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PubMed2001

Netrin signal is produced in leech embryos by segmentally iterated sets of central neurons and longitudinal muscle cells.

Abstract. Netrins are secreted molecules capable of attracting or repelling growing axons. They and their receptors, along with other netrin-interacting proteins, are widely conserved among animals from a broad range of phyla. We have raised and purified an antibody against a recently cloned leech netrin, which has allowed us to characterize embryonic netrin expression by cells in peripheral tissues and in the central nervous system. During early gangliogenesis, netrin expression was detected at particularly high levels in five bilateral pairs of central neurons. Towards the end of the period of axonal outgrowth, netrin expression was observed to be restricted to only six central neurons, comprising two bilateral pairs and two unpaired cells. A pair of netrin-producing central neurons, the bipolar cells, was identified by their expression of the antigen recognized by the monoclonal antibody Laz1-1. Double staining of sensory afferents from segmental sensilla with the monoclonal antibody Lan3-2 and the bipolar cells with the netrin antibody revealed that the terminals of these afferents grow up to the bipolar cells and turn anteriorly or posteriorly, without extending any further medially. Peripheral netrin expression was found to be restricted to longitudinal muscle cells in the ventral half of the body wall. Extracellular, secreted netrin was detected in a broad longitudinal stripe located symmetrically with respect to the ventral midline. The pattern of expression of netrin in leech embryos is consistent with observed expression patterns in other animals, suggesting that developmental netrin functions are conserved among all bilateral animals.

Animals↗
PubMed1999

Cell death in late embryogenesis of the leech Helobdella.

Cell death was characterized during stages 8 and 9 in the leech Helobdella with a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Using confocal analysis, the positions of dying cells were compared to rows of cells expressing the leech engrailed protein ht-en and to fluorescently marked cell lineages. Dying cells were present in diverse tissues. Some dying cells were in no obvious pattern, and others were in segmentally iterated patterns. Particular attention was paid to the ectoderm and mesoderm, where most of the cells examined died over a period equivalent to 1-4 h at 25 degrees C. Segmentally iterated rows of dying cells were observed in the mesoderm just beneath the nf-derived ht-en expressing cell rows at a time when ht-en expressing cells were beginning to disappear. The position of these dying cell rows was consistent with a role in the partial deterioration of the septum.

Animals↗
PubMed2001

Dorsal and snail homologs in leech development.

As part of an examination of how developmental mechanisms such as axis specification, cell fate specification, and segmentation have evolved, we have cloned homologs of the Drosophila melanogaster genes dorsal and snail from the glossiphoniid leech Helobdella robusta. Sequences from one dorsal-class gene (Hro-dl) and two snail-class genes (Hro-sna1 and Hro-sna2) were identified. Polyclonal antibodies were raised against the most conserved domains of HRO-DL and HRO-SNA1. Nuclear staining appeared for both proteins in mid-embryogenesis, in mesodermal and ectodermal precursors. During segmentation, segmentally iterated stripes of cells with strong HRO-DL staining appeared. The stripes of HRO-DL staining were first concentrated in the cytoplasm of cells, and later in the nuclei. Around this time, HRO-SNA levels also appeared in nuclei in segmentally iterated stripes. The localization of HRO-DL and HRO-SNA proteins raise the possibility that these genes are part of a conserved genetic pathway that, instead of specifying the dorsoventral axis and the mesoderm as in flies, might play a role in the diversification of cell types within segment primordia during leech development.

Amino Acid Sequence↗
PubMed2004

A trypanosome of Atlantic cod, Gadus morhua L., transmitted by the marine leech Calliobdella nodulifera (Malm, 1863) (Piscicolidae).

The fish leech, Calliobdella nodulifera, transmitted a trypanosome while feeding on laboratory-reared cod ( Gadus morhua). The flagellate body length increased from 43 microm 17 days post-infection (d.p.i.) to 57 microm 113 d.p.i. Characteristic features are cell striation, a nearly central nucleus and a short (3.5 microm) distance from the kinetoplast to the posterior end (PK). Following growth, the trypanosomes became increasingly slender, with fewer striae and a shorter flagellum, while the PK was unaffected. The trypanosomes differ morphologically from Trypanosoma murmanensis transmitted by the leech Johanssonia arctica.

Animals↗
PubMed2005

A new species of the medicinal leech (Oligochaeta, Hirudinida, Hirudo) from Transcaucasia and an identification key for the genus Hirudo.

A recent molecular phylogenetic study has suggested that the genus Hirudo contains a neglected species previously known as the orientalis coloration type of the medicinal leech Hirudo medicinalis. In this paper, the new species is formally described as Hirudo orientalis sp. n. It can most readily be identified by the grass green coloration of the dorsum, segmentally arranged pairs of black quadrangular or rounded dots on its paramarginal dorsal stripes and similarly arranged, but less regular light-colored markings on the predominantly black venter. It has medium-sized epididymes and an evenly coiled vagina. H. orientalis is known from Transcaucasia, Iran, and Uzbekistan. It is widely used in medicine as the "medicinal leech." Very little is known about its exact distribution, specific habitat, and conservation status. The paper contains an identification key to all species of the genus Hirudo.

Animals↗
PubMed2005

Coexistence of tonic spiking oscillations in a leech neuron model.

The leech neuron model studied here has a remarkable dynamical plasticity. It exhibits a wide range of activities including various types of tonic spiking and bursting. In this study we apply methods of the qualitative theory of dynamical systems and the bifurcation theory to analyze the dynamics of the leech neuron model with emphasis on tonic spiking regimes. We show that the model can demonstrate bi-stability, such that two modes of tonic spiking coexist. Under a certain parameter regime, both tonic spiking modes are represented by the periodic attractors. As a bifurcation parameter is varied, one of the attractors becomes chaotic through a cascade of period-doubling bifurcations, while the other remains periodic. Thus, the system can demonstrate co-existence of a periodic tonic spiking with either periodic or chaotic tonic spiking. Pontryagin's averaging technique is used to locate the periodic orbits in the phase space.

Action Potentials↗
PubMed1991

Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa.

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.

Amino Acid Sequence↗
PubMed1981

Long-term survival of glial segments during nerve regeneration in the leech.

Nerve injury that severs axons also disrupts ensheathing glial cells. Specifically, crushing or cutting the leech nerve cord separates the glial cell's nucleated portion from an anucleate recording, by intracellular injection of Lucifer Yellow dye and horseradish peroxidase (HRP) as tracers, and by electron microscopy. The nucleated portion of the glial cell did not divide, degenerate, or grow appreciably. The severed glial stump remained isolated from the nucleated portion but maintained its resting potential and normal morphology for months. Stumps typically began to deteriorate after 3 months. Small macrophage-like cells, or 'microglia' increased in number after injury and ensheathed axons, thus partially replacing the atrophying glial stump. Some axons in the nerve cord degenerated; the remainder appeared morphologically and physiologically normal. Thus, both nucleated and anucleate glial segments persisted throughout the one to two months required for axons to regenerate functional connections. Glial cells in the leech are therefore available to guide physically the growing axons or to contribute in other ways to nerve regeneration.

Animals↗
PubMed1982

Role of serotonin and cyclic AMP on facilitation of the fast conducting system activity in the leech Hirudo medicinalis.

In the nervous system of the leech Hirudo medicinalis it has been possible to study short-term plastic changes. Depression and facilitation have been demonstrated in the fast conducting system (FCS) activity; this pathway consists of a chain of electrically linked neurons present in each ganglion. In semi-intact animals or in preparation of nerve cord and segments of body wall, both electrical stimulation of peripheral roots and tactile stimulation of the skin induced, after repetitive stimulation (0.1/s) a prolonged decrement of FCS response. Strong nociceptive stimulation applied onto the head or the body wall produced a sustained facilitation of the waned response. The same potentiation has been observed by perfusing the isolated ganglion with serotonin (5 x 10(-5) M). Such a potentiation is abolished by preincubation with methysergide, an antagonist of serotonin, and with imidazole, a cAMP-phosphodiesterase activator. Such an effect is mimicked by an analog of cAMP, db-cAMP. Simultaneous recordings of both T neurons (intracellularly) and FCS firing discharge showed that, during FCS response decrement, the T cell activity remained unchanged and no modification of conductance occurred, excluding therefore a detectable involvement of sensory neurons in the depression. These results suggest that short-term plastic changes of the FCS of the leech are due to a prolonged potentiation of synaptic transmission as a result of serotonin-mediated increase in cAMP.

Animals↗
PubMed1982

Ionic mechanism of a hyperpolarizing 5-hydroxytryptamine effect on leech neuropile glial cells.

The ionic mechanism of a membrane effect of 5-hydroxytryptamine (5-HT) on neuropile glial (NG) cells in ganglia of the medicinal leech was investigated with conventional single-barrelled microelectrodes. Control experiments were made with double-barrelled ion-selective microelectrodes. 5-Hydroxytryptamine hyperpolarized the NG-cell membrane and increased the conductance considerably. Methysergide, a potent 5-HT antagonist, blocked the 5-HT-induced hyperpolarization completely. When leech ganglia were superfused with physiological bathing media free of 5-HT, the NG-cell membrane conductance returned to the original value, but the membrane potential recovered only partially from the hyperpolarization in most experiments. In glial membranes artificially depolarized by means of constant-current injection, the amplitude of the 5-HT response increased. The amplitude decreased with membrane hyperpolarization and reversed at - 73 mV, close to the potassium equilibrium potential. The reversal potential changed by 52 mV when the extracellular potassium concentration was altered by a factor of 10. We conclude that 5-HT increases the potassium conductance of NG-cell membranes.

Animals↗
PubMed1984

Procaine actions on tetrodotoxin sensitive and insensitive leech neurons.

Procaine (0.1-10 mM) was applied to two kinds of identified neurons in segmental leech ganglia. Both Retzius (R) cells and nociceptive (N) cells responded by dose-dependent reduction of maximum rates of de- and repolarization during action potentials. However, the N cells, which are more sensitive to tetrodotoxin than R cells, were also 3 times more sensitive to procaine. The prolongation of action potentials produced by procaine in R cells was enhanced by low Ca but antagonized by high Ca. This implies that the drug interfered with repolarization by affecting a Ca-dependent mechanism. In alkaline solution (pH 8.5), sensitivity of the R cell to procaine approached that of the N cell at pH 7.4 suggesting that the drug acted at an intracellular site after passage through membrane lipids in its uncharged form. The combined effects of TTX and procaine, in concentrations which produced about 50% inhibition of dV/dTdep in N cells when given separately, were found to be intermediate between those predicted by two models which assume identical and independent sites of action, respectively. These data extend our earlier observations regarding the existence of two types of Na channels in mature leech neurons. They imply that the differential sensitivity to procaine among these cells may be a consequence of variable access to an otherwise identical 'receptor' and that TTX and procaine act on separate sites which may interact with each other.

Animals↗
PubMed1985

Expression of surface antigens recognized by the monoclonal antibody lan 3-2 during embryonic development in the leech.

The monoclonal antibody lan 3-2 was used as a marker for the developmental expression of surface antigens which are specific for the two pairs of nociceptive neurons and a subset of axons in the adult CNS of the leech. In Haemopis embryos labeling of both nerve fibers and cell bodies with the antibody appears as expected for a metameric animal in a rostrocaudal temporal gradient from about day 5-6. Surprisingly, all central cell bodies are stained by the antibody in early development. However, later in embryogenesis around day 17 the staining intensity of most cells decreases except for the nociceptive cells, which remain antibody-positive, and the adult staining pattern gradually emerges. In addition to describing the central staining pattern, we show that specific peripheral neurons associated with the segmental sensilla also are antibody-positive during development. The distribution and developmental expression of the lan 3-2-positive antigens are compared between two phylogenetically different species of leeches and the diversity of the staining pattern of the monoclonal antibody is discussed.

Animals↗
PubMed1986

Barbiturates block divalent cation action potentials in leech nociceptive cells.

Phenobarbital (PNB), pentobarbital (PTB) and methohexital (MTX) decreased the maximum rate of depolarization Vmax and duration of divalent cation action potentials elicited in leech nociceptive neurons in Na+-free solutions containing the K+-channel blocker TEA, without significantly affecting resting membrane potential or conductance. The block of the divalent cation action potentials was reversible and dose-dependent, ED50 for inhibition of Vmax being 560 microM for MTX, 800 microM for PTB and 3000 microM for PNB. This order of potency correlated well with the ratio of unchanged/charged form of the drugs at physiological pH suggesting that in leech, as in other preparations, the non-ionized form was the active one. In Na+-containing Ringer, the 3 barbiturates depolarized and decreased membrane resistance in the lateral nociceptive cells, but not the medial nociceptive cells. These results provide additional information regarding the newly described pharmacological differences among closely related neurons. These membrane actions may be related to some of the excitatory properties described for other barbiturates in invertebrate and mammalian preparations.

Action Potentials↗
PubMed1986

Inhibitory connections between motor neurons modify a centrally generated motor pattern in the leech nervous system.

Both excitatory and inhibitory motor neurons innervate longitudinal body wall muscles in the leech Hirudo medicinalis. Each inhibitory motor neuron also centrally inhibits the excitatory motor neurons that innervate its same muscle field. This central inhibition is strong, probably monosynaptic, and largely a function of graded membrane potential changes in the inhibitory motor neurons. During leech swimming, both the excitatory and inhibitory motor neurons are rhythmically active. Here, we present evidence that the inhibitory motor neurons phasically inhibit the excitatory motor neurons during swimming, thereby augmenting the amplitude of membrane potential oscillations and the burst intensity of the excitors.

Animals↗
PubMed1990

Glutaminergic responses of neuropile glial cells and Retzius neurones in the leech central nervous system.

The effects of glutaminergic agonists on neuropile glial cells and on Retzius neurones in the central nervous system (CNS) of the leech, Hirudo medicinalis were investigated using double-barrelled ion-sensitive microelectrodes. In both types of cells, bath-application of L-glutamate (Glu), kainate (Ka) and quisqualate (Qui) elicited substantial membrane depolarizations which were accompanied by increases of the intracellular Na+ activity aiNa and by concomitant decrease of the intracellular K+ activity aiK. In the glial cells, these alterations of aiNa and aiK were preceded by a transient decrease of aiNa and an increase of aiK upon administration of Ka and Qui. In both glial cells and neurones, N-methyl-D-aspartate (NMDA) did not affect Em, aiK and aiNa. As found for Ka, the neuronal as well as the glial responses to glutaminergic agonists persisted during inhibition of synaptic transmission in high Mg2+, low Ca2+ solutions. The results indicate that leech neuropile glial cells have a Ka/Qui-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones.

Animals↗
PubMed1993

Biochemical evidence of angiotensin II-like peptides and proteins in the brain of the rhynchobdellid leech Theromyzon tessulatum.

The peptides contained in neurons localized in the brain of the leech Theromyzon tessulatum (Hirudinae, Rhynchobdellida) and showing an immunopositive reaction with an antibody directed against angiotensin II (AII), were purified by reversed-phase HPLC. Three AII-like peptides (P1, P2 and P3) which exhibited the same retention times and chromatographic behaviors as synthetic AVII (fragment 6-8 of AII), AIV (fragment 3-8 of AII) and AII, respectively, were resolved in brain extracts. An identification of the proteins immunoreactive to an anti-AII was performed at the level of both brain extracts and in vitro brain-translated RNA products. The protein detected at the level of the brain extracts (of a molecular mass of approximately 18 kDa) is multipeptidic as it is also recognized by two other antisera, a polyclonal one directed against gamma-MSH and a monoclonal one (Tt159) raised against a leech brain epitope. It could be the pro-AII-like precursor. The protein detected at the level of in vitro brain-translated RNA products (of a molecular mass of approximately 19 kDa) could be the prepro-AII-like precursor.

Angiotensin II↗
PubMed1994

Effects of glutamatergic agonists and antagonists on membrane potential and intracellular Na+ activity of leech glial and nerve cells.

The membrane potential of neuropile glial cells and Retzius neurones in the central nervous system of the leech Hirudo medicinalis was measured using electrolyte-filled single-barreled microelectrodes. Intracellular Na+ activity (aNai) was recorded with Na(+)-sensitive double-barreled microelectrodes. Bath-application of kainate, quisqualate and L-glutamate elicited concentration-dependent membrane depolarizations in both cell types as demonstrated by dose-response curves. The competitive quinoxalinedione antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the non-NMDA glutamate receptor inhibited the membrane depolarizations in neuropile glial cells completely, but in Retzius neurones only partially. These results confirm that leech neuropile glial cells have a kainate- and quisqualate-preferring non-NMDA glutamate receptor similar to that in the Retzius neurones. The initial decrease in aNai in neuropile glial cells in kainate- or quisqualate-containing solutions and the afterhyperpolarization in these glial cells and the Retzius neurones following the removal of both glutamate antagonists, were blocked in the presence of the cardiac glycoside ouabain (10(-4) M). In saline solutions containing 42.5 mM Li+ instead of Na+ the afterhyperpolarizations were blocked in neuropile glial cells and Retzius neurones. We conclude that the initial aNai changes and the afterhyperpolarization could be due to the stimulation of the electrogenic Na+/K+ pump in the glial and neuronal membranes.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1984

Embryonic origins of cells in the leech Helobdella triserialis.

To ascertain the embryonic origins of the cells in various tissues of the leech Helobdella triserialis, horseradish peroxidase (HRP) was injected as a cell lineage tracer into all identified blastomeres of the early embryo in turn, except for a few of the micromeres, and the resulting distribution of HRP-labeled cells was then examined in the late embryo. In this way it was found that in every body segment a topographically characteristic set of neurons in the ganglion and body wall and a characteristic territory of the epidermis is derived from each of the four paired ectodermal teloblasts N, O/P, O/P, and Q, whereas the muscles, nephridia, and connective tissue, as well as a few presumptive neurons in each segmental ganglion, are derived from the paired mesodermal teloblast, M. Each topographically characteristic, segmentally iterated set of neurons descended from a given teloblast is designated as a kinship group. However, the prostomial (nonsegmental) epidermis and the neurons of the supraesophageal ganglion were found to be derived from the a, b, c, and d micromere quartet to which the A, B, C, and D blastomeres give rise at the dorsal pole of the embryo. The superficial epithelium of the provisional integument, which covers the surface of the embryo midway through development and is sloughed off at the time of body closure, was found to be derived from the a, b, c, and d micromere quartet, as well as from other micromeres produced in the course of teloblast formation. The contractile fibers of the provisional integument were found to be derived from the paired M teloblast. These results demonstrate that development of the leech embryo proceeds according to a highly stereotyped pattern, in the sense that a particular identifiable blastomere of the early embryo regularly gives rise to a particular set of cells of the adult (or provisional embryonic) tissues.

Animals↗