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PubMed1969

Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech.

1. Experiments were made on single neurones and glial cells in the central nervous system of the leech to study the accumulation of K that occurs in the extracellular spaces around neurones as a result of impulse activity.2. The resting potential of a neurone is too insensitive to be used for the estimation of small changes in K concentration. The undershoot of the action potential, however, provided a reliable indicator of the K accumulation that occurs around a neurone during activity.3. After a single impluse the amplitude of the undershoot of a second action potential was decreased; the effect corresponded to a peak increase in K concentration of about 0.8 mM/l. immediately after the spike and declined exponentially with a time constant of about 100 msec. With trains of impulses the K concentration increased exponentially, again with a time constant of about 100 msec. The final value of K depended on the frequency and could build up to about double the normal concentration of 4 mM/l.4. The build-up of K was markedly reduced when the extracellular space surrounding a neurone was enlarged by removing its glial investment.5. Synchronous, repetitive activation of groups of neurones caused a slow depolarization of neighbouring glial cells in the C.N.S. of the leech, similar to that observed in amphibia and mammals. The change in glial membrane potential was also used to estimate the changes in K concentration and these values agreed with measurements derived from the undershoot.6. Increases of K concentration in the bathing fluid of the same order as those caused by neural firing markedly affected the frequency of ;spontaneous' neuronal discharges and synaptic potentials occurring within certain neurones in the C.N.S.7. The possible effects of physiologically occurring increases of K concentration on integration are discussed.

Action Potentials↗
PubMed1973

Conductance changes, an electrogenic pump and the hyperpolarization of leech neurones following impulses.

Following trains of impulses, sensory neurones in the C.N.S. of the leech show a prolonged hyperpolarization, which lasts for seconds or minutes. In the present investigation the mechanisms that underly this hyperpolarization have been studied by recording intracellularly. Two factors have been found to be responsible. One is the activity of an electrogenic pump (see Baylor & Nicholls, 1969b); the other is a long-lasting change in K conductance.1. Additional evidence that an electrogenic pump contributes to a slow after-hyperpolarization of leech sensory neurones is provided by the effects of injecting Na intracellularly. This leads to an increase in membrane potential that is blocked by the cardiac glycoside strophanthidin. Furthermore, after a train of impulses, reducing the K concentration in the external fluid characteristically reduces the hyperpolarizing action of the pump.2. The hyperpolarization following impulses is associated with a reduction of the cell membrane resistance that can persist for several minutes.3. Several lines of evidence suggest that the reduction in input resistance during the hyperpolarization is mainly due to an increased permeability to K. Thus, when the K concentration in Ringer fluid is reduced, the peak amplitude of the hyperpolarization following a train becomes larger. Furthermore, the conductance dependent part of the after-hyperpolarization has a reversal potential close to the equilibrium potential for K (E(K)). Substitution of Cl by SO(4) has little effect either on the after-hyperpolarization or on the conductance change following a train.4. Increased external Ca concentrations lead to a marked increase in the hyperpolarization that follows impulse activity. The enhanced hyperpolarization in high Ca is associated with a corresponding reduction in input resistance. The amplitude and duration of the hyperpolarization following a brief train of impulses can be increased by a factor of 5 or more in Ringer fluid containing 10 mM-Ca instead of the usual 1.8 mM. The hyperpolarization and resistance changes still occur in solutions containing 20 mM-Mg.5. To augment the hyperpolarization the increased concentration of Ca must be present during the train of impulses.6. The relative contributions of the K conductance increase and of the electrogenic pump for generating the hyperpolarization after impulse activity are different in the three types of sensory cell responding to touch, pressure and noxious stimulation.

Animals↗
PubMed1974

Persistent modification of synaptic interactions between sensory and motor nerve cells following discrete lesions in the central nervous system of the leech.

We have examined changes that develop in the synaptic interactions of sensory and motor nerve cells following surgical lesions to the central nervous system of the leech. In one type of operation an individual ganglion was isolated from the rest of the nervous system by severing all the incoming and outgoing fibres. During the next few weeks, marked changes appeared in synaptic interactions.1. In chronically isolated ganglia inhibitory potentials were recorded in the motoneurone which raises the skin into ridges (the AE cell) following impulses in sensory neurones that respond to pressure (P) or noxious (N) stimuli. In contrast the same AE cell in ganglia taken from normal animals shows excitatory synaptic potentials when the P or N sensory cells are stimulated.2. Another altered synaptic interaction in ganglia isolated by lesions was that between sensory cells responding to touch and a motoneurone that supplies longitudinal muscles (L cell). Instead of the pure, electrical coupling potential seen normally, a large, additional chemically mediated excitatory potential was also apparent.3. Some of the changes in synaptic interactions were not restricted to synapses within the isolated ganglion, but appeared gradually over the following year in successive ganglia along the length of the ventral nerve cord.4. Indirect evidence suggests that the altered synaptic potentials that became conspicuous after operations are also present but smaller and obscured in normal animals.5. It is concluded that some synapses in the leech nervous system are more readily changed than others by cutting the connectives. Furthermore, these changes influence in a predictable manner the way in which the animal behaves in response to mechanical stimuli.

Action Potentials↗
PubMed1978

Quantal analysis of transmitter release at an inhibitory synapse in the central nervous system of the leech.

The quantal nature of transmitter release has been analysed at central inhibitory synapses in the leech nervous system between an interneurone (HN) and a motoneurone (HE) that regulate the heartbeat. 1. Ganglia were bathed in leech Ringer fluid containing 20 mM-Mg and 1.8 mM-Ca and the membrane of the presynaptic HN interneurone was hyperpolarized by current injection. Under these conditions successive inhibitory potentials in the HE motoneurone, evoked by impulses in the HN interneurone, showed striking fluctuations in amplitude. 2. Assuming a Poisson distribution of the i.p.s.p.s and estimating the number of failures from the amplitude histograms of the observed responses, the mean size of the quantal unit was estimated as 0.25 +/- 0.015 mV (S.E. of mean, n = 26). When m, the mean number of quanta released per trial, was varied by changing the membrane potential of the presynaptic HN cell (Nicholls & Wallace, 1978), the experimentally observed amplitude distributions could be predicted by the Poisson theory. 3. An independent estimate of the unit size was obtained by noise analysis. A long subthreshold depolarizing pulse applied to the presynaptic HN interneurone evoked a sustained hyperpolarization of the HE motoneurone, apparently caused by an increase in the rate of on-going release of quanta by the HN cell terminals. From the mean change in membrane potential and the increase in variance, the size of the unit was calculated as 0.21 +/- 0.039 mV (S.E. of mean, n = 11). For ten pairs of cells an estimate of unit amplitude was made both from the Poisson analysis and the analysis of variance, again with good agreement. For these cells the estimated unit sizes were 0.24 +/- 0.023 mV (S.E. of mean, n = 10) from the failures and 0.21 +/- 0.043 m V (S.E. of mean, n = 10) from the noise. 4. A similar analysis was made of the inhibitory synaptic potentials evoked in one HN interneurone by stimulation of its contralateral homologue. Transmission again appeared to be qualtal; the mean unit amplitude from Poisson analysis was 0.31 +/- 0.022 mV (S.E. of mean, n = 19) and from the noise 0.29 +/- 0.027 mV (S.E. of mean, n = 3). 5. We conclude that transmitter is released from the terminals of the HN interneurone in quantal units that evoke miniature i.p.s.p.s of about 0.25 mV in the post-synaptic cells. Furthermore, modulation of transmission proudced by variation in the presynaptic resting potential and during presynaptic inhibition results from changes in the mean number of quanta released by each impulse.

Animals↗
PubMed1981

Membrane properties and selective connexions of identified leech neurones in culture.

1. Individual, identified neurones, dissected from the central nervous system of the leech and maintained in culture for several weeks, sprouted processes and formed synaptic connexions.2. The action potentials of isolated touch (T), pressure (P), nociceptive (N) cells and Retzius cells resembled those of their counterparts in situ, enabling them to be recognized unambiguously. Their input resistances were approximately 4 times greater than those of corresponding cells within the animal. In T, P and N cells trains of impulses were followed by a pronounced after-hyperpolarization, as in the animal.3. In certain cells, notably the L motoneurones, membrane properties became altered in culture. The current-voltage relation showed novel rectification and action potentials became much larger.4. Numerous neurites often extended for hundreds of micrometres from isolated neurones and ended in typical growth cones. Electron micrographs revealed that many fine axons were braided together to form thicker fascicles. Frequently, the processes were orientated between two neighbouring cells rather than at random. The fine structure of the cytoplasm, nucleus and organelles in cultured cells resembled those of their counterparts in situ. The glial cell that normally surrounds the neurones was, however, absent.5. Pairs of Retzius cells in culture usually became coupled electrically after about 6 days. Similarly L motoneurones became coupled in vitro. These junctions allowed current to pass in both directions and resembled those seen in the animal.6. Selective connexions were made by certain types of cells. Thus, P sensory neurones did not become coupled with Retzius cells but did develop electrical connexions with L motoneurones, as in the animal.7. Novel synaptic interactions not obvious in the animal could appear in culture. Retzius and L cells became electrically coupled and, in some instances where electrical coupling between Retzius cells failed to develop, chemically mediated inhibitory potentials became apparent.8. Isolated, identified leech neurones not only survive but regenerate processes and are capable of forming selective connexions in culture. The ability to define interactions between isolated pairs of cells offers the opportunity to explore in detail problems relating to synapse formation and cell-cell recognition.

Action Potentials↗
PubMed1982

Physiological responses, receptive fields and terminal arborizations of nociceptive cells in the leech.

The physiological responses, receptive fields and morphology of individual nociceptor (N) neurones have been studied in the leech. In each of the midbody ganglia there are four N cells (two on either side). Each N cell has a distinctive territory that it supplies in the periphery, on the surface or internally. 1. Both N cells respond selectively to noxious mechanical stimuli applied to the skin but not to touch, light, pressure or stretch. The receptive field of each cell is well defined and covers roughly the same area, extending from the dorsal midline to the ventral midline, with considerable overlap. 2. One of the N cells, situated more medially in the ganglion, also fires at high frequencies in response to mechanical stimulation, such as pinching or squeezing, of the connective tissue lining the viscera. In contrast, the other N cell (situated laterally in the ganglion) is activated by pressure or pinches applied to the opening of the excretory duct but not the gut. 3. Following injection of horseradish peroxidase into the soma, axons of N cells appear as unspecialized fine processes about 1 micrometer in diameter, in the dermis of the leech, deep to the layer of epidermal cells. In addition, at specific sites in the skin, the N cell situated laterally in the ganglion makes distinctive coiled terminals in association with the expanded dendrites of large neurones in the periphery, the functions of which are unknown. This finding raises the possibility that lateral N cells may perform some additional role as yet not understood.

Action Potentials↗
PubMed1985

A dual mechanism for intracellular pH regulation by leech neurones.

Neutral-carrier pH-sensitive micro-electrodes were used to investigate intracellular pH (pHi) in leech neurones. When used in snail neurones such electrodes gave very similar pHi values to those recorded simultaneously by recessed-tip glass micro-electrodes. Leech Retzius neurones superfused with a pH 7.4 HCO3--free physiological saline were found to have a pHi of 7.3, too high to be explained by a passive distribution of H+ or OH-. To investigate pHi regulation the pHi was decreased by one of three methods: by exposure to propionate, by adding and then removing NH4Cl or by exposure to CO2. Acidification by any method was followed by a recovery to normal pHi values within 15-20 min. In HCO3--free solutions, pHi recovery from acidification was blocked by removing external Na or by amiloride (2 mM). In solutions buffered with 2% CO2 and 11 mM-HCO3-, amiloride slowed but did not block pHi recovery. The anion exchange inhibitor SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) also slowed pHi recovery in the presence of HCO3-. In CO2/HCO3- solution the removal of external Na either slowed or blocked pHi recovery, and blocked it completely in the presence of amiloride. We conclude that in HCO3--free solutions pHi regulation is by a Na-H exchange system; but in the presence of HCO3- there is an additional mechanism which is probably a Na-dependent Cl-HCO3 exchanger.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Hyperpolarizing responses to stretch in sensory neurones innervating leech body wall muscle.

1. The membrane properties, morphology and physiological responses of peripherally located sensory neurones that innervate body wall muscle of the leech Hirudo medicinalis have been investigated using intracellular recording and dye injection techniques. 2. The peripheral neurones and their dendrites were visualized directly in whole mounts of the body wall by intracellular injection of horseradish peroxidase or Lucifer Yellow. They lie along the course of segmental nerves between the layers of longitudinal and oblique body wall muscle and within the sheath of the nerve. They have a distinctive morphology with two expanded, fan-shaped dendrites arranged in series separated by the cell body and a 300 micron long cylindrical process. Both dendrites are associated with longitudinal muscle of the ventral body wall but with separate bands of muscle fibres. The axons project into the ventral nerve cord and arborize within the ipsilateral half of the segmental ganglion. No processes extend across the mid-line of the ganglion or enter the connectives to neighbouring ganglia. 3. 'Resting' membrane potentials recorded from the peripheral cell body or from the axon as it entered the segmental ganglion ranged from -30 to -70 mV. The transmembrane potential recorded depended on the amount by which the body wall was stretched: the most hyperpolarized values were recorded from the most stretched preparations. Although the peripheral cell body can generate overshooting action potentials these are not actively propagated to the CNS. Rather, imposed voltage changes spread decrementally along the axon. Input resistances measured in the cell body ranged from 14 to 26 M omega. The space constant, estimated from the spread of hyperpolarizing current injected into the cell body, was 2.4 mm. 4. The response of the neurones to change in length of the longitudinal muscle recorded from the axon near its terminal arborization within the ventral nerve cord is a graded DC signal: the neurones thus relay information to CNS synapses in analogue form. Spiking activity recorded extracellularly in the anterior segmental nerve root in response to stretch of the body wall is due to activation of touch mechanosensory cells that innervate the skin. 5. Unlike stretch receptors innervating skeletal muscle in vertebrates or arthropods, the leech neurones respond to stretch of the body wall muscle with maintained hyperpolarizing potentials and to release of stretch with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1990

Intracellular chloride activity in glial cells of the leech central nervous system.

1. Chloride-sensitive double-barrelled microelectrodes were used to measure the intracellular Cl- activity (aicl) and membrane potential (Em) in neuropile glial cells of the leech, Hirudo medicinalis. 2. A close relation between the equilibrium potential for Cl- (ECl = -66.1 +/- 4.9 mV; mean +/- S.D.) and the resting potential (Em = -67.8 +/- 5.2 mV; n = 19) was observed in nominally CO2-HCO3(-)-free, HEPES-buffered solutions. A saline buffered with 2% CO2, 11 mM-HCO3- elicited a membrane hyperpolarization and a concomitant decrease of aCl. 3. Changes in ECl followed these of Em with a lag of less than 30 s in response to various extracellular K+ concentrations [( K+]o) or due to bath-application of carbachol or serotonin. 4. Introduction of a Cl(-)-free solution resulted in a transient depolarization indicating a substantial Cl- conductance and a rapid decrease of aiCl to an apparent value of 0.5-0.9 mM. 5. The loop diuretics furosemide (1 mM) and bumetanide (0.2 mM) did not affect the K(+)-induced changes of aiCl. 6. The results indicate a passive Cl- distribution across the membrane of leech neuropile glial cells as a result of a high Cl- conductance.

Animals↗
PubMed1995

Voltage-dependent clamp of intracellular pH of identified leech glial cells.

1. The intracellular pH (pHi) was measured in voltage-clamped, giant neuropile glial cells in isolated segmental ganglia of the leech Hirudo medicinalis, using double-barrelled, pH-sensitive microelectrodes and a slow, two-electrode voltage-clamp system. The potential sensitivity of the pHi regulation in these glial cells was found to be due to an electrogenic Na(+)-HCO3- cotransporter (Deitmer & Szatkowski, 1990). 2. In the presence of 5% CO2 and 24 mM HCO3- (pH 7.4), pHi shifted by 1 pH unit per 110 mV, corresponding to a stoichiometry of 2HCO3-: 1 Na+ of the cotransporter, while in Hepes-buffered CO2-HCO3(-)-free saline (pH 7.4), pHi changed by 1 pH unit per 274 mV. The potential sensitivity of pHi decreased at lower pHo, being 1 pH unit per 216 mV at external pH (pHo) 7.0. 3. Changing pHo between 7.8 and 6.6 induced pHi shifts with a slope of 0.72 pHi units per pHo unit in non-clamped, and of 0.80 pHi units per pHo unit in voltage-clamped cells, indicating that pHi largely followed pHo. The electrochemical gradient of H(+)-HCO3- across the glial membrane was around 56 mV, and remained almost constant over this pHo range. 4. The membrane potential-dependent and pHo-sensitive shifts of pHi were unaffected by amiloride, an inhibitor of Na(+)-H+ exchange. 5. The intracellular acidification upon lowering pHo could be reversed by depolarizing the membrane as predicted from a cotransporter, whose equilibrium follows the membrane potential by resetting pHi. 6. The results indicate that the pHi of leech glial cells is dominated by the electrogenic Na(+)-HCO3- cotransporter, and is hence a function of the membrane potential, and the Na+ and H(+)-HCO3- gradients, across the cell membrane.

Amiloride↗
PubMed1996

Sodium-magnesium antiport in Retzius neurones of the leech Hirudo medicinalis.

1. Intracellular free magnesium ([Mg2+]i) and sodium ([Na+]i) concentrations were measured in Retzius neurones of the leech Hirudo medicinalis using ion-sensitive microelectrodes. 2. The mean steady-state values for [Mg2+]i and [Na+]i were 0.46 mM (pMg, 3.34 +/- 0.23; range, 0.1-1.2 mM; n = 32) and 8.95 mM (pNa, 2.05 +/- 0.15; range, 5.1-15.5 mM, n = 21), respectively, at a mean membrane potential (Em) of -35.6 +/- 6.1 mV (n = 32). Thus, [Mg2+]i is far below the value calculated for a passive distribution (16.9 mM) but close to the equilibrium value calculated for a hypothetical 1 Na(+)-1 Mg2+ antiport (0.41 mM). 3. Simultaneous measurements of [Mg2+]i, [Na+]i and Em in Retzius neurones showed that an increase in the extracellular Mg2+ concentration ([Mg2+]o) resulted in an increase in [Mg2+]i, a parallel decrease in [Na+]i and a membrane depolarization, while a decrease in [Mg2+]o had opposite effects. These results are compatible with calculations based on a 1 Na(+)-1 Mg2+ antiport. 4. Na+ efflux at high [Mg2+]o still occurred when the Na(+)-K+ pump was inhibited by the application of ouabain or in K(+)-free solutions. This efflux was blocked by amiloride. 5. In the absence of extracellular Na+ ([Na+]o), no Mg2+ influx occurred. Mg2+ influx at high [Mg2+]o was even lower than in the presence of [Na+]o. Mg2+ efflux was blocked in the absence of [Na+]o. 6. The rate of Mg2+ extrusion was reduced by lowering [Na+]o, even if the Na+ gradient across the membrane remained almost unchanged. 7. Mg2+ efflux was blocked by amiloride (half-maximal effect at 0.25 mM amiloride; Hill coefficient, 1.3) but not by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). 8. No changes in intracellular Ca2+ and pH (pHi) could be detected when [Mg2+]o was varied between 1 and 30 mM. 9. Changing pHi by up to 0.4 pH units had no effect on [Mg2+]i. 10. The results suggest the presence of an electrogenic 1 Na(+)-1 Mg2+ antiport in leech Retzius neurones. This antiport can be reversed and is inhibited by low extracellular and/or intracellular Na+ and by amiloride.

Amiloride↗
PubMed1999

Symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech: a novel model for digestive tract associations.

Hirudo medicinalis, the medicinal leech, is applied postoperatively in modern medicine. Infections by Aeromonas occur in up to 20% of patients unless a preemptive antibiotic treatment is administered. The associated infections demonstrate the need for a better understanding of the digestive tract flora of H. medicinalis. Early studies reported the presence of a single bacterial species in the digestive tract and suggested that these bacteria were endosymbionts contributing to the digestion of blood. In this study, we cultivated bacteria from the digestive tract and characterized them biochemically. The biochemical test results identified the isolates as Aeromonas veronii biovar sobria. This species identification was supported by sequence comparison of a variable region of the genes coding for 16S rRNA. In a colonization assay, a rifampin-resistant derivative of a symbiotic isolate was fed in a blood meal to H. medicinalis. The strain colonized the digestive tract rapidly and reached a concentration similar to that of the native bacterial flora. For the first 12 h, the in vivo doubling time was 1.2 h at 23 degreesC. After 12 h, at a density of 5 x 10(7) CFU/ml, the increase in viable counts ceased, suggesting a dramatic reduction in the bacterial growth rate. Two human fecal isolates, identified as Aeromonas hydrophila and A. veronii biovar sobria, were also able to colonize the digestive tract. These data demonstrate that the main culturable bacterium in the crop of H. medicinalis is A. veronii biovar sobria and that the medicinal leech can be used as a model for digestive tract association of Aeromonas species.

Aeromonas↗
PubMed1991

Use of leeches in a case of severe periorbital haematoma.

A case of severe penetrating eye injury and resulting periorbital haematoma is described. Leeches applied to the eyelids enabled examination of the globe to be carried out and improved the surgical approach. Leeches provide a speedy and efficient means of reducing a periorbital haematoma.

Animals↗
PubMed2005

Bleeding due to a medicinal leech bite.

This paper reports a case of prolonged bleeding following application of leeches to treat chronic pain. The paper discusses the characteristics of the wounds and possible complication of prolonged bleeding following medicinal leech application. The principles of treatment are also described.

Adult↗
PubMed1987

Changes in the intracellular free calcium concentration of Aplysia and leech neurones measured with calcium-sensitive microelectrodes.

The intracellular free Ca concentration was measured in invertebrate neurones using single-barrelled and double-barrelled neutral-carrier microelectrodes. The electrodes were calibrated in solutions containing different Ca concentrations between 1 mM and 0.01 microM. The electrode responses were also tested at different ionic strengths and at varying Na concentrations. The electrodes responded with 25-30 mV per 10-fold change in Ca concentration between 1 mM and 1 microM and with 10-25 mV between 1 and 0.1 microM Ca. The intracellular free Ca concentration was measured to be between 0.1 and 0.7 microM in the neurones. The changes of intracellular Ca in identified voltage-clamped neurones of Aplysia californica were recorded during iontophoretic injections of Ca2+ or EGTA. The decrease of intracellular Ca following EGTA injection was correlated with the suppression of the Ca-dependent K current and with the reduction of Ca-induced inactivation of voltage-dependent Ca current. In identified neurones of the leech Hirudo medicinalis a reversible increase of intracellular Ca2+ was recorded after inhibition of the Na-K pump, either by addition of ouabain (0.5 mM) or by lowering the external K concentration (0.2 mM). This rise in intracellular Ca2+ did not occur, and was even reversed, in the absence of external Na, suggesting the existence of Na-Ca exchange across the leech neuronal membrane.

Animals↗
PubMed1987

Direct measurement of intracellular pH in identified glial cells and neurones of the leech central nervous system.

Neutral carrier pH-sensitive double-barrelled microelectrodes were used to investigate intracellular pH (pHi) in leech neuropile glial cells and in Retzius neurones. The mean pHi of the glial cells was 6.87 +/- 0.13 (+/- SD, n = 27) in HEPES-buffered saline (pHo 7.4) and 7.18 +/- 0.19 (n = 13) in solutions buffered with 2% CO2- 11 mM HCO3-. The distribution of H+ ions in both the glia and neurones was found not to be in electrochemical equilibrium. To investigate pHi regulation, the pHi was decreased by exposure to CO2 or by adding and then removing NH4Cl. Acidification by any method was followed by a recovery to normal pHi values within minutes. The pHi recovery from acidification in neuropile glial cells in HEPES-buffered saline and CO2-HCO3- buffered saline was, however, blocked by removing external Na. In HCO3(-)-free solutions the diuretic amiloride (2 mM) reduced the rate of pHi recovery. In the presence of HCO3-, the rate of acid efflux was stimulated; the stilbene 4-acetamido-4'-isothiocyanatostilbene-2,3'-disulfonic acid (SITS; 0.5 mM) slowed pHi recovery. In HEPES buffered and CO2-HCO3- buffered solutions pHi regulation in neurones was inhibited by removing external Na. In HCO3(-)-free solutions amiloride reduced the rate of pHi recovery considerably. In the presence of HCO3-, SITS or amiloride slowed but did not completely block pHi recovery. We conclude that leech glial cells and neurones have two mechanisms of pHi regulation, one being Na+-H+ exchange and the other Na+ and HCO3- dependent.

Animals↗
PubMed1995

Regulation of electrogenic Na+ transport across leech skin.

The dorsal integument of the medical leech Hirudo medicinalis exhibits a marked amiloride-sensitive Na+ absorption. With 20 mM Na+ in the apical solution, the transepithelial short-circuit current (Isc) was approximately 40% higher than with 115 mM Na+, whereas the transepithelial potential (VT) with 20 mM Na+ was -35.7 +/- 4.5 and -20.6 +/- 2.6 mV with 115 mM Na+. Amiloride (100 microM) inhibition at 20 mM apical Na+ was also significantly larger than with 115 mM Na+ in the solution. Benzamil (100 microM) showed additional inhibition after amiloride. Large transient overshooting currents occurred only when 115 mM Na+ was added after some minutes of Na(+)-free apical solution. Addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the serosal side in the presence of 115 mM apical Na+ nearly doubled Isc. This cAMP effect was reduced to only 20% in the presence of 20 mM Na+. Guanosine 3',5'-cyclic monophosphate (cGMP) slightly increased Isc, whereas ATP showed biphasic potency. Removal of calcium from the apical side resulted in a large stimulation of amiloride-sensitive Isc only in the presence of 115 mM Na+. When currents were activated with cAMP, a deprivation of Ca2+ modestly reduced the amiloride-sensitive Isc. The Na+ channel of leech integument was found highly selective for Na+ over other monovalent cations. The permeability ratio for Na+ over K+ was approximately 30:1; the selectivity relationship for the investigated cations was Na+ > Li+ > NH4+ > K+ approximately Cs+ approximately Rb+.

Amiloride↗
PubMed2003

Heartbeat control in leeches. I. Constriction pattern and neural modulation of blood pressure in intact animals.

Two tubular hearts propel blood through the closed circulatory system of the medicinal leech. The hearts are myogenic but are driven by a centrally generated motor pattern that controls heart rate and intersegmental coordination. In two consecutive papers, we address the question of how the motor pattern is translated into the pattern of diastole and systole of leech hearts. We imaged the constriction patterns of the hearts in quiescent intact animals. In one heart, systole progresses rear-to-front (peristaltic coordination mode), whereas systole occurs nearly simultaneously in the other heart (synchronous coordination mode) with regular switches between these two coordination modes. Intersegmental phase relations between heart segments do not vary with changes in the heartbeat period. The peristaltic heart drives blood forward through itself and then rearward through the other longitudinal vessels. The synchronous heart does not seem to contribute to rearward flow along the body axis and may support segmental circulation instead. Simultaneous monitoring of heart motor neuron discharge and the constriction of the corresponding heart segment in innervated, reduced preparations enabled us later to meld the constriction pattern with the fictive motor pattern described in the following paper. Current injections into one heart modulatory neuron while monitoring intravascular pressure from the corresponding heart showed that these neurons can acutely change diastolic and systolic pressure. However, they do not determine the different systolic pressure profiles associated with the two coordination modes, which appear to result from the constriction pattern.

Action Potentials↗