Medicinal Leech Hirudo Medicinalis Research Articles

Sensory and Neurosecretory Innervation of Leech Nephridia is Accomplished by a Single Neurone Containing FMRFamide

The neural control of the excretory system of the medicinal leech Hirudo medicinalis has been characterized morphologically and chemically using light and electron microscopy, immunocytochemistry and biochemistry. Immunoreactivity against RFamide-like peptides revealed elaborate neuronal aborizations of a neurone in the nephridium, around the urinary bladder sphincter and in the central nervous system. The processes arose from the nephridial nerve cell (NNC), a previously identified receptor neurone. Using a combination of reverse-phase high pressure liquid chromatography, radioimmunoassay and subsequent Edman degradation and mass spectrometry, authentic FMRFamide has been identified as the major peptide of the NNC. Sensory and neurosecretory innervation of the nephridia is thus accomplished by a single neurone, which is thought to modulate nephridial performance.

A lower bound on the detectability of nonassociative learning in the local bending reflex of the medicinal leech

Studies of neural mechanisms of learning and memory have focused on large changes at identified synapses. However, memory in distributed processing reflexes could involve widely distributed engrams characterized by small changes at every synapse in the network. To investigate this possibility, we used a neural network optimization algorithm to construct distributed engrams for nonassociative conditioning in a model of the local bending reflex of the medicinal leech (Hirudo medicinalis). The model comprised 4 sensory neurons, 10 to 40 interneurons, 8 motor neurons, and up to 480 connections. Synaptic connections in the model were first optimized to reproduce the amplitude and time course of motor neuron synaptic potentials recorded during local bending. This network, which represented the naive state before conditioning, was then reoptimized to the habituated or sensitized state. Following reoptimization, the memory for nonassociative learning was encoded by small changes dispersed across the entire network, and each change made only a small contribution to the learning. Moreover, because the changes were small, resolution of a few tenths of a millivolt, or 3-5% of an average synaptic potential, would be needed to account for half of the nonassociative learning. These results show how difficult distributed engrams can be to detect and provide a likely lower bound on the detectability of nonassociative learning in this and related networks.

A comparison of the performance of two sensory systems in host detection and location in the medicinal leech Hirudo medicinalis

1. 1. Mechanical and visual stimuli associated with surface waves were separated into their two components. 2. 2. Unfed leeches responded to the mechanical component of artificially generated surface waves by swimming towards their source. 3. 3. Leeches also responded to and orientated towards the source of the waves using visual cues derived from the same surface waves. 4. 4. Visual stimuli elicited fewer and less accurate responses.

Studies on the host specificity of the medicinal blood leechHirudo medicinalis L.

For the identification of host species of blood-sucking parasites, the suitability of disc-electrophoresis of the stomach contents was tested. Mammalian blood in the stomach of the medicinal blood leech Hirudo medicinalis gave satisfactory results. In the case of mixed blood samples from H. medicinalis, the identification of the host according to the electrophoretic patterns of the stomach contents failed as compared with an immunological method such as the Ouchterlony test. Medicinal blood leeches (H. medicinalis) collected in Istria Croatia, or bought in a pharmacy contained blood from cattle, horses, or frogs in their stomachs. Specimens of H. medicinalis from Lake Neusiedl or from the Seewinkel Austria, had sucked blood from mallards or frogs. Blood of cattle, mallards, and frogs was found in the stomachs of H. medicinalis coming from the National Park Kiskunsag Hungary. For the first time, horses were established as hosts for free-living specimens of H. medicinalis. A comparison of the weights of H. medicinalis bought in a pharmacy revealed that specimens containing frog blood in their stomachs weighed significantly less than those containing horse blood. These results confirmed the reports from Ssynewa (1944) concerning the breeding experiments. Probably, there is a change in hosts from the frog to warm-blooded animals during the life cycle of H. medicinalis. There were also significant differences in the weights of leeches as revealed by a comparison of the population from the Neusiedlersee with the leeches bought in the pharmacy.

Molecular basis for the inhibition of thrombin by hirudin.

The amino acid sequence of the 65 residue major form of hirudin is shown in Figure 1. Hirudin was originally isolated from the salivary glands of the European medicinal leech Hirudo medicinalis by Markwardt1. The anticoagulant properties of leech saliva were, however, first described over 100 years ago by Haycraft2. The N-terminal region of the hirudin contains 6 cysteines that form three disulphide bridges: Cys6′-Cysl4′, Cys16′-Cys28′ and Cys22′-Cys39′. (Residues in hirudin are distinguished from those in thrombin by the use of primed numbers and the numbering of the sequence of thrombin is that of Bode et al.3 which is based on chymotrypsin numbering). The C-terminal region of hirudin is particularly rich in acidic residues (Figure 1) and the acidic nature of this region of hirudin is important for the formation of the thrombin-hirudin complex as will be discussed later. One of the charged residues found in this region of the natural hirudin is a sulphated tyrosine residue at position 63. This post-translational modification is not, however, found in recombinant molecules expressed in Escherichia coli or yeast. More than 20 different isoforms of hirudin have been isolated from H. medicinalis and sequenced4–6 and in each of these isoforms the position of the disulphide bridges and the acidic nature of the C-terminal region is conserved. The residues that are conserved between the different isoforms are indicated in Figure 1 and many of these residues are involved in the binding of hirudin to thrombin7–9.

Mesenchyme of embryonic reproductive ducts directs process outgrowth of Retzius neurons in the medicinal leech

In the two segments of the medicinal leech ( Hirudo medicinalis) that contain the male (segment 5) and the female (segment 6) reproductive ducts, the paired Retzius (Rz) neurons are distinguished by several unique properties. For example, the muscles and glands of the body wall are the primary peripheral targets of Rz neurons in standard segments [Rz(X)], whereas the muscles and glands of the reproductive ducts are the primary peripheral targets of Rz neurons in the two reproductive segments [Rz(5,6)]. In this paper, we show that organogenesis and differentiation, which generate an epithelial tube surrounded by mesenchymal cells, occur in the embryonic reproductive ducts at approximately the time when Rz processes first contact these structures. The growth cones leading one branch of the posterior axon of Rz(5,6) contact the duct mesenchymal cells. Following initiation of this contact, these posterior growth cones enlarge and send out numerous filopodia. Secondarily, growth cones leading the anterior axon of each Rz(5,6) also modify their shapes and trajectories. When embryonic reproductive ducts were transplanted into posterior (nonreproductive) segments, the branch of the posterior Rz axon near the ectopic reproductive tissue produced enlarged growth cones and extended several secondary branches into the mesenchyme of the ectopic tissue. This result suggests that the reproductive mesenchyme is attractive to, and can modify the growth of, all Rz neurons. The behavior of Rz(5,6) growth cones suggests that the reproductive mesenchyme cells provide guidance cues that control the location in which Rz axons elaborate their peripheral arborization and form synapses, and that the mesenchyme may also stimulate the production of a densely branched arbor.

Development of segment- and target-related neuronal identity in the medicinal leech.

The rhythmic pumping of the paired heart tubes in the medicinal leech Hirudo medicinalis offers an excellent system for studying the development of a simple behavior in terms of its neuronal and muscular components. The present experiments examined the development of identified heart excitor (HE) motor neurons during normal embryogenesis. Using intracellular impalements and dye-filling, we found that the HE motor neurons could be identified at an early stage of development and that they initially elaborated axonal arborizations in inappropriate target fields in the ventral body wall. These inappropriate projections were retracted as those at the appropriate target (developing heart tube muscle) extended. This remodelling occurred at least 4 days before the HEs acquired the adult phenotype of being driven to fire action potentials in a rhythmic pattern. Although the HEs exhibited centrally driven rhythmic oscillations late in embryogenesis, at earlier stages they exhibited largely a tonic discharge interrupted by bursts of inhibitory potentials in a periodic, but not a rhythmic, pattern. We also found what appeared to be non-rhythmic HE homologs in anterior and posterior segments where HE neurons have not been previously described. These homologs may project along similarly patterned guidance cues early in development, since they are at first indistinguishable from the definitive HEs, but they continued to elaborate both lateral and medial body wall projections over the same period that definitive HEs were expanding their arborizations over the developing heart tube and retracting their body wall projections. In both adult and embryonic leeches the homologs exhibited mostly tonic activity that was interrupted by pronounced, but non-rhythmic, hyperpolarizing postsynaptic potentials. Thus, there appears to be early segmental specification directing the final phenotype of the iterated neuron that, in most segments, becomes the HE motor neuron.

Protease inhibitors in the alimentary tract of the medicinal leech Hirudo medicinalis: in vivo and in vitro studies.

In individual leeches the flux of labeled serum through the digestive tract was monitored to measure the rate of digestion. A mean value of 10 mg of the original serum (or 2-3 mg of the contents of the foregut) per individual per day was found, which was constant during 10 weeks. On average the serum remained in the intestinum for 20 days. Occurrence and concentrations of eglin and bdellin, specific proteinase inhibitors of Hirudo, were analyzed after various periods following the ingestion of a meal. In the foregut they were present immediately after feeding. Their quantities increased several-fold within a few weeks. In the intestinum the tests for these inhibitors were always negative. The inhibition of the proteolytic activity of intestinum preparations by eglin, bdellin, and foregut extract was tested in vitro. Using azo-albumin as (an unspecific) substrate, inhibition by eglin was maximally 25% and by bdellin 60%. When the quantitative relations presumably representing in vivo conditions were applied, only a slight inhibition of proteolysis occurred. A hypothetical role of the inhibitors in the preservation of the blood stored in the foregut is discussed.

Old and New Natural Products as the Source of Modern Antithrombotic Drugs

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic ( ALLIUM SATIVUM) has been shown to inhibit platelet aggregation IN VIVO and IN VITRO; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech ( HIRUDO MEDICINALIS), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.

The structure of a natural population of the medicinal leech, Hirudo medicinalis, at Dungeness, Kent

SUMMARY 1. A hot branding method was used to mark medicinal leeches (Hirudo medicinalis L.) from five samples (four in 1984, one in 1985) at agravel pit at Dungeness in Kent. Analysis of capture-recapture data by the Fisher-Ford and Jolly methods gave similar results and indicated a population in excess of 10000 (0.112 leeches per m2) in 1984 and 1985. Successive estimates were each higher than the last suggesting a thriving community. 2. The population structure was skewed towards the lower weight classes. This may indicate good recruitment of hatchlings, as also indicated by the presence of cocoons, or the failure to capture heavier satiated or gravid leeches because of their relative unresponsiveness to the sampling method involving water disturbance, or their complete absence from the water during cocoon laying. Gravid leeches were always above 2 g in weight. Recaptured marked leeches revealed that they migrated throughout the site, indicating active foraging for hosts.

Development of serotonin-induced ion currents in identified embryonic Retzius cells from the medicinal leech (Hirudo medicinalis)

Retzius cells and the cutaneous baroreceptive P-cells of adult medicinal leeches (Hirudo medicinalis) respond to 5-HT by Cl- as well as by monovalent cation conductances (Drapeau et al., 1989). However, chemical synaptic connections between Retzius cells (Liu and Nicholls, 1989) as well as Retzius cells and P-cells in culture (Drapeau and Sanchez-Armass, 1988) are mediated by postsynaptic Cl- currents in response to 5-HT release from the presynaptic cell. It was the aim of the present study to find out whether the response, which has been shown to be involved in synaptic transmission or whether the cation current, which has so far only been observed upon extrasynaptic 5-HT application, dominates during embryogenesis. Currents induced by transmitter application with a fast perfusion system were measured with a single-electrode voltage clamp and patch pipettes in whole-cell configuration. Control experiments, performed on Retzius cells from adult leeches after 1 d in culture, showed Cl- currents in response to 5-HT application in all cells investigated. Ninety percent of the cells tested showed additional 5-HT-elicited Na+, K+, or both currents. However, the conductance of cation currents amounted only to 10% of the Cl- currents. Embryonic Retzius cells were identified in dissociated cultures by their violet-blue fluorescence acquired by preincubation of intact ganglia or embryos in culture medium containing the autofluorescent 5-HT analog 5,7-dihydroxytryptamine. The first responses to 5-HT were measured at embryonic day 10 (E10), after voltage-activated Ca2+ and Na+ currents had already developed. 5-HT application induced exclusively Cl- currents until E16. This suggests that the receptor-channel complex, which also mediates synaptic responses, is expressed first in measurable quantities. Hence, there is currently no indication that extrasynaptic receptors dominate in immature cells in this preparation.

Isolation of thrombin inhibitor from the leech Hirudinaria manillensis

The leech Hirudinaria manillensis belongs to the same family as the medicinal leech Hirudo medicinalis, which has been widely used for the study of hirudin, a specific thrombin inhibitor. A similar inhibitor has now been isolated from the heads of the Hirudinaria leech by acetone/acid extraction and further purified to near homogeneity by ion exchange chromatography followed by affinity chromatography on thrombin-agarose and reverse phase HPLC. The purified material was recovered at about 10-15% yield and had a specific activity of about 12,000-14,000 ATU/mg, similar to other hirudin variants. The inhibitor was shown to be homogenous by sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of 8 M urea with an apparent molecular mass of about 7000 daltons under reducing conditions. Comparison of the anticoagulant effect on human plasma by partial thromboplastin time assay have shown that the inhibitor from Hirudinaria has similar potency as hirudin variant 1 at equivalent dosage. However, it does not cross-react with monoclonal antibodies towards recombinant hirudin variant 1. Comparison of the N-terminal amino acid sequence up to residue 25 also indicates differences at positions 2, 13, 17 and 24 between the two thrombin inhibitors. These findings indicate that the primary anticoagulant present in the leech Hirudinaria is a potent thrombin inhibitor (Bufrudin) with biological activity similar to hirudin, but differs in its structural and immunological properties.

Two forms of sensitization of the local bending reflex of the medicinal leech

Sensitization of the local bending reflex of the medicinal leech Hirudo medicinalis was studied in a semi-intact preparation in which behavioral and electrophysiological recordings were made simultaneously. 1. Sensitization of local bending could be produced in two ways: by repeated stimulation of the mechanoreceptor sensitive to pressure (the P cell), and by stimulation of the mechanoreceptor sensitive to noxious stimuli (the N cell). 2. Both forms of sensitization produced a central neuronal change, measured as an increase in the number of stimulus-evoked action potentials in cell 3 (an excitor of dorsal longitudinal muscles). 3. Intracellular stimulation of serotonin-containing neurons 21 and 61 mimicked the sensitizing stimuli, but stimulation of the Retzius cell, which also contains serotonin, did not. 4. Stimulation of the Leydig cell, which releases octopamine, decreased the strength of local bending.

Secretion of hementin and other antihaemostatic factors in the salivary gland complex of the giant amazon leech Haementeria ghilianii

Biological observations were made on the antihaemostatic activity in the saliva of the giant Amazon leech Haementeria ghilianii. Particular emphasis was placed on determining where the fibrino(geno)lytic enzyme hementin is produced and secreted in the salivary gland complex. Hungry third-fed Amazon leeches produce about 650 units of hementin, with by far the majority of the activity (75%) in the two posterior glands. The two anterior glands contain much less hementin (12%), and a small amount (12%) resides in the proboscis itself, presumably in the salivary gland ductules. In the anterior gland, hementin appears to be produced by only certain cells. The secretion of hementin is confined to the lumen of the proboscis. Secretions in the proboscis lumen are rich in antihaemostatic activity, as evidenced by fibrinogenolysis (hementin), prolongation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin clotting time (TCT) and Atroxin clotting time, and inhibition of collagen-induced platelet aggregation. In contrast, no anti-haemostatic, including hementin, activity was detectable at the tip. Suprisingly, the wound from the bite by the Amazon leech is not associated with prolonged bleeding. This is in marked contrast to the wound by the European medicinal leech Hirudo medicinalis which bleeds for an average of ten hours. The absence of bleeding is compatible with the interpretation, based on the above findings, that during feeding the Amazon leech does not inject antihaemostatic factors into the host, or at least not in the vicinity of the wound.

In vivo investigations on the metabolism of amino acids in the medicinal leech Hirudo medicinalis

1. 1. Leeches were fed test solutions which contained 14C-labelled alanine, aspartate or glutamate and, as a reference base, 3H-inulin to analyse the absorption and subsequent metabolic transformation of these amino acids. 2. 2. Soon after the ingestion a pronounced shift of the 3H/ 14C ratio in the contents of the fore-gut occurred which indicated the absorption. The uptake of alanine proceeded particularly rapidly, while that of glutamate was comparatively slow. 3. 3. A large proportion of the ingested amino acids was degraded to CO 2 within a few days. 4. 4. In all experiments the distribution of the label exhibited a characteristic pattern: very high radioactivity in malate, much less in succinate, fumarate, alanine, aspartate and glutamate. In addition a certain proportion of the label glycogen regularly appeared in glycogen, whereas virtually none was found in lipids.

Mechanism of potassium uptake in neuropile glial cells in the central nervous system of the leech

1. Ion-selective double-barreled microelectrodes (ISME) were used to measure intracellular K+ (aKi), Na+ (aNai), and Cl- (aCli) activities of neuropile glial (NG) cells in the central nervous system of the medicinal leech Hirudo medicinalis. Ion fluxes were induced by an increase in extracellular K+ concentration [( K+]o) and analyzed to elucidate the ionic mechanism of the K+ uptake occurring under such conditions. 2. In addition, the K+ concentration of the extracellular space of the nerve cell body region (NCBR) and the neuropile (N) was measured with neutral carrier K(+)-ISME. In normal saline (4 mM K+), a concentration of 4.2 mM was measured in both extracellular spaces. No differences between the K+ concentration of the bathing fluid and the extracellular spaces were found at higher (i.e., 10 and 40 mM) K+ concentrations. 3. In normal saline, the mean membrane potential (Em) was -68 mV, and the mean aKi, aNai, and aCli were found to be 77, 10, and 7 mM, respectively. The corresponding equilibrium potentials were -81, 56, and -66 mV. The chloride equilibrium potential (ECl) was similar to Em, and it is concluded that chloride is passively distributed across the NG cell membrane. 4. When [K+]o was transiently increased 10-fold (i.e., to 40 mM), aKi and a Cli increased transiently by 22 and 25 mM, respectively, and the membrane depolarized to -28 mV, which was similar to both K+ equilibrium potential (EK) and ECl. The KCl uptake was accompanied by a transient decrease in aNai to 5 mM. 5. After incubation for at least 1 h in Na(+)-free saline, NG cells accumulated K+ in the absence of extracellular Na+ to levels similar to those observed in the presence of Na+. Therefore the uptake of K+ was not dependent on external--and probably also internal--Na+. 6. Changes in cell volume induced by the increase in [K+]o were estimated by loading NG cells with choline and monitoring its intracellular concentration with Corning-K(+)-ISME. In saline containing 40 mM K+, NG cell volume increased to approximately 150% of its volume in normal saline. 7. It is concluded that the mechanism of K+ uptake in NG cells is by passive KCl and water influx, which causes cell swelling.

The medicinal leech, Hirudo medicinalis (L.) (Hirudinea: Gnathobdellae), at Dungeness, Kent

WILKIN, P. J., 1989. The medicinal leech, Hirudo medicinalis (L.) (Hirudinea: Gnathobdellae), at Dungeness, Kent. An account of the ecology and conservation of the medicinal leech (Hirudo medicinalis (L.)) at Dungeness, Kent, is presented.

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis.

The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCPB. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCPA and SCPB.

Controlling the medicinal leech

A simple method is presented for applying a medicinal leech ( Hirudo medicinalis) to the precise area in which the clinician desires the leeching to take place.

Leeches: Objective Monitoring of Altered Perfusion in Congested Flaps

In plastic and reconstructive surgery, there has been new interest in the use of the medicinal leech (Hirudo medicinalis) for the treatment of venous congestion. To the best of our knowledge, no study has been performed to objectively demonstrate the benefits of leeching a congested flap. Doppler laser perfusion monitoring (DLPM) was used to record blood flow changes in pig rump flaps compromised by venous occlusion. With the application of leeches to nine congested pig rump flaps, significant increases in blood flow were demonstrated both clinically and by DLPM. It is concluded from this study that DLPM can demonstrate objective improvement in microcirculatory perfusion with leeching.