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PubMed1998

Movements and stepwise fusion of endodermal precursor cells in leech.

At the four-cell stage, embryos of glossiphoniid leeches comprise identified blastomeres A, B, C and D. Subsequent cleavages of the A, B and C quadrants yield three large, yolk-rich endodermal precursor cells, macromeres A"', B"' and C"'. Eventually, these cells generate the epithelial lining of the gut via cellularization of a multinucleate syncytium. Meanwhile, cleavage in the D quadrant generates ten teloblasts that give rise to segmental mesoderm and ectoderm via stem cell divisions. Here we show that, during cleavage, macromeres A"', B"' and C"' shift clockwise relative to the D quadrant, while C"' comes to envelop the nascent teloblasts. During gastrulation, derivatives of the teloblasts undergo epibolic movements over the surface of the A"', B"' and C"' macromeres to form the germinal plate, from which segmental tissues arise. We find that the three macromeres fuse in a stepwise manner to initiate formation of the multinucleate syncytium; cell C"' fuses about 25 h after the fusion of A"' and B"', and the teloblasts fuse with the macromere-derived syncytium later still. When macromeres are biochemically arrested by microinjecting them with the A chain of ricin, a further difference among the macromeres is revealed. Biochemical arrest of A"' or B"' slightly retards the rate of germinal plate formation, but arrest of C"' frequently accelerates this process.

Animals↗
PubMed1996

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots.

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

Amino Acid Sequence↗
PubMed1998

Pleated septate junctions in leech photoreceptors: ultrastructure, arrangement of septa, gate and fence functions.

The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular "vacuole" that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial sections from photoreceptors infiltrated with colloidal lanthanum. The septa form tortuous interseptal corridors that are spatially continuous, and open ended basally and apically. Individual septa seem to be impermeable to lanthanum; interseptal corridors form the only diffusional pathway for this ion. The junctions form no diffusion barrier for the electron-dense tracer Ba2+, but they hinder the diffusion of various hydrophilic fluorescent dyes as demonstrated by confocal laser scanning microscopy (CLSM) of live cells. Even those dyes that penetrate gap junctions do not diffuse beyond the septate junctions. The aqueous diffusion pathway within the septal corridors is, therefore, less permeable than the gap-junctional pore. Our morphological results combined with published electrophysiological data suggest that the septa themselves are not completely tight for small physiologically relevant ions. We also examined, by CLSM, whether the septate junctions create a permeability barrier for the lateral diffusion of fluorescent lipophilic dyes incorporated into the peripheral membrane domain. AFC16, claimed to remain in the outer membrane leaflet, does not diffuse beyond the junctional region, whereas DiIC16, claimed to flip-flop, does. Thus, pleated septate junctions, like vertebrate tight junctions, contribute to the maintenance of cell polarity.

Animals↗
PubMed2002

Ultrastructure of electrical synapses between nociceptive and anterior pagoda neurons in the CNS of the leech (Whitmania pigra).

We have used electron microscopy to measure quantitatively the morphology of electrical synapses in a circuit that has been proposed to account for the positional discrimination of the leech. Injection of a presynaptic nociceptive sensory neuron and the postsynaptic anterior pagoda neuron with HRP showed gap junctions in the neuropil. After double labeling, La(3+)-treated ganglia revealed labeled gap junctions from 2.0 to 3.5 nm wide. Between the labeled axon terminals, there were innexons with diameters of 8 to 10 nm. The innexon's central pore diameter was 2 nm, and the mean of the center-to-center distance between two innexons was 30 nm. Except for the gap junction areas of nociceptive sensory neuron axon terminals, the other ultrastructural parameters measured by freeze fracture were similar to those of samples labeled with HRP and filled with La(3+). These data suggested that the gap width, innexon diameter, and its central pore do not on their own account for the mechanism of positional discrimination, which may depend rather on the differences in distribution and number of gap junctions.

Animals↗
PubMedDate not supplied

The A-like potassium current of a leech neuron increases with age in cell culture.

Single leech neurons isolated and maintained in culture sprout and form electrical and chemical synapses, as they do in vivo, retaining most of the electrical properties of the intact membrane. However, some cells, such as Retzius, Anterior Pagoda (AP) cells and motoneurons, exhibit consistent changes of biophysical characteristics, which mimic those induced by axotomy in vivo and are reversed after reconnection. To improve our understanding of the mechanisms involved in these alterations and of their physiological significance, we investigated the early changes in outward currents developed by cultured AP neurons, using the patch-clamp technique in the whole-cell recording configuration. Different currents were isolated and a differential sensitivity to the time spent in culture and to internal calcium was observed. Three potassium currents were dissected: an A-like current, a delayed rectifier and a third unidentified component. The A-like potassium current was significantly increased with neuronal age in cell culture and was a function of the internal Ca(2+) concentration, whereas the two other potassium currents remained unchanged. Intracellular recordings performed from axotomized neurons of cultured ganglia revealed clear-cut alterations in spike adaptation, which might be due to changes of the A-like current.

Animals↗
PubMed2006

Systems-level modeling of neuronal circuits for leech swimming.

This paper describes a mathematical model of the neuronal central pattern generator (CPG) that controls the rhythmic body motion of the swimming leech. The systems approach is employed to capture the neuronal dynamics essential for generating coordinated oscillations of cell membrane potentials by a simple CPG architecture with a minimal number of parameters. Based on input/output data from physiological experiments, dynamical components (neurons and synaptic interactions) are first modeled individually and then integrated into a chain of nonlinear oscillators to form a CPG. We show through numerical simulations that the values of a few parameters can be estimated within physiologically reasonable ranges to achieve good fit of the data with respect to the phase, amplitude, and period. This parameter estimation leads to predictions regarding the synaptic coupling strength and intrinsic period gradient along the nerve cord, the latter of which agrees qualitatively with experimental observations.

Algorithms↗
PubMed1990

Amino acid sequence of ghilanten: anticoagulant-antimetastatic principle of the South American leech, Haementeria ghilianii.

This study reports the amino acid sequence of ghilanten, an anticoagulant-antimetastatic principle of the hematophagous leech, Haementeria ghilianii. Ghilanten consists of 119 amino acids with twenty cysteines and a consensus sequence for heparin-binding at its carboxyl-terminus. Arginine-34 represents the reactive residue involved in the active-site inhibition of trypsin and Factor Xa. Immunoreactivity data suggest that heterogeneity among ghilantens is due in part to amino acid substitutions at their carboxyltermini.

Amino Acid Sequence↗
PubMed1975

Transfer of radioactive material between electrically coupled neurons of the leech central nervous system.

Intracellular application of tritiated precursors by means of microiontophoresis was performed on nerve cells in isolated segmental ganglia of the leech ventral nerve cord. Incorporation as well as intra- and interneuronal transport were studied by autoradiography after injection of fucose, glucosamine, glycine, leucine, orotic acid and uridine. With several minutes of intraneuronal injection the precursors were incorporated into macromolecules. Depending upon the tracer used, the radioactive material was distributed in a specific pattern over the cell somata and then released into the nerve processes. After application of orotic acid and uridine a transport of radioactive material, presumably RNA, could be observed in the processes of the injected neurons at a distance of about 200-500 mum. Fucose and glucosamine injection resulted in the most extended labeling of the nerve cell processes, indicating a transport rate of about 11 mm/day. When the radiochemicals were injected into one of the two electrically coupled giant nerve cells -- the so-called Retzius cells (Rc) -- a specific labeling not only of the injected Rc but also of the coupled but not injected Rc was found. Injection of protein or glycoprotein precursors into one Rc produced heavy labeling of both Rcs including their processes; a slight labeling of other ganglion compartments was only found after increasing the dosage of the amino acids glycine and leucine. With orotic acid and uridine this interneuronal transfer was confined to the electrically coupled Rc twin. Intracellular injection of one Rc with puromycin followed by injection of amino acids or fucose into the same Rc or into the coupled Rc resulted in an inhibition of precursor incorporation within the puromycin-injected Rc and an exclusive labeling of the coupled Rc, thus indicating that the precursors themselves were transferred. It is suggested that after microiontophoretic application an interneuronal transfer of relatively low molecular weight material takes place, probably across the low-resistance junction through which the Rcs are electrically coupled.

Animals↗
PubMed1981

Long term survival of enucleated segments of glial cytoplasm in the leech Macrobdella decora.

Enucleated cytoplasmic segments of the giant connective glial cell (GCGC) survive morphologically intact for at least 10 weeks in the leech Macrobdella decora. Enucleated GCGC segments isolated from regenerating nerve axons show some degenerative changes after 4 weeks compared to GCGC segments which surround intact or regenerating nerve axons. Survival of GCGC cytoplasm is associated with an increase in the number of microglia. Relatively few (10-30%) nerve axons degenerate after severance from their cell body.

Animals↗
PubMed1982

External ions and membrane potential of leech neuropile glial cells.

In ion-substitution experiments supplemented by measurements of the membrane conductance, the membrane potential of neuropile glial (NG) cells in the CNS of th medicinal leech has exhibited a dependence of the external concentration of both potassium and chloride. The membrane potential was largely dependent on the external potassium concentration, as may be inferred from the change in potential as the potassium concentration of the bathing solution was changed. The external potassium concentrations had been corrected to allow for the discrepancy between intra- and extraganglionic levels found with ion-selective electrodes. A transient membrane depolarization was recorded when the chloride in the bathing medium was replaced by sulphate or glucuronate. The restoration of the normal membrane potential following the return to chloride-based saline was preceded by a transient hyperpolarization. After transfer to low-chloride solutions, the transient depolarization of the NG cell membrane was followed by a steady-state hyperpolarization. The amplitude of the steady-state hyperpolarization depended on the concentration of chloride in the bathing medium. The membrane conductance decreased in low-chloride solutions.

Animals↗
PubMed1983

Potassium activity in leech neuropile glial cells changes with external potassium concentration.

The effect of the external K+ concentration on the intracellular K+ activity of neuropile glial cells and of sensory neurons in the central nervous system of the leech (Hirudo medicinalis L.) was determined directly with double-barreled ion-sensitive microelectrodes. As the external K+ concentration was raised, the intracellular K+ activity of the neuropile glial cells increased considerably, and was accompanied by the uptake and/or intracellular synthesis of an, as yet, unidentified substance. In contrast, the intracellular K+ activity of sensory neurons was unchanged when the external K+ concentration was increased. These neurons appeared not to accumulate a second substance.

Animals↗
PubMed1983

The Na+-K+ pump in neuropile glial cells of the medicinal leech.

The membrane potential of neuropile glial (NG) cells in the central nervous system of the medicinal leech and the K+ concentration in extracellular spaces (ECS) of the neuropile were measured under various experimental conditions to determine properties of a glial Na+-K+ pump. The ganglia were exposed to K+-free saline thereby loading the NG cells with intracellular Na+. Their membranes hyperpolarized transiently when the K+-free solution was replaced by a bathing medium with normal (= 4 mM) K+ concentration. The hyperpolarization increased in amplitude with time of exposure to K+ -free solution and could be abolished by ouabain or by replacing Na+ with Li+. The transient membrane hyperpolarization could not be attributed to K+ depletion in the ECS of the neuropile or to changes in membrane input conductance. In a (bathing) medium containing 5 X 10(-4) M ouabain, the K+ concentration in the ECS increased transiently, and the NG cell membrane depolarized rapidly. This short-term depolarization (duration 2-3 min) was followed by a second long-term depolarization (duration 15 min) of the NG cell membrane, which reached a steady-state 20 min after ouabain application. In a bathing medium with elevated external K+ concentrations, the amplitude of the membrane depolarization was enhanced by ouabain. This depolarizing ouabain effect was a result of K+ accumulation in the ECS. We conclude that the Na+-K+ pump does not contribute directly to the resting membrane potential of NG cells and is not directly involved in K+ homeostasis at the cellular level.

Animals↗
PubMed1984

Effects of tetraethylammonium-chloride and divalent cations on the afterhyperpolarization following repetitive firing in leech neurons.

In leech Retzius cells, repetitive activity evoked a prolonged Ca2+-dependent after-hyperpolarization (PAH) (30-60 s) accompanied by an increase in input conductance. PAH persisted in Retzius cells, as well as in nociceptive (N) cells, when Sr2+ but not Mg2+ was substituted for Ca2+. In the presence of tetraethylammonium-chloride (TEA) or Ba2+, PAH was replaced by a Ca2+-dependent, Mg2+-blockable depolarization which was present in the order N greater than R. Careful study of the differences in such phenomena in identified cells may improve our understanding of the differential susceptibility of various neurons to hyperexcitability.

Animals↗
PubMed1984

6-Hydroxydopamine produces lesions of serotonin-containing Retzius cells in the leech nervous system.

The neurotoxin 6-hydroxydopamine (6-OHDA) is reported to selectively ablate neurons which contain catecholamines. Leeches were injected with 6-OHDA over a 4-month period in an attempt to destroy their identified dopamine neurons. Ganglionic Retzius cells (RZ), which contain serotonin, were the first to be rendered brown, misshapen, and non-fluorescent. Continued injections of 6-OHDA had similar, but less substantial, morphological and histochemical effects on the dopamine-containing neurosomata within the anterior roots. Toxin treatment reduced RZ serotonin by 75-90%, but these depleted cells retained normal electrophysiological properties. Serotonin and dopamine within ganglia, as well as dopamine within anterior roots, were reduced significantly by 6-OHDA.

Animals↗
PubMed1984

Response to axotomy of an identified leech neuron, in vivo and in culture.

Membrane properties of identified leech neurons with non-spiking somata were studied after axotomy. Two distinct procedures were used: the section of ganglionic roots in vivo and the isolation of cell somata in culture. The results indicated that axotomized neurons progressively developed the excitability of somatic membrane, both in vivo and in culture.

Animals↗
PubMed1985

The embryonic development of peripheral neurons in the body wall of the leech Haemopis marmorata.

The appearance of peripheral neurons within the skin during embryonic development of the leech is described. These neurons were labeled using a monoclonal antibody, Lan3-6, which recognizes antigens in both the cell body and the axons of these cells. Within the 5 annuli that are found in each midbody segment, peripheral neurons first label in the middle and last in the most anterior and posterior ones. In each annulus, the number of cells labeled is initially 4 and increases as development proceeds. By the end of embryogenesis, all annuli show approximately equal numbers of Lan3-6 labeled neurons. The development of peripheral neurons in the skin of the rear sucker is also described.

Animals↗
PubMed1985

Identified GABAergic inhibitory motor neurons in the leech central nervous system take up GABA.

Physiologically identified inhibitory motor neurons in the leech central nervous system were double-labeled by intracellular injection of the dye Lucifer Yellow and gamma-amino-[3H]butyric acid ([3H]GABA) uptake to demonstrate that these inhibitory neurons are among the approx. 35 neurons per abdominal ganglion previously shown to take up GABA. GABA uptake into the inhibitory motor neurons is stimulated by electrical activity in the neurons.

Action Potentials↗
PubMed1986

Probing structural homologies in cell-specific glycoproteins in the leech CNS.

Three monoclonal antibodies (mAbs) raised against the leech CNS recognize surface antigens on small sets and subsets of neurons or on glial cells. On immunoblots, they all recognize proteins of 130 kDa molecular weight. In addition, they each bind up to several different lower molecular weight forms. The 130 kDa polypeptides recognized by these mAbs are not major proteins on Coomassie blue-stained gels. They behave as glycoproteins on lentil lectin columns but are not major Concanavalin A-binding molecules. These molecules therefore represent a group of lower abundance, cell-type-specific antigens. Structural relationships between these antigens were explored using immunoprecipitation. The glial cell antigen was immunopurified, however, a fraction of the neuronal antigens co-precipitate. The co-precipitation of neuronal antigens raises the possibility that different neuronal antigenic determinants are carried on the same protein molecule. Such a protein may be modified to carry either one or both neuronal determinants, and could serve as a tag to physiologically delineate subsets of neurons nested within larger neuronal sets.

Animals↗