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PubMed1999

Phylogeny of the leech family Glossiphoniidae based on mitochondrial gene sequences and morphological data.

The phylogenetic relationships of the Glossiphoniidae (Rhynchobdellida) were investigated using morphological characters and the mitochondrial genes cytochrome c oxidase subunit I and nicotinamide adenine dinucleotide dehydrogenase subunit 1. Thirty-five taxa representing 10 of the 23 currently recognized glossiphoniid genera were sampled, including more than 70% of known North American species, as well as others from Europe, South America, Africa, and a species endemic to Lake Baikal. Outgroup taxa included species from the Piscicolidae and Ozobranchidae. Cladistic analysis resulted in 1 most-parsimonious tree. Subfamily distinctions, i.e., Haementeriinae, Theromyzinae, and Glossiphoniinae, that have been based on eye morphology and reproductive biology are not corroborated. Results also provide insights into several problematic genus-level classifications. For example, relationships of Placobdella and Haementeria are clarified and elimination of Desserobdella may be necessary. Bloodfeeding from vertebrates is seen to be a primitive characteristic that has been lost twice within the clade. The hypothesis that the biannulate leech, Oligobdella biannulata, represents an important transitional form is re-evaluated in a phylogenetic context.

Amino Acid Sequence↗
PubMed1999

Pharmacological characterization of the response of the leech pharynx to acetylcholine.

In this study we document the sensitivity of the leech pharynx to acetylcholine and begin to characterize the acetylcholine receptor mediating this response by examining the effects of selective cholinergic agonists and antagonists on the contractile behavior of the pharynx. The order of potency derived from the EC50 of each agonist was (+/-)epibatidine > acetylcholine (in the presence of physostigmine) >> McN A-343 >> carbachol > nicotine. However, when response amplitude was considered, the order of potency to the tested agonists was (+/-)epibatidine >> nicotine >> McN A-343 >> carbachol > acetylcholine. Acetylcholine-induced contractions of the pharynx were antagonized by d-tubocurarine, but not by alpha-bungarotoxin, alpha-conotoxin M1, or mecamylamine. Application of high concentrations of hexamethonium (1 mM) augmented the acetylcholine-induced contractions. However, this augmentation was apparently due to inhibition of acetylcholinesterase by hexamethonium. The muscarinic antagonist atropine produced complex actions and apparently acted as a mixed agonist/antagonist. Atropine by itself produced an increase in basal tonus and increased the frequency and amplitude of phasic contractions. Atropine increased the peak tension of the acetylcholine-induced response; however, it reduced the amplitude of both the acetylcholine-induced increase in basal tonus and integrated area. Based on the pharmacological profile of the pharyngeal acetylcholine response, we conclude that the acetylcholine receptor mediating the response is a nicotinic receptor. However, the responsiveness of the pharynx to muscarinic agents diverges from that of a classical nicotinic receptor.

Acetylcholine↗
PubMed2000

Kinematics and modeling of leech crawling: evidence for an oscillatory behavior produced by propagating waves of excitation.

Many well characterized central pattern generators (CPGs) underlie behaviors (e.g., swimming, flight, heartbeat) that require regular rhythmicity and strict phase relationships. Here, we examine the organization of a CPG for leech crawling, a behavior whose success depends more on its flexibility than on its precise coordination. We examined the organization of this CPG by first characterizing the kinematics of crawling steps in normal and surgically manipulated animals, then by exploring its features in a simple neuronal model. The behavioral observations revealed the following. (1) Intersegmental coordination varied considerably with step duration, whereas the rates of elongation and contraction within individual segments were relatively constant. (2) Steps were generated in the absence of both head and tail brains, implying that midbody ganglia contain a CPG for step production. (3) Removal of sensory feedback did not affect step coordination or timing. (4) Imposed stretch greatly lengthened transitions between elongation and contraction, indicating that sensory pathways feed back onto the CPG. A simple model reproduced essential features of the observed kinematics. This model consisted of an oscillator that initiates propagating segmental waves of activity in excitatory neuronal chains, along with a parallel descending projection; together, these pathways could produce the observed intersegmental lags, coordination between phases, and step duration. We suggest that the proposed model is well suited to be modified on a step-by-step basis and that crawling may differ substantially from other described CPGs, such as that for swimming in segmented animals, where individual segments produce oscillations that are strongly phase-locked to one another.

Animals↗
PubMed1999

Simultaneous transmission of Trypanosoma mukasai, Babesiosoma mariae and Cyrilia nili to fish by the leech Batracobdelloides tricarinata.

Trypanosoma mukasai (HOARE, 1932), Babesiosoma mariae (HOARE, 1930) and Cyrilia (= Haemogregarina) nili (WENYON, 1909) were concurrently transmitted from Tilapia nilotica to Clarias lazera using the leech vector Batracobdelloides tricarinata. Concurrent transmission was more successful in immature Clarias lazera in which prepatent periods were shorter and patency longer than in mature fish.

Animals↗
PubMed2000

Enhancement of glutamate uptake transport by CO(2)/bicarbonate in the leech giant glial cell.

Glutamate uptake into glial cells via the excitatory amino acid transporter (EAAT) is accompanied by an influx of sodium and acid equivalents into the cells. The sodium-bicarbonate cotransport (NBC) in glial cells moves sodium and base equivalents across the glial membrane in both directions. We have studied possible interactions between these two electrogenic transporters in the giant glial cell of isolated ganglia of the leech Hirudo medicinalis. Changes in membrane potential, membrane current, intracellular sodium, and intracellular pH evoked by aspartate (1 mM), an EAAT agonist, were measured both in the absence and in the presence of CO(2)/bicarbonate. When 5% CO(2) and 24 mM bicarbonate was added to the saline (at constant pH 7.4), the aspartate-induced membrane current was increased, while the change in intracellular sodium was decreased. The acid influx evoked by aspartate was enhanced by CO(2)/bicarbonate but, because of the increased intracellular CO(2)/bicarbonate-dependent buffering power, the change in intracellular pH was decreased. 4,4'-Diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 0.5 mM), which inhibits the NBC, reversed the effects of CO(2)/bicarbonate on the aspartate-induced current and pH change. Our results suggest that the NBC helps counteract dissipation of the sodium and the acid-base gradients induced by the EAAT, enhancing the rate and capacity of glutamate uptake by glial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Disruption of peripheral target contact influences the development of identified central dendritic branches in a leech motor neuron in vivo.

Retrograde signaling from target tissues has been shown to influence many aspects of neuronal development in a number of developmental systems. In these experiments using embryonic leeches (Hirudo medicinalis), we examined how depriving a neuron of contact with its peripheral target affects the development of the cell's central arborization. We focused our attention on the motor neuron cell 3, which normally stimulates dorsal longitudinal muscle fibers to contract. At different locations in the periphery and in embryos of several different stages, we cut the nerve containing the growing axon of cell 3. This surgery led to dramatic overgrowth of cell 3's central dendritic branches, which normally accept synaptic contacts from other neurons, including the inhibitory motor neuron cell 1. When cell 3's peripheral axon was cut relatively early in development, its overgrown central branches eventually retracted. However, cells that were disrupted later in development retained their overextended branches into adulthood. In addition, if the axon was cut close to the ganglion early in development, depriving the cell of contact with any dorsal tissues, the central branches failed to retract and were instead retained into adulthood. Unlike cell 3, the central branches of cell 1, which has the same peripheral target muscles as cell 3, remained unchanged following all axotomy protocols. These results suggest that in at least some neurons contact with peripheral targets can influence development of the central processes that normally mediate synaptic contacts.

Aging↗
PubMed2000

Mechanisms of Mg2+ influx, efflux and intracellular 'muffling' in leech neurones and glial cells.

Mg2+ is known to influence conductance and gating properties of a multitude of ion channels and is thus able to modulate synaptic transmission. Therefore, a tight regulation of the intracellular free Mg2+ concentration ([Mg2+]i) in neurones and glial cells is crucial for maintaining the functions of central nervous systems. [Mg2+]i is regulated through the balance of Mg2+ influx and Mg2+ efflux, together with heavy damping of [Mg2+]i changes through intracellular buffering and sequestration. To investigate the mechanisms involved in [Mg2+]i regulation, neurones and glial cells from the central nervous system of the leech Hirudo medicinalis proved to be an ideal model system. The present article summarizes the evidence for a Mg2+ influx pathway which is distinct from that for Ca2+, for a dual regulation of Mg2+ efflux (a 1 Na+/1 Mg2+ antiport and a Na(+)-independent Mg2+ efflux mechanism), for pH-dependent Mg2+ buffering through ATP and other intracellular Mg2+ binding components and for the involvement of mitochondria in intracellular Mg2+ sequestration.

Animals↗
PubMed2001

Non-associative learning and serotonin induce similar bi-directional changes in excitability of a neuron critical for learning in the medicinal leech.

In studies of the cellular basis of learning, much attention has focused on plasticity in synaptic transmission in terms of transmitter release and the number or responsiveness of neurotransmitter receptors. However, changes in postsynaptic excitability independent of receptors may also play an important role. Changes in excitability of a single interneuron in the leech, the S-cell, were measured during non-associative learning of the whole-body shortening reflex. This interneuron was chosen because it is known to be necessary for sensitization and full dishabituation of the shortening response. During sensitization, S-cell excitability increased, and this enhancement corresponded to facilitation of the shortening reflex and increased S-cell activity during the elicited response. During habituation training, there was a decrement in both the shortening reflex and the elicited S-cell activity, along with decreased S-cell excitability. Conversely, dishabituation facilitated both the shortening response and S-cell activity during shortening, with an accompanying increase in S-cell excitability. Bath application of 1-10 micrometer serotonin (5HT), a modulatory neurotransmitter that is critical for sensitization, for full dishabituation, and for associative learning, increased S-cell excitability. S-cell excitability also increased after stimulation of the serotonergic Retzius cells. However, focal application of serotonin onto the S-cell soma hyperpolarized the interneuron, and bath application of a lower dose of serotonin (0.1 micrometer) decreased excitability. The observed changes in postsynaptic excitability appear to contribute to non-associative learning, and modulatory neurotransmitters, such as serotonin, evidently help regulate excitability. Such changes in S-cell excitability may also be relevant for more complex, associative forms of learning.

Animals↗
PubMed2001

Excitotoxicity of lathyrus sativus neurotoxin in leech retzius neurons.

The effects of Lathyrus sativus neurotoxin were studied on the cell membrane potential and cellular cation composition in Retzius nerve cells of the leech Haemopis sanguisuga, with ion-selective microelectrodes using liquid ion-exchangers. Bath application of 10(-4) mol/l Lathyrus sativus neurotoxin for 3 min depolarized the cell membrane potential and decreased the input resistance of directly polarized membrane in Retzius neurons. At the same time the cellular Na+ activity increased and cellular K+ activity decreased with slow but complete recovery, while the intracellular Ca2+ concentration was not changed. Na+-free Ringer solutions inhibited the depolarizing effect of the neurotoxin on the cell membrane potential. Zero-Ca2+ Ringer solution or Ni2+-Ringer solution had no influence on the depolarizing effect of the neurotoxin on the cell membrane potential. It is obvious that the increase in membrane conductance and depolarization of the cell membrane potential are due to an influx of Na+ into the cell accompanied by an efflux of K+ from the cell.

Amino Acids, Diamino↗
PubMed1975

[Electrical characteristics of the sensory neurons of the medical leech].

The electrical characteristics of three types of sensory neurons (N, T and P) were determined in the leech Hirudo medicinalis. Statistically significant differences were found between the values of tau, Cs and Rs in the neurons of N- and T-types. The same parameters show the least variation coefficients. The value of time constant (tau) is 17.000-46 ms, the resistivity of the soma membrane TRs) is 19.50 kphi-cm2 in T-neurons and 35 kphi-cm2 in N-neurons. The surface of the neurons estimated by their capacitance (Cs) can be 4-10 times larger than the surface of a sphere of the same size. The possible influence of electrical characteristics of the neurons on the peculiarities of their electrical reactions is discussed.

Action Potentials↗
PubMed2002

[Inheritance of reproductive traits of medicinal leeches (Hirudo medicinalis L.)].

The phenotype variability and inheritance of reproductive traits were investigated in the medicinal leech. Distribution parameters were determined for the following traits: batch size (X = 4.3 +/- 0.2, sigma = 1.7, CV = 40%, As = 0.23 +/- 0.25, Ex = 0.19 +/- 0.51), number of juveniles in a cocoon (X = 10.9 +/- 0.3, sigma = 4.6, CV = 42%, As = 0.31 +/- 0.15, Ex = 0.23 +/- 0.30), and juvenile weight (X = 32.0 +/- 0.3, sigma = 14.9, CV = 47%, As = 1.38 +/- 0.05, Ex = 3.32 +/- 0.11. A nonlinear negative correlation between the number of juveniles in a cocoon and their weight was found (correlation ratio R = 0.86). It was shown that the environmental variance dominated over the genotypic one in the structure of phenotypic variance of the traits studied. The genetic variability is determined mainly by additive gene interactions and, to a small extent, intralocus dominance. The narrow-sense heritability, h2, for batch size was 0.35-0.40; for the number of juveniles in a cocoon, 0.35; for juvenile weight, 0.42.

Animals↗
PubMed2002

Using optical flow to characterize sensory-motor interactions in a segment of the medicinal leech.

Activation of motoneurons innervating leech muscles causes the appearance of a two-dimensional vector field of deformations on the skin surface that can be fully characterized using a new technique (Zoccolan et al., 2001) based on the computation of the optical flow, the two-dimensional vector field describing the point displacements on the skin. These vector fields are characterized by their origin (i.e., the singular point) and by four elementary components that combine linearly: expansion (or compression), rotation, longitudinal shear, and oblique shear. All motoneurons can be classified and recognized according to the components of the deformations they elicit: longitudinal motoneurons give rise almost exclusively to longitudinal negative shear, whereas circular motoneurons give rise to both positive longitudinal shear and significant negative expansion. Oblique motoneurons induce strong oblique shear, in addition to longitudinal shear and negative expansion. Vector fields induced by the contraction of longitudinal, circular, and oblique fibers superimpose linearly. Skin deformations can therefore be attributed rather reliably to the contraction of distinct longitudinal, circular, and oblique muscle fibers. We compared the deformation patterns produced by touching the skin with those produced by intracellular stimulation of P, T, and N cells: vector fields resulting from the activation of P cells were almost identical to those produced by mechanical stimulation. Therefore, motor responses triggered by light or moderate touch are almost entirely mediated by excitation of P cells, with minor contributions from T and N cells.

Action Potentials↗
PubMed2002

[Predicting efficiency of selection on reproductive traits in the medicinal leech Hirudo medicinalis L].

A set of selection measures for increasing reproduction efficiency in Hirudo medicinalis has been developed. The optimal values of reproductive traits corresponding to the highest progeny number were determined and recommended. The probability of correlated selection response in traits "number of threads in a cocoon" and "weight of threads" was estimated. Based on earlier results on phenotypic variation and heritability of reproductive traits in medicinal leech, efficiency of different selection modes was predicted.

Animals↗
PubMed1999

[Research on anti-thrombosis effect of ethanol extract from beheaded leech (EEBL)].

OBJECTIVE: To study the anti-thrombosis effect of EEBL and its possible mechanism. METHOD: Using a thrombosis model induced by collagen-adrenaline in mice and an artery-vein bypass in rats. The effect of EEBL on erythrocyte membrane and thrombocyte membrane fluidity were measured by fluorescence polarization. RESULT: EEFL inhibited the thrombosis induced by collagen-adrenaline in mice and the thrombosis in artery-vein bypass in rats significantly. EEBL also enhanced in vitro erythrocyte membrane fluidity and thrombocyte membrane fluidity markedly. CONCLUSION: The anti-thrombosis effect of EEBL may possibly be associated with the enhancement of fluidity of erythrocyte membrane and thrombocyte membrane. There exist anti-thrombosis chemical constituents in the body of beheaded leech.

Animals↗
PubMed2002

Bursting in leech heart interneurons: cell-autonomous and network-based mechanisms.

Rhythmic activity within the heartbeat pattern generator of the medicinal leech is based on the alternating bursting of mutually inhibitory pairs of oscillator heart interneurons (half-center oscillators). Bicuculline methiodide has been shown to block mutual inhibition between these interneurons and to cause them to spike tonically while recorded intracellularly (Schmidt and Calabrese, 1992). Using extracellular recording techniques, we show here that oscillator and premotor heart interneurons continue to burst when pharmacologically isolated with bicuculline, although the bursting is not robust in some preparations. We propose that a nonspecific leak current introduced by the intracellular microelectrode suppresses endogenous bursting activity to account for the discrepancy with results using intracellular recording. A two-parameter bifurcation diagram (E(leak) vs g(leak)) of a mathematical model of a single heart interneuron shows a narrow stripe of parameter values where bursting occurs, separating large zones of tonic spiking and silence. A similar analysis performed for a half-center oscillator model outlined a much larger area of bursting. Bursting in the half-center oscillator model is also less sensitive to variation in the maximal conductances of voltage-gated currents than in the single-neuron model. Thus, in addition to ensuring appropriate bursting characteristics such as period, phase, and duty cycles, the half-center configuration enhances oscillation robustness, making them less susceptible to random or imposed changes in membrane parameters. Endogenous bursting, in turn, ensures appropriate bursting if the strength of mutual inhibition is weakened and limits the minimum period of the half-center oscillator to a period near that of the single neuron.

Action Potentials↗
PubMed1999

[The comparison of contents of uracil, xanthine and hypoxanthine in five species of leech].

Three constituents(uracil, xanthine, hypoxanthine) were isolated from five species of leech and determined by reversed phase HPLC. The column employed was Shim-pack CLC-ODS C18(150 mm x 6 mm). The mobile phase was 0.05 mol/l ammonium phophate dibasic solution(pH = 8.4). The flow rate was 0.8 ml/min and detection was effected at 254 nm. This method is accurate, rapid and reproducible. Analytical data for five species and Whitmania pigra samples from different places were given.

Animals↗
PubMed2003

Modulation of spike-mediated synaptic transmission by presynaptic background Ca2+ in leech heart interneurons.

At the core of the rhythmically active leech heartbeat central pattern generator are pairs of mutually inhibitory interneurons. Synaptic transmission between these interneurons consists of spike-mediated and graded components, both of which wax and wane on a cycle-by-cycle basis. Low-threshold Ca2+ currents gate the graded component. Ca imaging experiments indicate that these low-threshold currents are widespread in the neurons and that they contribute to neuron-wide changes in internal background Ca2+ concentration (Ivanov and Calabrese, 2000). During normal rhythmic activity, background Ca2+ concentration oscillates, and thus graded synaptic transmission waxes and wanes as the neurons move from the depolarized to the inhibited phases of their activity. Here we show that in addition to gating graded transmitter release, the background Ca2+ concentration changes evoked by low-threshold Ca2+ currents modulate spike-mediated synaptic transmission. We develop stimulation paradigms to simulate the changes in baseline membrane potential that accompany rhythmic bursting. Using Ca imaging and electrophysiological measurements, we show that the strength of spike-mediated synaptic transmission follows the changes in background Ca2+ concentration that these baseline potential changes evoke and that it does not depend on previous spike activity. Moreover, we show using internal EGTA and photo-release of caged Ca2+ and caged Ca2+ chelator that changes in internal Ca2+ concentration modulate spike-mediated synaptic transmission. Thus activity-dependent changes in background Ca2+, which have been implicated in homeostatic regulation of intrinsic membrane currents and synaptic strength, may also regulate synaptic transmission in an immediate way to modulate synaptic strength cycle by cycle in rhythmically active networks.

Action Potentials↗
PubMed2003

[Effect of the antibodies to gangliosides on the Retzius neuron electrical activity and the functional activity of sodium channels for incoming current in leech].

In the present study, the effect of antibodies to gangliosides on the Retzius neurons of the leech was investigated to study the spike activity and the functional activity of the Na-channels which generate the spike. A forty-minute incubation of the Retzius neurons in a 20% solution of antiganglioside serum in a Ringer solution provoked appearance of a double spike (a spike with two parts) connected with a decrease of the speed of the activation of the tetrodotoxin (TTX)-sensitive Nachannels. The high frequency synaptic activation of the neuron (10 Hz during 10 minutes) under the plasticity exchange of the gate system of the TTX-sensitive Na-channels. As a result of this, there was a disturbance of the habituation of the Retzius neuron to the high-frequency stimulation.

Action Potentials↗