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Extracellular potassium in neuropile and nerve cell body region of the leech central nervous system.

Potassium-sensitive double-barrelled microelectrodes were used to measure the potassium content of extracellular spaces in leech ganglia, both intact and with the ganglion capsule opened. When the ganglion capsule was opened, the extracellular concentrations of potassium in the ganglion were similar to that of the bathing medium (4 mM). With intact ganglia the extracellular potassium concentration in the neuropile averaged 6.3 +/- 0.7 mM and in the nerve cell body region 5.8 +/- 0.6 mM. The potential measured in these parts of the ganglion was between +2 and -8 mV, averaging -1.9 mV. The change of potassium concentration in the extracellular spaces following increase or decrease in the concentration of potassium ions in the bath declined exponentially. This rate of change, which would be expected of a first-order diffusion process, was found in both the neuropile and the nerve cell body region. In a medium containing 5 x 10(-4) M ouabain, the potassium concentration in both parts of the ganglion increased transiently by an average of 3.8 +/- 1.0 mM in the neuropile and 1.2 +/- 0.4 mM in the nerve cell body region. Negatively charged polyelectrolytes in extracellular spaces of leech ganglia could affect the distribution of potassium ions to give a Donnan distribution. It is also possible, that the endothelial layer influences the extracellular potassium concentration in a ganglion under resting conditions.

Animals↗

Hoarseness due to leech ingestion.

This paper presents a case of hoarseness caused by the pharyngolaryngeal localization of a leech. This pathological lesion is extremely rare in western European countries, but is more frequent in endemic areas. Possibly lethal dyspnoea, haemoptysis or haematemesis can be the revealing symptoms. When the diagnosis is suspected simple examination under anaesthesia and removal of the leech will effect a cure.

Animals↗

Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis.

A DNA containing the coding sequence for the proteinase inhibitor protein hirudin from the leech Hirudo medicinalis has been obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides. The 226 bp synthetic gene carries signals for the translation initiation and termination. Fragment synthesis was performed by the Khorana ligation method as well as by the fill-in method. Efficiencies of these two methods are compared. The synthetic gene was expressed in E. coli as a fusion protein with beta-galactosidase under the control of the lac-promoter as well as a non-hybrid protein under the control of the lambda PL-promoter. The non-hybrid expression product was shown to have similar biological properties as the authentic protein isolated from the leech.

Amino Acid Sequence↗

Propagation of voltage transients in arborized neurites of Retzius cells of the leech in culture.

Propagation of electrical signals is studied in Retzius cells of the leech in culture using voltage-sensitive fluorescent dyes at a spatial resolution of 8 x 8 microns 2 and 14 x 14 microns 2 and at a sampling interval of 0.12 ms. The neurons are stimulated by a microelectrode impaled in the soma. Action potentials of a half width of 2-3 ms are triggered close to the end of the primary neurite dissociated from the leech. They propagate back to the soma at invariant half width at a velocity of 50-230 microns/ms. They pervade extended aborized secondary neurites which are grown on extracellular matrix protein. Their width is enhanced up to a factor two. The velocity is around 100-150 microns/ms such that delays up to 3.5 ms are observed. Accordingly the neuritic trees are not isopotential. The features of propagation are found to be incompatible with passive spread.

Action Potentials↗

A protein from the salivary glands of the giant Amazon leech with high sequence homology to a nicotinic acetylcholine receptor subunit.

A gene coding for a soluble protein with homology to the beta subunit of the nicotinic acetylcholine receptor from goldfish was isolated from a cDNA library of Haementeria ghilianii salivary glands. Comparison of the leech protein sequence with the database showed that the N terminus has high homology with the extracellular portion of acetylcholine receptor beta subunits, whilst the C terminus, highly charged, has homology to proteins which may be involved in chelating divalent cations. The leech protein was expressed in mammalian cells and the product compared to the native protein. Both proteins are glycosylated and form polymers which are disrupted by heat but not by reducing agents. A role for this protein in salivary gland secretion is suggested.

Amino Acid Sequence↗

Multiple forms of long-term potentiation and long-term depression converge on a single interneuron in the leech CNS.

Long-term potentiation (LTP) of synaptic transmission was observed in two types of synapses that converge on the same postsynaptic neuron in the leech CNS. These synapses were made by identifiable sensory neurons, the mechanosensory touch (T-) and pressure (P-) cells, onto the S-cell, an interneuron critical for certain forms of learning. Changes in both the T-S and P-S synapses appear to be activity dependent because LTP was restricted to inputs that had undergone tetanization; however, properties of synaptic plasticity at the T-S and P-S connections differ considerably. At the P-S synapse, LTP was induced in the tetanized synapse but not in the nontetanized synapse tested in parallel. P-S LTP was blocked by the NMDA receptor antagonist dl-2-amino-5-phosphono-valeric acid (AP-5) or by lowering the extracellular concentration of glycine, an NMDA receptor (NMDAR) co-agonist. P-S LTP was strongly affected by the initial amplitude of the synaptic potential at the time LTP was induced. Smaller amplitude synapses (<3.5 mV) underwent robust potentiation, whereas the less common, larger amplitude synapse (>3.5 mV) depressed after tetanization. At the T-S synapse, tetanization simultaneously induced homosynaptic LTP in the tetanized input and heterosynaptic long-term depression (LTD) in the input made by a nontetanized T-cell onto the same S-cell. Interestingly, AP-5 failed to block homosynaptic LTP at the T-S synapse but did prevent heterosynaptic LTD. T-S LTP was not affected by the initial EPSP amplitude. Thus, leech neurons exhibit synaptic plasticity with properties similar to LTP and LTD found in the vertebrate nervous system.

Animals↗

Molecular basis of gap junctional communication in the CNS of the leech Hirudo medicinalis.

Gap junctions are intercellular channels that allow the passage of ions and small molecules between cells. In the nervous system, gap junctions mediate electrical coupling between neurons. Despite sharing a common topology and similar physiology, two unrelated gap junction protein families exist in the animal kingdom. Vertebrate gap junctions are formed by members of the connexin family, whereas invertebrate gap junctions are composed of innexin proteins. Here we report the cloning of two innexins from the leech Hirudo medicinalis. These innexins show a differential expression in the leech CNS: Hm-inx1 is expressed by every neuron in the CNS but not in glia, whereas Hm-inx2 is expressed in glia but not neurons. Heterologous expression in the paired Xenopus oocyte system demonstrated that both innexins are able to form functional homotypic gap junctions. Hm-inx1 forms channels that are not strongly gated. In contrast, Hm-inx2 forms channels that are highly voltage-dependent; these channels demonstrate properties resembling those of a double rectifier. In addition, Hm-inx1 and Hm-inx2 are able to cooperate to form heterotypic gap junctions in Xenopus oocytes. The behavior of these channels is primarily that predicted from the properties of the constituent hemichannels but also demonstrates evidence of an interaction between the two. This work represents the first demonstration of a functional gap junction protein from a Lophotrochozoan animal and supports the hypothesis that connexin-based communication is restricted to the deuterostome clade.

Amino Acid Sequence↗

Changes in gene expression at the precursor --> stem cell transition in leech.

The glossiphoniid leech, Theromyzon trizonare, displays particularly large and accessible embryonic precursor/stem cells during its early embryonic cleavages. We dissected populations of both cell types from staged embryos and examined gene expression profiles by differential display polymerase chain reaction methodology. Among the approximately 10,000 displayed cDNA fragments, 56 (approximately 0.5%) were differentially expressed at the precursor --> stem cell transition; 29 were turned off (degraded, precursor-specific); and 27 were turned on (transcribed, stem cell-specific). Several putative differentially expressed cDNAs from each category were confirmed by Northern blot analysis on staged embryos. DNA sequencing revealed that 19 of the cDNAs were related to a spectrum of genes including the CCR4 antiproliferation gene, Rad family members, and several transcriptional regulators, while the remainder encoded hypothetical (10) or novel (27) sequences. Collectively, these results identify dynamic changes in gene expression during stem cell formation in leech and provide a platform for examining the molecular aspects of stem cell genesis in a simple invertebrate organism.

Animals↗

Host specificity in Metamera sillasenorum, n. sp., a gregarine parasite of the leech Helobdella triserialis with notes on transmission dynamics.

Eugregarines of the suborder Septatorina are apicomplexan parasites that are found mainly in arthropods. Some exceptions are species in the Metameridae that contains the only 5 septate gregarines recorded from annelids. The type genus is Metamera Duke, 1910 with 2 species, Metamera schubergi Duke, 1910, in European Glossiphonia complanata, and Metamera reynoldsi Jones, 1943, from North American G. complanata. Over the summers of 1995-1998, in Keith County, Nebraska, septate gregarines were found in the glossiphoniid leech Helobdella triserialis. The gregarines were determined to be a new species of Metamera, herein named Metamera sillasenorum. Measurements of size and body proportions of over 600 gregarines and 50 oocysts showed differences from measurements of M. schubergi and M. reynoldsi, and secondary septa in the deutomerite were rarely observed. Field observations indicated that M. sillasenorum is probably host specific. In the laboratory, leeches also exhibited strong feeding preferences; e.g., H. triserialis and G. complanata consumed only snails, whereas Helobdella stagnalis consumed only oligochaetes. Infection experiments demonstrated that freshwater snails ingest the oocysts and are required as mechanical vectors. Oocysts were passed unaltered through the snails' intestines. Glossiphonia complanata did not become infected regardless of heavy exposure to oocysts, although only 5 G. complanata were used in the experiments. The results show that host specificity of M. sillasenorum is most likely due to a combination of host-feeding habits and host-parasite compatibility.

Animals↗

Observations on the leech Placobdella ornata feeding from bony tissues of turtles.

The leech Placobdella ornata was observed feeding from the blood sinuses of the plastron and carapace bones of Chelydra serpentina and Chrysemys picta. Evidence of successful feeding included blood upwelling from the point of attachment and gastric ceca of the leeches freshly filled with blood after removal. There was an apparent preference for the sulci between scales of the shell.

Animals↗

A new species of glossiphoniid leech from Rana pretiosa (Amphibia: Ranidae) in Oregon.

A new species of ectoparasitic glossiphoniid leech was found feeding on frogs in the Nature Center Pond and elsewhere in Deschutes County, Oregon. The new species of Placobdella resembles the southern alligator leech, Placobdella multilineata Moore, 1953, notwithstanding their vast geographic separation in North America. The new species is readily distinguished by possessing subdivided annuli, by its papillation and pigmentation patterns as well as by the arrangement of ovarian tissues. There is strong evidence of nocturnality and of the potential for parasitizing humans.

Animals↗

Leech extracellular hemoglobin: two globin strains that are akin to vertebrate hemoglobin alpha and beta chains.

Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1; 15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21-30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of "two globin strains in annelid hemoglobin", which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin alpha and beta chains.

Amino Acid Sequence↗

Generalization of habituation and intrinsic sensitization in the leech.

Using the shortening reflex of the medicinal leech Hirudo medicinalis we examined stimulus generalization of habituation learning. Preparations received mechanosensory stimulus at two positions on the leech body wall, one site used to carry out habituation training and a second novel site to test for generalization of habituation. After training, the specific mechanosensory neurons activated by each stimulus were assessed using intracellular recordings. As expected, the closer the two sites were to each other, the greater the degree of generalization of habituation at the novel site and the more sensory cells were shared. However, a form of behavioral facilitation was observed at the trained site that resembled behavioral sensitization, but differed from the standard sensitization process in several respects. (1) Facilitation was induced by stimulation of the novel site before habituation training at the trained site, although the stimulus intensity at the novel site was equivalent to the training stimuli and was not the strong, noxious stimuli that normally induce sensitization. (2) The magnitude of the facilitating effect was proportional to the proximity of the novel and trained stimulation sites. (3) Although behavior at the trained site was facilitated, behavior at the novel site was habituated, indicating that the induced behavioral facilitation did not generalize throughout the animal, as normally occurs during sensitization, but was limited to a single stimulus-response pathway.

Analysis of Variance↗

A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech.

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.

Amino Acid Sequence↗

Nitric oxide influences injury-induced microglial migration and accumulation in the leech CNS.

Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. The aim of this study was to determine whether NO plays a role in microglial migration and accumulation at lesions in which NO is generated by a rapidly appearing endothelial nitric oxide synthase (eNOS) in leeches. Immunohistochemistry and cytochemistry demonstrated active eNOS before and throughout the period of microglial accumulation at the lesion. Decreasing NO synthesis by application of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (1 mM) significantly reduced microglial accumulation, whereas its inactive enantiomer N(w)-nitro-D-arginine methyl ester (1 mM) resulted in microglial accumulation similar to that in crushed controls. Increasing NO with the donor spermine NONOate (SPNO) (1 mM) also inhibited accumulation, but not in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5, 5-teramethylimidazoline-oxyl-3-oxide (50 microM). The effect of SPNO was reversed by washout. SPNO application reduced average microglial migratory speeds and even reversibly arrested cell movement, as measured in living nerve cords. These results suggest that NO produced at a lesion may be a stop signal for microglia to accumulate there and that it can act on microglia early in their migration. Thus, NO may assume a larger role in nerve repair and recovery from injury by modulating accumulation of microglia, which appear to be important for axonal regeneration.

Animals↗

Chemical and thermal stimuli have short-lived effects on the retzius cell in the medicinal leech.

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi-intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding.

Action Potentials↗

[The results of a study of the species composition of fish parasites in the river basins of northeastern European Russia. Leeches (Hirudinea), mollusks (Mollusca), crayfish (Crustacea) and arachnids (Arachnida)].

The report contains the results of faunistic study of parasitic Hirudinea, Mollusca, Crustacea and Arachnida occurring on fishes in the region of the S. Dvina, Mezen' and Pechora river basins. The parasite fauna in the first basin is represented by 3 species of leeches, 4 species of molluscs, 13 species of crustaceans and 1 species of water mites, in the second basin--4, 1, 11, 0; in the third basin--2, 7, 7, 1 species respectively. Total number of parasites in the region investigated: 4 leech species, 5 molluscs species, 16 crustacean species and 1 water mite species.

Animals↗

Intracellular Ca2+ dynamics during spontaneous and evoked activity of leech heart interneurons: low-threshold Ca currents and graded synaptic transmission.

In oscillatory neuronal networks that pace rhythmic behavior, Ca(2+) entry through voltage-gated Ca channels often supports bursting activity and mediates graded transmitter release. We monitored simultaneously membrane potential and/or ionic currents and changes of Ca fluorescence (using the fluorescence indicator Ca Orange) in spontaneously active and experimentally manipulated oscillator heart interneurons in the leech. We show that changes in Ca fluorescence in these interneurons during spontaneous bursting and evoked activity reflect the slow wave of that activity and that these changes in Ca fluorescence are mediated by Ca(2+) entry primarily through low-threshold Ca channels. Spatial and temporal maps of changes in Ca fluorescence indicate that these channels are widely distributed over the neuritic tree of these neurons. We establish a correlation between the amount of transmitter released, as estimated by the integral of the postsynaptic current, and the change in Ca fluorescence. In experiments in which we were able to record presynaptic low-threshold Ca currents, associated IPSCs, and presynaptic changes in Ca fluorescence from fine neuritic branches of heart interneurons near their region of synaptic contact with their contralateral partner, there was a close association between the rise in Ca fluorescence and the rise of the postsynaptic conductance. The changes in Ca fluorescence that we record at the end of fine neuritic branches appear to reflect changes in [Ca(2+)](i) that mediate graded synaptic release in leech heart interneurons. These results indicate that widely distributed low-threshold Ca currents play an important role in generating rhythmic activity and in mediating graded transmitter release.

Animals↗