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PubMed2004

Confocal and video imaging of cytoskeleton dynamics in the leech zygote.

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.

Actins↗
PubMed1995

Leech therapy in digital replantation.

This article presents a protocol for the perioperative care of patients undergoing digital replantation, which is the most common microsurgical procedure performed today. Venous congestion, a common complication of digital replantation, often has been treated through surgical exploration and creation of arteriovenous anastomosis. Leech therapy, however, is experiencing a resurgence among surgeons as an alternative method for treating venous congestion. This article discusses the anatomical, physiological, and clinical indications and methods of leech therapy in digital replantation.

Amputation, Traumatic↗
PubMed2001

The ouabain-induced [Ca2+]i increase in leech Retzius neurones is mediated by voltage-dependent Ca2+ channels.

In leech Retzius neurones the inhibition of the Na+/K+ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca2+]i). To elucidate the mechanism of this increase we investigated the changes in [Ca2+]i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na+/K+ pump in bathing solutions of different ionic composition. The results show that Na+/K+ pump inhibition induced a [Ca2+]i increase only if the cells depolarized sufficiently in the presence of extracellular Ca2+. Specifically, the relationship between [Ca2+]i and the membrane potential upon Na+/K+ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca2+ channels by raising the extracellular K+ concentration. It is concluded that the [Ca2+]i increase caused by inhibiting the Na+/K+ pump in leech Retzius neurones is exclusively due to Ca2+ influx through voltage-dependent Ca2+ channels.

Animals↗
PubMed1997

Voltage-dependent Ca2+ influx into identified leech neurones.

We determined the relationships between the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of six different neurones in the leech central nervous system: Retzius, 50 (Leydig), AP, AE, P, and N neurones. The [Ca2+]i was monitored by using iontophoretically injected fura-2. The membrane depolarization evoked by raising the extracellular K+ concentration ([K+]o) up to 89 mM caused a persistent increase in [Ca2+]i, which was abolished in Ca(2+)-free solution indicating that it was due to Ca2+ influx. The threshold membrane potential that must be reached in the different types of neurones to induce a [Ca2+]i increase ranged between -40 and -25 mV. The different threshold potentials as well as differences in the relationships between [Ca2+]i and EM were partly due to the cell-specific generation of action potentials. In Na(+)-free solution, the action potentials were suppressed and the [Ca2+]i/Em relationships were similar. The K(+)-induced [Ca2+]i increase was inhibited by the polyvalent cations Co2+, Ni2+, Mn2+, Cd2+, and La3+, as well as by the cyclic alcohol menthol. Neither the polyvalent cations nor menthol had a significant effect on the K(+)-induced membrane depolarization. Our results suggest that different leech neurones possess voltage-dependent Ca2+ channels with similar properties.

Action Potentials↗
PubMed1997

Identification and characterization of the leech CNS cannabinoid receptor: coupling to nitric oxide release.

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in leech (Theromyzon tessulatum and Hirudo medicinalis) central nervous system. The anandamide binding site is monophasic and of high affinity exhibiting a Kd of approximately 32 nM with a Bmax of 550 fmol/mg protein in both animals. These sites are highly select as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release. A deduced amino acid sequence (153 residues) analysis from a 480 pb amplified RT-PCR fragment cDNA exhibits a 49.3% and 47.2% sequence identity with human and rat cannabinoid receptors (CB1R), respectively. Thus, the leech cannabinoid receptor may be a G-protein coupled receptor with seven transmembrane domains as in CB1R. Moreover, this sequence exhibits highly conserved regions, particularly in the putative transmembrane domains 1 and 2. The presence of a cannabinoid receptor in these organisms indicates that this signaling system has been conserved during evolution.

Amino Acid Sequence↗
PubMed1998

ATP-inhibited K+ channels and membrane potential of identified leech neurons.

The effect of the ATP-inhibited K+ channel on the membrane potential of leech Retzius neurons was analyzed using electrolyte-filled single-barrelled microelectrodes. The membrane potential was independent of the external nutrient supply during a period of 11 h, probably because the internal energy reserves were sufficient. The K+ channel activator HOE 234 ((3S,4R)-3-hydroxy-2, 2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6-phenylsulfonylchromane hemihydrate, 500 microM) induced a membrane hyperpolarization. In the presence of HOE 234, action potentials occurred with a reduced after-hyperpolarization and were discharged in bursts, possibly because of an inhibition of Ca2+ channels. The blocker of ATP-inhibited K+ channels tolbutamide did not significantly alter the membrane potential. In the absence of tolbutamide, the metabolic inhibitors iodoacetate, azide and cyanide (10 mM) evoked membrane hyperpolarizations, but in the presence of 1 mM tolbutamide their hyperpolarizing actions were reduced or abolished while membrane depolarizations were intensified. We conclude that ATP-inhibited K+ channels in the soma membrane of leech Retzius neurons provide coupling of cellular metabolism to electrical activity and ionic fluxes.

ATP-Binding Cassette Transporters↗
PubMed1989

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.

Animals↗
PubMed1990

Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii.

The fibrinogenolytic enzyme hementin, present in extracts of the posterior salivary glands of the giant leech Haementeria ghilianii, was isolated by ultrafiltration, high-performance ion-exchange chromatography and subsequent reversed-phase liquid chromatography. Approximately 100 micrograms (1 nmol) of hementin, present at less than 0.5% in the crude leech salivary extract, was brought to about 90% purity in three steps. Hementin migrated at an Mr of about 73,000 on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and at 82,000 on reducing SDS-PAGE. The amino terminal sequence was determined to be TTLTE-PEPDL. The amino terminal sequences of two inactive proteins that partially coeluted with hementin in the first chromatographic step were also determined.

Amino Acid Sequence↗
PubMed2003

Ca(2+)-dependent and Ca2+ release-dependent excitation in leech photoreceptors: evidence from a novel "inside-out" cell model.

We have developed a novel, electrophysiologically intact and light-sensitive "inside-out" cell model (IOCM) of microvillar photoreceptors of the leech Hirudo medicinalis. Light responses recorded from the IOCM with sharp microelectrodes are depolarizations with amplitudes of up to 50-60 mV. In darkness, graded elevations of the free Ca(2+) concentration in the "intracellular medium" (ICM) reversibly increase the conductance of the microvillar membrane leading to Ca(2+)-induced graded voltage changes up to approximately 50 mV. The threshold for Ca(2+)-induced voltage changes is approximately 0.06 microM, EC(50) is approximately 1.2 microM, and saturation occurs at approximately 20 microM free Ca(2+). Small Ca(2+) elevations (<0.6 microM) produce discrete waves of depolarization resembling quantum bumps. Stimulating IOCMs with short (20-ms) and long (5-s) light stimuli produces transient light responses (repolarization within ca. 200 ms) in an ICM containing only 10nM free Ca(2+). At 0.44 microM free Ca(2+) in the ICM, the microvillar membrane depolarizes by 10-20 mV and responses to 5-s light steps have an initial transient component and a plateau component, similar to responses in intact cells. Generation of the plateau component in IOCMs is suppressed by heparin and cyclopiazonic acid (CPA), agents that block inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3))-induced Ca(2+) release from and Ca(2+) uptake into the endoplasmic reticulum (ER). These results indicate that there is a Ca(2+)-dependent conductance in the microvillar membrane and that the light-induced Ins(1,4,5)P(3)- and Ca(2+) release-mediated intracellular Ca(2+) elevation in leech photoreceptors contributes to the generation of the receptor potential, particularly the plateau component of responses to long steps of light.

Action Potentials↗
PubMed1997

Caffeine mediates cation influx and intracellular Ca2+ release in leech P neurones.

We investigated the effect of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) of leech P neurones by using the fluorescent indicator Fura-2. Caffeine induced a [Ca2+]i increase that was strongly reduced, but not abolished, in Ca(2+)-free solution. The effect of caffeine on [Ca2+]i was dose-dependent: while 5 mM caffeine evoked a persistent [Ca2+]i increase that could be elicited repetitively, 10 mM caffeine or more induced a transient [Ca2+]i increase that was strongly reduced upon subsequent applications at the same concentration. Surprisingly, the cells remained fully responsive to a moderately increased caffeine concentration. The caffeine-induced [Ca2+]i increase was not blocked by millimolar concentrations of La3+, Mg2+, Cd2+, Zn2+, Co2+, Ni2+, or Mn2+. While La3+ and Mg2+ had no effect on the caffeine response, the other cations caused irreversible changes in the Fura-2 fluorescence. The inhibitors of intracellular Ca2+ pumps-thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ)--had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, but they reduced it in Ca(2+)-free solution. Ryanodine had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, and also in Ca(2+)-free solution it seemed to be largely ineffective. Caffeine evoked complete fluctuations of the membrane potential. The effect in Ca2+ free and in Na(+)-free solution suggests that the depolarizing response components were mainly due to Na+ influx, while Ca2+ reduced the Na+ influx and/or activated mechanisms which re- or hyperpolarize the cells. It is concluded that leech P neurones possess caffeine-sensitive intracellular Ca2+ stores, as well as caffeine-sensitive ion channels, in the plasma membrane that are activated by a voltage-independent mechanism. The plasma membrane channels are permeable to various divalent cations including Ca2+, and possibly also to Na+.

Animals↗
PubMed1988

Discriminative conditioning alters food preferences in the leech, Haemopis marmorata.

The feeding behavior of the carnivorous leech, Haemopis marmorata, was aversively trained in a discriminative classical conditioning task. Two conditioned stimuli were used: One consisted of a food (chicken or liver) paired with an unconditioned stimulus of quinidine (bitter chemical); the other consisted of the alternate food presented in an unpaired relationship with the quinidine. Training consisted of alternating exposures to the two conditioned stimuli. Testing consisted of the simultaneous presentation of a conditioned stimulus food and a neutral food, beef. The percentages of responding to the conditioned stimuli were tabulated. Haemopis could discriminate between the conditioned stimuli. As a result of pairing a food with quinidine, the leeches selectively reduced their preference for that paired food, while they did not alter their preference for the unpaired food.

Animals↗
PubMed2001

Cloning and expression analysis of a cDNA that encodes a leech hemerythrin.

We report the cDNA sequence of a leech hemerythrin. A cDNA was isolated from a Theromyzon tessulatum cDNA library and encodes a 120 amino acid protein of about 14 kDa. The predicted protein contains the hemerythrin signature sequence and the iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin. The protein displayed the highest identity to myohemerythrin, a non-heme iron-binding protein described in sipunculids. Expression analysis indicated that the mRNA is widely expressed in leech and is stage specific in appearance, being absent after the two first blood meals, appearing after the last blood meal during the period preceding oogenesis and disappearing after egg laying.

Amino Acid Sequence↗
PubMed2000

Destabilase from the medicinal leech is a representative of a novel family of lysozymes.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

Amino Acid Sequence↗
PubMed1997

Intracellular Ca2+, Na+ and H+ transients evoked by kainate in the leech giant glial cells in situ.

The membrane responses to the glutamate receptor agonist kainate and the subsequent changes in intracellular Ca2+, H+ and Na+ concentration were measured in giant glial cells of the leech central nervous system using ion-selective microelectrodes and microfluorimetry of Fura-2. The membrane depolarization or membrane inward current of exposed neuropile glial cells in situ, evoked by 2-20 microM kainate, were reversibly blocked by 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX), (50-100 microM) and by Ni2+ (2 mM), but not by methoxyverapamil (D600, 500 microM), which blocked voltage-gated Ca2+ influx. Local iontophoretic application of kainate on to the somatic membrane of single neuropile glial cells in situ, resulted in CNQX-sensitive depolarization and rises in intraglial Ca2+ concentration similar to those observed with bath-application of the agonist, indicating the presence of non-N-methyl-D-aspartate-type (NMDA) glutamate receptors in the somatic membrane of these cells. In voltage-clamped glial cells bath-application of kainate (5-10 microM) evoked inward currents and an increase in the membrane conductance,. while the intracellular Ca2+ increased (up to 200 nM). This increase in Ca2+i was not affected by substitution of Na+ by Li+, indicating that it is not due to reversed Na+/Ca2 exchange following intracellular Na+ accumulation. The intracellular Na+ concentration increased (up to 40 mM), and the intracellular pH decreased (0.2-0.3 pH units) in voltage-clamped glial cells following bath application of kainate. All these changes of the concentration of intracellular cations were reversibly suppressed by CNQX and Ni2+. The results indicate that Ca2+, Na+ and H+ enter leech neuropile glial cells presumably through non-selective cation channels, activated by the non-NMDA glutamate receptor agonist kainate.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Biochemical identification and ganglionic localization of leech angiotensin-converting enzymes.

We demonstrate the presence of a membrane and soluble form of leech Theromyzon tessulatum angiotensin-converting enzyme (ACE). Four steps in the purification of this enzyme include gel-permeation, captopril-sepharose affinity and anion-exchange chromatography followed by a reverse-phase HPLC. The peptidyl dipeptidases (of approximately 120 and approximately 100 kDa) are glycosylated enzymes hydrolysing the Phe8-His9 bond of angiotensin I, exhibiting the same specific activity and Km whereas the soluble ACE exhibits a higher catalytic efficiency. This hydrolysis is inhibited by the ACE-specific antagonist captopril. Western blot analysis of a polyclonal antiserum raised against the first 11 amino-acid residues of the membrane ACE and the N-terminal sequence of the soluble molecule also demonstrates the presence of two ACE enzymes. Anti-ACE immunocytochemistry also supports the presence of two forms of ACE. This material is found in neurons and glia. We demonstrate for the first time the cellular localization and biochemical characterization of ACEs in the central nervous system of an invertebrate. Thus, the leech brain may represent a simple model for the study of these enzymes.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2002

Membrane responses of the leech giant glial cell to the peptide transmitter myomodulin.

A myomodulin peptide has been suggested to mediate the response of the giant glial cells to stimulation of the Leydig interneuron in the central nervous system of the leech Hirudo medicinalis [Eur. J. Neurosci. 11 (1999) 3125]. We have now studied the glial response to the endogenous leech MM peptide (GMGALRL-NH(2), MMHir). The peptide evokes a membrane outward current (EC(50) approximately 2 microM), which neither desensitizes nor shows any sign of run-down, and elicits a K(+) conductance increase of the glial cell membrane. The peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) enhances the glial current response, suggesting the presence of endogenous extracellular peptidases.

Animals↗
PubMed2001

Identified neurons and leech swimming behavior.

Since the experiments of Nicholls and Baylor, the initial characterization of identified neurons has provided significant insight into the circuitry transforming mechanosensory input into the motor output of swimming. From physiological characterization of only a small percentage of cells within the leech CNS, we have gained important information about how the decision to swim is processed and how the rhythmic motor pattern is generated. While many of the synaptic connections in the swim-generating circuit have been identified, the elucidation of the biophysical and biochemical mechanisms underlying these connections has only recently begun. The observation that constant input can result in variable motor output suggests that, in addition to describing a cell's identity in terms of structure and function, factors such as behavioral context and the "internal state" of the nervous system must also be considered. As circuits controlling other behaviors become known, one can examine the interactions between these networks to understand issues of behavioral choice at the level of identified neurons. The leech CNS has expanded our understanding of how the nervous system produces behavior and continues to serve as an excellent model in this endeavor.

Animals↗
PubMed1999

Serotonin induces four pharmacologically separable contractile responses in the pharynx of the leech Hirudo medicinalis.

Stimulation of the serotoninergic innervation of the leech pharynx or application of serotonin to the isolated pharynx induced four distinct types of contractile activity: an increase in basal tonus, large phasic contractions of 10-15 s in duration, smaller phasic contractions occurring at approximately 1 Hz, and a relaxation after washout of serotonin. Application to the isolated pharynx of the selective serotonin agonists (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin, N-(3-trifluoromethylphenyl)piperazine, 1-(m-chlorophenyl)-piperazine, (+/-)-2,5-dimethoxy-4-iodoamphetamine, 2-methyl-5-hydroxytrypamine, alpha-methyl-5-hydroxytryptamine, and 5-methoxytryptamine induced distinct types of pharyngeal contractile activity. The results of this study suggest that the leech pharynx possesses more than one type of serotonin receptor.

5-Methoxytryptamine↗