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PubMed2006

Effects of intracellular pH and Ca2+ on the activity of stretch-sensitive cation channels in leech neurons.

The effects of intracellular pH and calcium on the activity of the leech mechanosensitive cation channels have been studied. These channels exhibited two activity modes denoted as spike-like (SL) and multiconductance (MC). In the absence of mechanical stimulation, acidification of the intracellular side of membrane patches from 7.2 to 6.2 reversibly increased the mean channel open time as well as the opening frequency in the SL mode. Channels in MC mode were activated by a pH(i) reduction from 7.2 to 6.2, but were inhibited at pH(i) 5.5. Unlike MC mode, SL mode was strongly activated by intracellular Ca(2+). Fura-2 imaging experiments showed that intracellular calcium was induced to increase by hypotonic cell swelling. The major component of this response did not require extracellular calcium. A component of the swelling-induced calcium response was sensitive to blockers of stretch-sensitive cation channels. The results indicate that the two activity modes of mechanosensitive channels of leech neurons respond differently to changes of intracellular pH and calcium. The sensitivity of the channel to micromolar concentrations of internal free calcium, along with its permeability to this ion, is consistent with a role in the amplification of mechanically induced Ca(2+) signals in leech neurons.

Action Potentials↗
PubMed1998

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system.

Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity.

Animals↗
PubMed1999

The production and elimination of supernumerary blast cells in the leech embryo.

Different species of leech vary greatly in body size but all have 32 body segments. It is unclear how the development of this precise number of segments is regulated, although it is known that the teloblasts of the early leech embryo initially produce more than the required numbers of segment founder cells (blast cells). We used fluorescent dextrans to show that the M teloblast of the Helobdella robusta embryo produces a variable number of additional (supernumerary) cells. These cells fail to enter the germinal band (which contains cells of all lineages and gives rise to the adult leech), but detach from its posterior end and disappear. Our observations suggest that some suffer an increase in membrane permeability while others fuse with the M teloblasts, but that they do not undergo apoptosis. The supernumerary cells of different lineages detach from the germinal band at different times, suggesting that detachment is not triggered by a global signal acting simultaneously on all lineages. We tested the hypothesis that the elimination of the supernumerary m blast cells results from a requirement of m blast cells for close interactions with cells of the other lineages for their survival, a condition that would not be achieved by the last-born m blast cells that fail to enter the germinal band. We cultured isolated M teloblasts and found that they do produce blast cells that themselves divide, indicating that cells of the M lineage can survive in the absence of any interactions with cells of the other lineages.

Animals↗
PubMed2000

The leech hunchback protein is expressed in the epithelium and CNS but not in the segmental precursor lineages.

We are interested in identifying the regulatory genes involved in segmental pattern formation in annelids. The Drosophila segmentation gene hunchback (hb) is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. Here, the protein expression pattern of Leech Zinc Finger II (LZF2), a leech orthologue of hb is characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the Caenhorhabditis elegans hb homologue hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation.

Animals↗
PubMed2004

Genetic differentiation between two species of the medicinal leech, Hirudo medicinalis and the neglected H. verbana, based on random-amplified polymorphic DNA.

The medicinal leech is one of the few parasitic invertebrates widely used in medicine and as a scientific model object. Because of a dramatic decline in its natural populations, it is subject to considerable conservation effort. Despite all attention, there is confusion regarding the taxonomic status of different morphological forms. The prevailing view is that all varieties of medicinal leech in Europe represent the same species, Hirudo medicinalis. However, the present study based on RAPD molecular markers demonstrates that a second European taxon, H. verbana, forms a distinct species. Phenetic clustering and principal coordinate analysis of eight populations revealed the same basic structure, reflecting taxonomic rather than geographic subdivision. Variation between species explained 60% of the total molecular variance (phiCT=0.60, P<0.001). Both taxa displayed a significant number of specific RAPD markers. Conversely, no specific fragment supporting the geographic association of both taxa was found. Since the stronghold of commercially exploited medicinal leech populations in southeastern Europe and Turkey belongs to H. verbana, most medicinal and scientific applications probably use this species, not H. medicinalis. Appropriate taxonomic correction of international conservation conventions and legislation is essential.

Animals↗
PubMed2006

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum.

The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp-Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1-->6 is homologous to antistasin, a leech anticoagulant, and Cys7-->12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.

Animals↗
PubMed2002

Evidence against the presence of NMDA receptors at a central glutamatergic synapse in leeches.

The N-methyl- D-aspartate (NMDA) receptor, able to detect the coincidence of pre- and postsynaptic events, is considered to be the molecular analogue of associative learning. Associative learning is well known in leeches, particularly for reflexive shortening. The neuronal circuits underlying shortening have been documented and include neurons that release glutamate. Is this type of learning in leeches also mediated by NMDA receptors? The synapse between the P sensory neuron and the motoneuron-like AP cell was examined and: (1) NMDA failed to elicit a response in the AP cell, (2) the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid affected synaptic transmission only at high, non-specific levels, and (3) the antagonist for the glycine-binding site 7-chloro-kynurenic acid at 20 microM did not inhibit transmission. Therefore, there are evidently no NMDA receptors at the P to AP synapse, suggesting other mechanisms of associative learning in leeches.

2-Amino-5-phosphonovalerate↗
PubMed2005

Repair and regeneration of functional synaptic connections: cellular and molecular interactions in the leech.

A major problem for neuroscience has been to find a means to achieve reliable regeneration of synaptic connections following injury to the adult CNS. This problem has been solved by the leech, where identified neurons reconnect precisely with their usual targets following axotomy, re-establishing in the adult the connections formed during embryonic development. It cannot be assumed that once axons regenerate specific synapses, function will be restored. Recent work on the leech has shown following regeneration of the synapse between S-interneurons, which are required for sensitization of reflexive shortening, a form of non-associative learning, the capacity for sensitization is delayed. The steps in repair of synaptic connections in the leech are reviewed, with the aim of understanding general mechanisms that promote successful repair. New results are presented regarding the signals that regulate microglial migration to lesions, a first step in the repair process. In particular, microglia up to 900 microm from the lesion respond within minutes by moving rapidly toward the injury, controlled in part by nitric oxide (NO), which is generated immediately at the lesion and acts via a soluble guanylate cyclase (sGC). The cGMP produced remains elevated for hours after injury. The relationship of microglial migration to axon outgrowth is discussed.

Animals↗
PubMed1983

Microglial movement to sites of nerve lesion in the leech CNS.

The small glial cells in the central nervous system of the leech, Hirudo medicinalis, have been studied using two histological stains. Weak silver carbonate, a classic stain for vertebrate microglia, can selectively stain these small glial cells and shows that they are morphologically similar to vertebrate microglia. Feulgen's DNA-specific stain is useful for counting the compact and distinctive microglial nuclei. In uninjured connectives, which link segmental ganglia, there are 134 +/- 28 microglia per 210 micron of connective length. Within 24 h after the nerve cord is crushed leech microglia aggregate at the site of injury. This increase in cells, seen both in vivo and in culture, is approximately 5-fold. Although cells do not continue to accumulate at the injury site after the first day, their numbers continue to vary with time in the regions immediately adjacent to the crush for at least one week. A second crush made 24 h after the first shows that leech microglia are capable of responding to repeated injury.

Animals↗
PubMed1984

Intersegmental coordination of leech swimming: comparison of in situ and isolated nerve cord activity with body wall movement.

Recordings of motoneuron activity during swimming, obtained from leech ventral nerve cords in situ, were compared with films of swimming leeches and with recordings of motoneuron activity from isolated nerve cords. It was found that the intersegmental phase lag in body movement is greater than the phase lag of in situ neuronal activity, which is in turn greater than the phase lag of isolated nerve cord activity. We conclude that peripheral neuronal or mechanical effects, as well as sensory feedback to the central pattern generator, contribute to the movement pattern of the intact swimming leech.

Animals↗
PubMed1992

Effects of serotonin and carbachol on glial and neuronal rubidium uptake in leech CNS.

Effects of serotonin (5-HT) and carbachol on Rb uptake (used as a K marker) in leech neuron and glia were studied by electron probe microanalysis (EPMA). Hirudo medicinalis ganglia were perfused 60 s in 4 mM Rb substituted normal leech Ringer's with and without 5-HT (dosage range 5-500 microM) or carbachol (range 10-1000 microM), quench frozen cryosectioned, and subjected to EPMA to determine elemental mass fractions and cell water content. Both 5-HT and carbachol altered leech neuron and glial cell elemental distribution and water content. In glial cells, a dose-dependent increase in Rb uptake was observed following 5-HT (control: 26 +/- 2 microM; 5 microM: 47 +/- 4; 50 microM: 62 +/- 4; 500 microM: 82 +/- 11 mmol/kg dry wt. +/- S.E.M.) and carbachol (10 microM: 35 +/- 3; 100 microM: 52 +/- 3; 1000 microM: 68 +/- 3 mmol/kg dry wt. +/- S.E.M.). In neurons, 5-HT and carbachol had small effects. 5-HT decreased glial and neuronal cell water content. Carbachol decreased neuronal (but not glial) water content by approximately the same amount (mean decrease 9%) regardless of dose. Both 5-HT and carbachol affected glial cell K-accumulating properties, providing evidence that certain neurotransmitters may modulate invertebrate glial cells' K clearance function.

Animals↗
PubMed1996

Putative nitric oxide synthase (NOS)-containing cells in the central nervous system of the leech, Hirudo medicinalis: NADPH-diaphorase histochemistry.

The presence and distribution of putative nitric oxide synthase (NOS)-containing cells in whole-mount preparations of the central nervous system of the leech, Hirudo medicinalis, were studied using NADPH-diaphorase (NADPH-d) histochemistry. Specific staining occurred mainly in somata of some central neurones but NADPH-d-reactive branches and terminals were found in peripheral nerves and connectives: neuropile areas were stained weakly or unstained. Intense staining was located in many neurones on the ventral side of the segmental ganglia, including primary sensory neurones, motoneurones and interneurones, and in the anterior root ganglion. The sex ganglia contained some extra NADPH-d-positive cells. Head and tail ganglia and the dorsal side of the segmental ganglia showed less staining. Specific activity was not detected in salivary glands, crop or intestine. Controls using beta-NADPH or nitro blue tetrazolium (NBT) alone or with NBT plus alpha-NADPH, beta-NAD+, beta-NADH or beta-NADP+ did not induce specific staining. A potential NOS inhibitor, 2,6-dichlorophenol-indophenol (DPiP) at 10(-3) M, totally abolished NADPH-d-positive staining. Long-term fixation did not change the pattern of distribution of NADPH-d-positive cells. We conclude that (i) fixative-resistant NADPH-diaphorase is a characteristic marker of 12-15% of neurones in the leech CNS, and (ii) the specific distribution of the putative NOS-containing neurones suggests that NO may be a natural signal molecule in leeches.

Animals↗
PubMed1983

The medicinal leech and its use in plastic surgery: a possible cause for infection.

Recent interest in the use of the medicinal leech (Hirudo medicinalis) in microsurgical practice prompted us to undertake a bacteriological study. Aeromonas hydrophila was consistently cultured from 9 leeches that we examined. This organism is pathogenic to man and we consider that there is a significant risk of infection, particularly in those procedures where the vascularity of the tissues is compromised. There are several pharmacological preparations which have similar properties to the secretion of the medicinal leech and we submit that these non-infective preparations may be worthy of investigation.

Aeromonas↗
PubMed1987

Differentiation of the O and P cell lines in the embryo of the leech. II. Genealogical relationship of descendant pattern elements in alternative developmental pathways.

The p blast cells are a group of embryonic precursors found in the ectodermal cell layer of the leech germinal band. Each p blast cell normally undergoes the same invariant sequence of cell divisions and gives rise to a precisely defined set of uniquely identifiable neuronal and epidermal descendants in the mature leech. In the present paper, various of the p blast cell progeny were injected with a fluorescent lineage tracer in order to characterize the cellular composition of their descendant clones, and the results show that there is a stereotyped segregation of descendant cell fates through the first three p blast cell divisions. Previous work has shown that neurons and epidermal specializations which normally descend from the p blast cell will arise from a different precursor--the o blast cell--in response to ablation of the neighboring P cell line and that if the o blast cell is at a certain stage of differentiation when the ablation is performed it will produce only a subset of the normal P descendants. Comparison with the present findings indicates that under those conditions the o blast cell clone is not simply recapitulating a branch of the normal p blast cell lineage, but rather manifests an alternative lineage in which P descendants exhibit an abnormal genealogical relationship. Thus, even though normal leech development comprises a nearly invariant cell lineage, lineage relationships are open to considerable reorganization under experimental conditions.

Animals↗
PubMed1996

Metabolism of angiotensins by head membranes of the leech Theromyzon tessulatum.

Angiotensins (angiotensin I, angiotensin II, angiotensin II-amide) have been isolated in leeches and such peptides are involved in diuresis in these animals. To explore possible inactivation mechanisms of these peptides, angiotensins were incubated with head membranes of the leech T. tessulatum. Membranes derived from head parts of this leech are very rich in peptidases. They contain endopeptidase-24.11-like enzyme (NEP-like) associated with a battery of exopeptidase. The way that angiotensins are degraded by the combined attack of these membrane peptidases has been investigated. The contribution of individual peptidases was assessed by adding inhibitors (phosphoramidon, captopril and amastatin) to the membrane fractions, when they were incubated with the peptides. In the case of angiotensin I, the primary attack was performed by a combined action of the NEP-like and the ACE-like enzymes, followed by aminopeptidase attacks. Angiotensin II and III were hydrolyzed by NEP-like enzyme at the same Tyr-Ile bond, whereas the N-terminal arginine residue of angiotensin III was removed by an arginyl aminopeptidase. These results show that angiotensins are efficiently degraded by membranes and that NEP-like enzyme plays a key role in this process.

Amino Acid Sequence↗
PubMed1989

Effects of anoxia, hyperoxia, and salinity on neurons in the leech Nephelopsis obscura (Erpobdellidae): RNA redistribution by fluorescence histochemistry.

The histochemical distribution of cytoplasmic RNA in ganglion cells of the freshwater leech Nephelopsis obscura has been studied using the fluorochrome acridine orange as a marker of nucleic acids. Two series of experiments, employing 50 adult animals, involved changes in oxygen tension in the water and changes in salinity. Normal leech neurons exhibit finely granular orange fluorescence uniformly distributed throughout the cytoplasm, with a perinuclear ring of especially strong fluorescence. After exposure to anoxic (0% O2), hypoxic (20% O2), or hyperoxic (200% O2) conditions at 20 degrees C for 1-15 days, the orange cytoplasmic fluorescence is no longer uniformly distributed; the redistribution is generally toward the periphery, leaving the perinuclear zone without RNA fluorescence, but irregular zones of cytoplasm devoid of RNA also occur not as a gradient. Leeches exposed to salinity of, or greater than, 2.5 ppt for 15 days exhibit similar changes. These alterations are confirmed by electron microscopy. Seasonal fluctuations in oxygen tension and salinity of lake water affect the distribution and abundance of organisms. The acridine orange method provides one measure of stress to the nervous system in freshwater invertebrates that might be applicable to ecological studies as well as to metabolic studies of individual animals.

Acridine Orange↗
PubMed1995

Arterial antithrombotic effect of piyavit, the novel pharmacological preparation from the medicinal leech, and of its components, prostanoids and enzyme destabilase.

Piyavit, the novel pharmacological preparation from the medicinal leech (Hirudo medicinalis) contained the leech saliva, produces the potent arterial antithrombotic effect examined on experimental of Laser Induced Thrombus formation. Administrated orally into rats or injected subcutaneously as water extract, non-diluted or in 1600 times diluted, it inhibits statistically significant comparing with control platelet thrombus stimulated by laser beams. Its components, prostanoid fraction and purified enzyme destabilase, endo-epsilon-(gamma-Glu)-Lys-isopeptidase, also inhibit thrombus formation in statistically different manner, comparing with control. All the tested preparations inhibit platelet aggregation induced by ADP. The dependence of arterial antithrombotic effect on the leech prostanoid is discussed.

Administration, Oral↗
PubMed1992

Effects of acid Ca2+ Ringer on passive electrical properties and intracellular ion activities in leech Retzius neuron.

1. A significant drop in effective input resistance of the free membrane and an increase in effective coupling resistance in acid Ca2+ Ringer (complete replacement of Na+ with Ca2+, pH 4) compared to control medium has been obtained in leech Retzius neurons. 2. In neutral Ca2+ Ringer (pH 7.2), effective input resistance increased while effective coupling resistance did not change. In acid sodium, leech Ringer (pH 4) effective input resistance increased while coupling resistance decreased. 3. Ten millimolar manganese and 10 mmol tetraethylammonium did not block conductance changes obtained in acid Ca2+ Ringer. 4. Intracellular activity of Na+ decreased, cellular activity of Cl- increased and intracellular K+ activity was unchanged in both acid and neutral Ca2+ Ringer. 5. The main difference was intracellular acidification in acid Ca2+ Ringer while intracellular pH was unchanged in neutral Ca2+ Ringer. 6. We discuss the possibility that in acid Ca2+ Ringer, intracellular acidification in leech neurons may be responsible for accompanying conductive changes.

Animals↗