of healthy leech, the bacterial diversities were analyzed by 16S rDNA sequence analysis of the bacteria of muscle and intestinal tract of Whitmania pigra, the environment water and sediment of cultivating the diseased Wh. pigra in high temperature by high-throughput sequencing to determine the possible pathogenic bacteria of bacterial diseases of Leech in high temperature. The results showed that the original sequence reached over 83 000, and the effective sequences accounted for more than 87%. The GC contents ranged from 52% to 54% and the bacterial diversities were abundant. Bacterial relative abundance analysis showed that the bacterial content of Proteobacteria, Bacteroidetes, and Firmicutes was the most abundant in all treatments. Compared with healthy leech muscles and intestines, the muscle and intestinal tract of pathogenic leech relative abundance of Bacteroides, Pseudomonas, and Desulfovibrio was significantly increased, and it was abundant in water and sediment of diseased leeches, Lead to the possibility that the pathogenic bacteria of this bacterial disease may be Bacteroides, Pseudomonas, Desulfovibrio.

Evaluation of processed borax as antidote for aconite poisoning

Aconite root is very poisonous; causes cardiac arrhythmias, ventricular fibrillation and ventricular tachycardia. There is no specific antidote for aconite poisoning. In Ayurveda, dehydrated borax is mentioned for management of aconite poisoning. The investigation evaluated antidotal effect of processed borax against acute and sub-acute toxicity, cardiac toxicity and neuro-muscular toxicity caused by raw aconite. For acute protection Study, single dose of toxicant (35mg/kg) and test drug (22.5mg/kg and 112.5mg/kg) was administered orally, and then 24h survival of animals was observed. The schedule was continued for 30 days in sub-acute protection Study with daily doses of toxicant (6.25mg/kg), test drug (22.5mg/kg and 112.5mg/kg) and vehicle. Hematological and biochemical tests of blood and serum, histopathology of vital organs were carried out. The cardiac activity Study was continued for 30 days with daily doses of toxicant (6.25mg/kg), test drug (22.5mg/kg), processed borax solution (22.5mg/kg) and vehicle; ECG was taken after 1h of drug administration on 1TB, 15th and on 30th day. For neuro-muscular activity Study, the leech dorsal muscle response to 2.5µg of acetylcholine followed by response of toxicant at 25µg and 50µg doses and then response of test drug at 25µg dose were recorded. Protection index indicates that treated borax gave protection to 50% rats exposed to the lethal dose of toxicant in acute protection Study. Most of the changes in hematological, biochemical parameters and histopathological Study induced by the toxicant in sub-acute protection Study were reversed significantly by the test drug treatment. The ventricular premature beat and ventricular tachyarrhythmia caused by the toxicant were reversed by the test drug indicate reversal of toxicant induced cardio-toxicity. The acetylcholine induced contractions in leech muscle were inhibited by toxicant and it was reversed by test drug treatment. The processed borax solution is found as an effective protective agent to acute and sub-acute aconite poisoning, and aconite induced cardiac and neuro-muscular toxicity. Processed borax at therapeutic dose (22.5mg/kg) has shown better antidotal activity profile than five times more than therapeutic dose (112.5mg/kg).

Mechanisms underlying rhythmic locomotion: dynamics of muscle activation

We have studied the dynamical properties of tension development in leech longitudinal muscle during swimming. A new method is proposed for modeling muscle properties under functionally relevant conditions where the muscle is subjected to both periodic activation and rhythmic length changes. The 'dual-sinusoid' experiments were conducted on preparations of leech nerve cord and body wall. The longitudinal muscle was activated periodically by injection of sinusoidal currents into an identified motoneuron. Simultaneously, sinusoidal length changes were imposed on the body wall with prescribed phase differences (12 values equally spaced over 2π radians) with respect to the current injection. Through the singular value decomposition of appropriately constructed tension data matrices, the leech muscle was found to have a multiplicative structure in which the tension was expressed as the product of activation and length factors. The time courses of activation and length factors were determined from the tension data and were used to develop component models. The proposed modeling method is a general one and is applicable to contractile elements for which the effects of series elasticity are negligible.

Serotonin modulates muscle function in the medicinal leechHirudo verbana

The body wall muscles of sanguivorous leeches power mechanically diverse behaviours: suction feeding, crawling and swimming. These require longitudinal muscle to exert force over an extremely large length range, from 145 to 46 per cent of the mean segmental swimming length. Previous data, however, suggest that leech body wall muscle has limited capacity for force production when elongated. Serotonin (5-HT) alters the passive properties of the body wall and stimulates feeding. We hypothesized that 5-HT may also have a role in allowing force production in elongated muscle by changing the shape of the length-tension relationship (LTR). LTRs were measured from longitudinal muscle strips in vitro in physiological saline with and without the presence of 10 µM 5-HT. The LTR was much broader than previously measured for leech muscle. Rather than shifting the LTR, 5-HT reduced passive muscle tonus and increased active stress at all lengths. In addition to modulating leech behaviour and passive mechanical properties, 5-HT probably enhances muscle force and work production during locomotion and feeding.

Identification, Isolation and Expansion of Myoendothelial Cells Involved in Leech Muscle Regeneration

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.

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 microM). 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.

Elliot S Valenstein, The war of the soups and the sparks: the discovery of neurotransmitters and the dispute over how nerves communicate, New York, Columbia University Press, 2005, pp. xviii, 237, illus., £19.50, $31.00 (hardback 0-231-13588-2).

Elliot S Valenstein, The war of the soups and the sparks: the discovery of neurotransmitters and the dispute over how nerves communicate, New York, Columbia University Press, 2005, pp. xviii, 237, illus., £19.50, $31.00 (hardback 0-231-13588-2).

Dynamics and reproducibility of a moderately complex sensory-motor response in the medicinal leech.

Local bending, a motor response caused by mechanical stimulation of the leech skin, has been shown to be remarkably reproducible, in its initial phase, despite the highly variable firing of motoneurons sustaining it. In this work, the reproducibility of local bending was further analyzed by monitoring it over a longer period of time and by using more intact preparations, in which muscle activation in an entire body segment was studied. Our experiments showed that local bending is a moderately complex motor response, composed of a sequence of four different phases, which were consistently identified in all leeches. During each phase, longitudinal and circular muscles in specific areas of the body segment acted synergistically, being co-activated or co-inhibited depending on their position relative to the stimulation site. Onset and duration of the first phase were reproducible across different trials and different animals as a result of the massive co-activation of excitatory motoneurons sustaining it. The other phases were produced by the inhibition of excitatory and activation of inhibitory motoneurons, and also by the intrinsic relaxation dynamics of leech muscles. As a consequence, their duration and relative timing was variable across different preparations, whereas their order of appearance was conserved. These results suggest that, during local bending, the leech neuromuscular system 1) operates a reduction of its available degrees of freedom, by simultaneously recruiting groups of otherwise antagonistic muscles and large populations of motoneurons; and 2) ensures reliability and effectiveness of this escape reflex, by guaranteeing the reproducibility of its crucial initial phase.

Highly Variable Spike Trains Underlie Reproducible Sensorimotor Responses in the Medicinal Leech

The nervous system of the leech is a particularly suitable model to investigate neural coding of sensorimotor responses because it allows both observation of behavior and the simultaneous measurement of a large fraction of its underlying neuronal activity. In this study, we used a combination of multielectrode recordings, videomicroscopy, and computation of the optical flow to investigate the reproducibility of the motor response caused by local mechanical stimulation of the leech skin. We analyzed variability at different levels of processing: mechanosensory neurons, motoneurons, muscle activation, and behavior. Spike trains in mechanosensory neurons were very reproducible, unlike those in motoneurons. The motor response, however, was reproducible because of two distinct biophysical mechanisms. First, leech muscles contract slowly and therefore are poorly sensitive to the jitter of motoneuron spikes. Second, the motor response results from the coactivation of a population of motoneurons firing in a statistically independent way, which reduces the variability of the population firing. These data show that reproducible spike trains are not required to sustain reproducible behaviors and illustrate how the nervous system can cope with unreliable components to produce reliable action.

Using Optical Flow to Characterize Sensory-Motor Interactions in a Segment of the Medicinal Leech

Activation of motoneurons innervating leech muscles causes the appearance of a two-dimensional vector field of deformations on the skin surface that can be fully characterized using a new technique (Zoccolan et al., 2001) based on the computation of the optical flow, the two-dimensional vector field describing the point displacements on the skin. These vector fields are characterized by their origin (i.e., the singular point) and by four elementary components that combine linearly: expansion (or compression), rotation, longitudinal shear, and oblique shear. All motoneurons can be classified and recognized according to the components of the deformations they elicit: longitudinal motoneurons give rise almost exclusively to longitudinal negative shear, whereas circular motoneurons give rise to both positive longitudinal shear and significant negative expansion. Oblique motoneurons induce strong oblique shear, in addition to longitudinal shear and negative expansion. Vector fields induced by the contraction of longitudinal, circular, and oblique fibers superimpose linearly. Skin deformations can therefore be attributed rather reliably to the contraction of distinct longitudinal, circular, and oblique muscle fibers. We compared the deformation patterns produced by touching the skin with those produced by intracellular stimulation of P, T, and N cells: vector fields resulting from the activation of P cells were almost identical to those produced by mechanical stimulation. Therefore, motor responses triggered by light or moderate touch are almost entirely mediated by excitation of P cells, with minor contributions from T and N cells.

Dystrophin-associated proteins in obliquely striated muscle of the leech Pontobdella muricata (Annelida, Hirudinea).

The distribution of dystrophin-associated proteins (beta-dystroglycan, alpha-, beta-, gamma- and delta-sarcoglycan, alpha-syntrophin and sarcospan) were studied in obliquely striated muscle of the leech Pontobdella muricata. Western blot analysis and immunohistochemical electron microscopy, using various polyclonal antibodies, were employed. Western blot analysis of all of these antibodies showed a single band, with approximately the same molecular weights as similar proteins detected in vertebrate muscles. The immunoelectron microscopy study confirmed specific immunogold labelling in the membrane of muscle cells. Since all dystrophin complex components have similar molecular weights and the same localisation in leech as in vertebrate skeletal muscle, we assume that these proteins have similar properties in leech and vertebrate muscle. The presence of these molecules in annelid muscles, together with a short version of dystrophin (previously described as IDLp-140) is of particular interest since phylogenetic and functional studies on this material could help to shed new light on the role and function of this complex in the muscle membrane.

Mapping motor neuron activity to overt behavior in the leech. I. Passive biomechanical properties of the body wall.

As an initial step in constructing a quantitative biomechanical model of the medicinal leech (Hirudo medicinalis), we determined the passive properties of its body wall over the physiological range of dimensions. The major results of this study were: 1. The ellipsoidal cross section of resting leeches is maintained by tonic muscle activation as well as forces inherent in the structure of the body wall (i.e., residual stress). 2. The forces required for longitudinal and circumferential stretch to maximum physiological dimensions were similar in magnitude. Cutting out pieces of body wall did not affect the passive longitudinal or circumferential properties of body wall away from the edges of the cut. 3. The strain (i.e., the percentage change in dimension of different body segments when subject to the same force was identical, despite differences in muscle cross-sections. 4. Serotonin, a known modulator of tension in leech muscles, affected passive forces at all physiological muscle lengths. This suggests that the longitudinal muscle is responsible for at least part of the passive tension of the body wall. 5. We propose a simple viscoelastic model of the body wall. This model captures the dynamics of the passive responses of the leech body wall to imposed step changes in length. Using steady-state passive tensions predicted by the viscoelastic model we estimate the forces required to maintain the leech at any given length over the physiological range.

Possible existence of dopaminergic receptors on cholinergic nerve terminals in the guinea-pig Auerbach's plexus

In the Auerbach's plexus of guinea-pig ileum lower concentrations of oxotremorine (Oxo-T) produced an increase, whereas higher concentrations of Oxo-T caused inhibition of evoked acetylcholine (ACh) release, measured by bioassay using dorsal muscle of leech. Dopamine inhibited the increase in evoked release of ACh induced by Oxo-T as a function of its concentration and this inhibitory effect was nullified in presence of pimozide, the dopamine receptor antagonist. The results demonstrate existence of presynaptic dopamine receptors having inhibitory influence on excitatory presynaptic muscarinic receptors on regulation of ACh release. However, no physiological role of dopamine could be observed on ACh release in this preparation.

An identified cell is required for the formation of a major nerve during embryogenesis in the leech.

Investigations of the cues by which axonal growth cones navigate long distances to their targets have revealed the use of a rich and complex diversity of cellular and extracellular information. In the present study we describe one of the most conceptually simple pathfinding cues: a single identified cell in the leech, Hirudo medicinalis, that may guide axons several hundred micrometers to innervate a particular target. One of the stereotyped nerves of H. medicinalis is a "sex nerve" that projects from the anterior root of ganglion 6 [SNA (6)] to the male reproductive structures in the adjacent anterior segment. The pathway for SNA (6) is completely underlain by a single peripheral cell, here called the axonal runway cell (ARC), before axons enter the pathway. The ARC is apparently a nonneuronal cell that stains with a monoclonal antibody that recognizes leech muscle cells. The importance of the ARC for establishing SNA(6) was tested by ablating it before axons entered the pathway. When the ARC was killed either by physical disruption with a microelectrode, or by photoablation after filling it with the fluorescent dye Lucifer yellow, SNA(6) always failed to form, whereas all other nerves formed normally. Killing other peripheral cells in proximity to the ARC did not interfere with SNA(6) formation. Ablation of possible "pioneer neurons" for SNA(6) also did not prevent its formation. These results show that formation of a particular nerve requires only a single cell to serve as a guide for outgrowing processes.

Proton magnetic resonance spectroscopy of leech muscle and nervous system.

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.

Gangliogenesis in leech embryos: migration of neural precursor cells

In the metameric CNS of leeches, identified neurons occupy highly stereotyped positions in each segmental ganglion. Although many of the neural precursor cells arise near their definitive positions, some arise outside the prospective domain of the segmental ganglia and thus must migrate into the CNS. Here, we report the results of an analysis of the role of cell migration in gangliogenesis in the leech Theromyzon rude. Segmental ganglia of the ventral nerve cord arise as laterally thickened sheets of tissue lying astride the ventral midline. Particular identified circular and longitudinal muscle fibers, visualized by indirect immunofluorescence using a monoclonal antibody against leech muscle, outline the presumptive ganglionic territories even before the ganglionic rudiments become morphologically distinct and serve as anatomical landmarks to which the cell movements are related. Cell lineage tracers microinjected into precursor blastomeres are used to visualize migratory cells. Small groups of neural precursor cells that arise outside the prospective ganglionic territories migrate with stereotyped timing along stereotyped pathways to reach their definitive positions, and each group of migratory cells gives rise to a stereotyped subset of the cells in a ganglion. No segmental or regional differences are observed in any aspect of cell migration studied here, supporting the view that segmental differences in the architecture of the leech CNS arise only after the initial condensation of the ganglionic rudiments.

Acetylcholine antirelease effect of morphine and its modification by calcium

The effects of morphine (10 mg/kg) and calcium (10 mg/kg) were studied on the neocortical release of acetylcholine (ACh) in ketamine-anesthetized rats. Morphine decreased ACh release as measured by the dorsal leech muscle as well as by an enzymatic assay. Calcium was ineffective alone but antagonized the action of morphine. This study supports the hypothesis that the ACh antirelease action of narcotics is mediated through an interaction with calcium.

Isotonic and isometric responses of different tonic muscles to agonists and antagonists.

1 With isotonic recording the percentage of muscle shortening as compared with the maximal possible shortening, and with isometric recording the percentage of developed tension were determined. In relatively 'thick' muscles, such as dorsal leech muscle, frog rectus abdominis or protractor pharynx of holothuria (0.3-0.8 mm thick), the concentrations of a full agonist (carbachol) producing a given percentage of tension, (e.g. 50%) are about 5 times greater than the concentrations, producing the same percentage of shortening. In 'thin' muscles the difference between the percentage of shortening and tension is either small (retractor dentis of the sea urchin, 0.1 mm thick, response to carbachol) or absent (guinea-pig ileum, 0.06 mm thick, responses to methylfurmethide). The possible mechanism underlying this difference is discussed. 2 With partial agonists (dodecamethonium and heptamethonium) the fractional tension of the frog rectus abdominis is always less than the fractional shortening and the correlation between shortening and tension is the same as in the case of full agonists. 3 The blocking activity of (+)-tubocurarine on the frog rectus abdominis is the same in isotonic and in isometric conditions. 4 On the frog rectus abdominis the alkylating agent, decamethonium mustard, does not produce any 'parallel shift' of the dose-response curve for carbachol, the only result of alkylation being a decrease in maximal response, which is more pronounced in isometric than in isotonic conditions. The degree of decrease is in accordance with the correlation between percentage of shortening and percentage of tension in the absence of alkylating agent. Probably this muscle does not possess any 'spare receptors'. 5 On the frog muscle the dose-isometric response curve for acetylcholine (ACh) is shifted toward greater concentration about 33-fold as compared with the dose-isotonic response curve but after the inhibition of cholinesterases the shift is only about 6-fold. The same shift (5-fold) is observed for carbachol, which is not hydrolysed by cholinesterases. The results with ACh are due to the fact, that after cholinesterase inhibition the sensitivity to ACh increases in isotonic conditions only 13-fold, but in isometric conditions it increases 71-fold. Probably under isometric conditions, when the muscle remains in the extended state, the rate of hydrolysis of ACh is much greater than under isotonic conditions when the muscle is shortened during contraction.

The effect of curare on the release of acetylcholine from mammalian motor nerve terminals and an estimate of quantum content.

Curarized and non-curarized rat hemidiaphragm muscles were indirectly stimulated in vitro. 2. The fluid bathing the active curarized muscles was eluted through a dextran gel (Sephadex G-10), effecting a complete separation of ACh from curare. The acetylcholine fraction was then assayed on an isometric leech muscle preparation. 3. Prostaglandin (PGE1) in a concentration fifteen times that estimated to be released from the skeletal muscle preparation did not affect the response of leech muscle to ACh. 4. The amount of ACh released by curarized muscles (4-9 X 10(-18) mole/impulse-junction) was not significantly different from that released by non-curarized muscles (4-6 X 10(-18) mole/impulse-junction). These quantities are similar to those obtained by previous workers. It is concluded that curare in a paralytic dose does not affect the output of ACh from motor nerve terminals stimulated at low frequencies. 5. Spontaneous release of ACh from non-curarized muscles was estimated at 0-45-0-65 p-mole/min. hemidiaphragm. It is calculated that only 2% of this amount could give rise to post-synaptic electrical events, the remainder having a non-synaptic source. 6. The number of molecules of 'quantal' ACh released by stimulated muscle is calculated as 2-5 X 10(6)/impulse-junction, taking account of the non-synaptic release. The number of ACh molecules in one quantum was estimated to be 6250, an amount that could be easily accommodated in one synaptic vesicle.

Blocking action of snake venom polypeptides on cholinergic mechanisms of the leech dorsal muscle

The blocking action of α-polypeptides isolated from the venom ofBungarusmulticinctus andNajanaja siamensis was investigated in experiments on the isolated dorsal muscle of the leech. These neurotoxins (NTs), in a concentration of 1×10−5 g/ml, did not block responses to monoquaternary cholinomimetics (acetylcholine, carbachol, nicotine, monocholine ester of succinic acid). The ability of NT to block responses to biquaternary cholinomimetics depended on the length of their molecule. Only responses to dicholine esters of malonic, succinic and glutaric acids were blocked by NT. Dicholine esters of adipic, pimelic, suberic, azelaic, and sebacic acids retained the whole of their activity after treatment of the leech muscle with NT. The possible causes of the selective action of NT are discussed.