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At least 757 records · Page 42Linked to original sources
PubMed1990

Electrophysiological measurements of volume changes in leech neuropile glial cells.

Double-barrelled microelectrodes, sensitive to quaternary ammonium ions, were used for simultaneous measurements of the intracellular free concentrations of choline ([Ch]i) or tetramethylammonium ([TMA]i) as well as membrane potential (Em) in neuropile glial cells of the leech, Hirudo medicinalis. Bath application of Ch or TMA (5 mM, 1 min) resulted in a transient membrane depolarization accompanied by a long-lasting (0.5-1 h) intracellular accumulation of these compounds to levels of between 5 and 15 mM. Changes in [Ch]i or [TMA]i were used for the calculation of changes in relative cell volume. Elevation of the extracellular K+ concentration [( K+]e) from 4 to 9, 15, 21, 27.5, or 40 mM elicited a membrane depolarization and a reversible cell swelling by about 7.5, 14, 18.5, 27 and 50%, whereas reduction of [K+]e to 1.5 mM as well as bath application of serotonin (5-HT) produced a membrane hyperpolarization and a concomitant shrinkage by about 6 and 14.3%, respectively. The measured alterations in cell volume were compared with calculated data based on the assumption of an osmotic equilibrium disturbed by potential-dependent changes of the intracellular Cl- concentration. The results indicate, that K(+)- and serotonin-induced changes in the cell volume of the neuropile glial cells are due to passive KCl and water fluxes.

Animals↗
PubMed1992

Evidence for glial control of extracellular pH in the leech central nervous system.

Double-barrelled pH-sensitive microelectrodes were used to measure the intracellular pH (pHi) of neuropil glial cells and the pH of extracellular spaces (pH(e)) within isolated, intact ganglia of the leech Hirudo medicinalis. By application of a weak acid (propionate, 40 mM) or a weak base (ammonium, 20 mM) the total buffer capacity was estimated by changes in glial pHi and in pH(e). The buffering power of glial cells and in the extracellular spaces was increased by up to threefold in the presence of CO2/bicarbonate. The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3-0.5 mM) reversed this increase in buffering power both in the glial cells and in the extracellular spaces. Inhibitors of the carbonic anhydrases reduced the CO2/bicarbonate-dependent increase in extracellular buffering power. The results suggest that extracellular H+ buffering dependent upon the availability of bicarbonate is linked to DIDS-sensitive bicarbonate transport across the glial membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Mechanisms of pH recovery from intracellular acid loads in the leech connective glial cell.

We used double-barrelled, neutral carrier, pH-sensitive microelectrodes to study the mechanisms by which the intracellular pH (pHi) is regulated in the connective glial cells of the medicinal leech. In HEPES-buffered, nominally CO2/HCO3(-)-free solutions the recovery of pHi from intracellular acidosis is Na(+)-dependent and reduced by at least half in the presence of amiloride, suggesting the action of Na+:H+ exchange. The rate of pHi recovery by this mechanism can be increased by raising the extracellular buffering power or by increasing extracellular pH. The presence of CO2/HCO3(-)-greatly increases the rate of pHi recovery from intracellular acidosis. This CO2/HCO3(-)-stimulated recovery is also dependent on external Na+, largely Cl(-)-independent, inhibited by DIDS, and accompanied by membrane hyperpolarization. This is consistent with it being mediated by the electrogenic cotransport of Na+ and HCO3- into the cells. A Cl(-)-dependent component to Na(+)- and HCO3(-)-dependent regulation is most easily explained by the added presence of a Na(+)-dependent exchange of HCO3- and Cl-.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Independent changes of intracellular calcium and pH in identified leech glial cells.

The intracellular Ca2+ (Ca2+i) and the intracellular pH (pHi) were measured in identified neuropile glial cells in the central nervous system of the leech Hirudo medicinalis, using the fluorescent dye fura-2, and double-barrelled, neutral carrier, pH-sensitive microelectrodes. Different stimuli were used to elicit Ca2+i and/or pHi changes, such as application of ammonium, high external K(+)-concentration, and low external pH. Ammonium (20 mM) and high external K+ (20 mM), which depolarized the glial membrane by 20-30 mV, evoked rapid and large rises of Ca2+i. In contrast to the Ca2+i changes, amplitude and direction of the pHi changes were dependent on the presence of CO2/HCO3- in the saline. The addition of CO2/HCO3-, and the subsequent reduction of external pH from 7.4 to 7.0, had no effect on Ca2+i, but caused significant changes of pHi. The results suggest that the ammonium- and K(+)-induced Ca2+i rises were due to the membrane depolarization leading to a Ca2+ influx through voltage-gated Ca2+ channels in the glial membrane, while the pHi changes resulted from movements of ammonia and from the activation or inhibition of the Na(+)-HCO3- cotransporter. This indicates that changes of intracellular Ca2+ and pH can occur independently of each other, suggesting that the homeostasis of these ions is not necessarily interrelated in these glial cells.

Ammonium Chloride↗
PubMed1993

Single ion channel currents in neuropile glial cells of the leech central nervous system.

The patch-clamp technique was used to investigate the activity of single ion channels in neuropile glial (NG) cells in the central nervous system (CNS) of the medicinal leech, Hirudo medicinalis. We found evidence for two distinct Cl- channels that could be distinguished by their basic electrical properties and their responses to different inhibitors on single ion channel currents. In the inside-out configuration in symmetrical Cl- solutions, these channels showed current-voltage relationships with slight outward rectification, mean conductances of 70 and 80 pS, and reversal potentials near 0 mV. Significant permeability to Na+, K+, or SO4(2-) could not be detected. The open-state probability of the 70 pS Cl- channel increased with membrane depolarization, whereas the open-state probability of the 80 pS Cl- channel was voltage-independent. The application of the stilbene derivative DIDS (100 microM) to the cytoplasmic side of the glial cell membrane blocked both Cl- channels. The activity of the 70 pS channel was blocked irreversibly by DIDS, whereas the activity of the 80 pS channel reappeared after wash-out of DIDS. Both channels were blocked reversibly by 1 mM Zn2+. K+ channels could only be observed occasionally in the soma membrane of the NG cells. We have characterized a 60 pS K+ channel with a high selectivity for K+ over Na+. The low density of K+ channels in the soma membrane may indicate a non-uniform distribution of this channel type in NG cells.

Action Potentials↗
PubMed1994

Fura-2 signals evoked by kainate in leech glial cells in the presence of different divalent cations.

The glutamate-agonist kainate evokes Ca2+ transients in both neurones and glial cells. Owing to the membrane depolarization elicited by kainate, a Ca2+ influx could occur through voltage-gated Ca2+ channels or through the kainate-gated cation channels directly. We have measured ratio signals of the calcium indicator dye fura-2, injected into giant glial cells of the leech Hirudo medicinalis, as response to kainate (5-20 microM) in the presence of different divalent cations. The responses to kainate increased during the first 2-4 kainate applications, both in unclamped and in voltage-clamped cells. The fura-2 fluorescence ratio (F350/F380) still increased when Ca2+ was replaced by Ba2+ but was suppressed in Ca(2+)-free saline and in the presence of Ni2+ (2 mM). Co2+ and Mn2+ (2 mM) also reduced the kainate-induced fura-2 fluorescence signals, due to entry of these divalent cations into the cells and subsequent quenching the fluorescence of the intracellular dye. It is concluded that Ni2+ blocks the kainate-induced membrane depolarization and Ca2+ transient but apparently does not enter the cells, while Ba2+, Co2+, and Mn2+ appear to permeate the membrane, presumably through the kainate-gated channels.

Animals↗
PubMed1994

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors.

The effect of glutamatergic agonists on the intracellular free Ca2+ concentration ([Ca2+]i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca2+]i levels of both cell types were almost the same (glial cells: 58 +/- 30 nM, n = 51; Retzius neurones: 61 +/- 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca2+]i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca2+ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca2+. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strength of DNQX. In Retzius neurones the agonist-induced [Ca2+]i increase was abolished after replacing extracellular Na+ by organic cations or by mM amounts of Ni2+, whereas in glial cells the [Ca2+]i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca2+ influx is predominantly mediated by voltage-dependent Ca2+ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.

Animals↗
PubMed1994

The pseudoblastema in the wound healing process of the leech Hirudo medicinalis L. (Hirudinea): changes in cell junctions.

The role of the connective tissue cells and their migratory behavior have been investigated in the formation and evolution of the pseudoblastema during wound healing in the leech Hirudo medicinalis. In H. medicinalis the healing process shows first a flow of cells that effectively seal the wound and form a temporary cellular clump, the pseudoblastema, which contributes to the phagocytotic process and apparently regenerates the extracellular matrix. The migratory cells forming the pseudoblastema are connective tissue cells known as vasocentral cells, which, when in a resting state, are associated with the vasofibrous cells. During the formation and evolution of the pseudoblastema several changes affect vasocentral cell junctions. At rest, vasocentral cells do not show cell to cell junctions but they show adhering junctions in contact with the extracellular matrix. These junctions disrupt during the migratory phase. When vasocentral cells regroup in the pseudoblastema, adhering junctions are formed between them, and adhering junctions making contact with the matrix appear again. As the pseudoblastema evolves, cell to cell adhering junctions become more conspicuous and undergo other changes. During the next stage of retraction, close contacts develop between pseudoblastema cells and neighboring nondamaged muscle fibers, which probably serve as points of anchorage for the approaching movement of the wound edges. Finally, cell to cell and cell to matrix junctions disappear and the pseudoblastema disintegrates.

Animals↗
PubMed1988

Ultrastructure of the anterior salivary gland cells of the giant leech, Haementeria ghilianii (Annelida, Hirudinea).

The giant anterior salivary gland cells from the large mammalian blood-sucking, glossiphoniid leech, Haementeria ghilianii, can be subdivided into three morphologically and functionally distinct regions: 1) a soma, responsible for the synthesis and storage of secretory products; 2) a long cell process, responsible for the storage and intracellular transport of the secretory vesicles; and 3) the site of exocytosis at the process terminal. The giant somata are densely packed with secretory vesicles. Deep plasmalemmal invaginations invade the soma and form an extensive system of extracellular lacunae. The rough endoplasmic reticulum (ER) and the Golgi apparatus are organized in the cell periphery, near the highly branched nucleus, and along the lacunae. The somata taper into long processes extending over several centimeters to the proboscis tip. These contain secretory vesicles through their whole length. In the process periphery, the vesicles are completely ensheathed by a concentric subplasmalemmal smooth ER cisterna. This originates deeply within the soma and extends through the whole cell process to its terminal. The ER provides support for up to several hundred longitudinally oriented microtubules. Secretion occurs at the very tip of the cell processes, each of which terminates at the proboscis tip at the base of a cuticular pore. We found synapses close to the sites of exocytosis, providing morphological evidence for neuronal control of secretion.

Animals↗
PubMedDate not supplied

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to serine (pro)enzymes: a comparative thermodynamic study.

The binding of the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis to serine (pro)enzymes belonging to the chymotrypsin and subtilisin families has been investigated from the thermodynamic viewpoint, between pH 4.5 and 9.5 and from 10 degrees C to 40 degrees C. The affinity of eglin c for the serine (pro)enzymes considered shows the following trend: Leu-proteinase [the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves] greater than human leucocyte elastase congruent to human cathepsin G congruent to subtilisin Carlsberg congruent to bovine alpha-chymotrypsin greater than bovine alpha-chymotrypsinogen A congruent to porcine pancreatic elastase congruent to bovine beta-trypsin. The serine (pro)enzyme-inhibitor complex formation is an entropy-driven process. On increasing the pH from 4.5 to 9.5, the affinity of eglin c for the serine (pro)enzymes considered increases thus reflecting the acid pK shift of the invariant hystidyl catalytic residue from approximately to 6.9 in the free serine proteinases and bovine alpha-chymotrypsinogen A to congruent to 5.1 in the serine (pro)enzyme-inhibitor complexes. Considering the known molecular models, the observed binding behaviour of eglin c was related to the inferred stereochemistry of the serine (pro)enzyme-inhibitor contact regions.

Animals↗
PubMed1983

Cross-reactivities of monoclonal antibodies between select leech neuronal and epithelial tissues.

Three monoclonal antibodies, originally studied because of their neuron-specific staining in the leech central nervous system, are characterized further here, both immunocytochemically and biochemically, with Western blot staining using the central nervous system and peripheral tissues. The three antibodies react both with neurons and select epithelial tissue in the central nervous system, gut, and penis. Antibody Lan3-8 reacts with neurons in the nerve cord and gut but with epithelial cells in the penis; it binds to a 65K molecule in all three tissues. Lan3-2 and Laz2-369 are considered as a related pair because in the central nervous system the former stains all (four) and the latter generally only half (two) of the neurons in a standard midbody ganglion responding to nociceptive stimulation. In the gut, both antibodies label patches of epithelial cells and Laz2-369 stains a previously unknown type of gut neuron. While a given antibody stains different bands in gut and central nervous system immunoblots, comparing the bands of both antibodies for the same tissue extract makes it apparent that there are similarities in the molecular species that both antibodies recognize. For each monoclonal antibody, the histologically identified tissue antigens need to be correlated with proteins identified on Western blots. Of particular interest are the broad 130K bands to which Lan3-2 and Laz2-369 bind. The question is raised whether the molecular species in these bands represent a family of proteins that serve a specific nociceptive cell function.

Animals↗
PubMed2003

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS.

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.

Animals↗
PubMed2001

Calcium transients in subcompartments of the leech Retzius neuron as induced by single action potentials.

Regional Ca(2+) influx into neurons plays an essential role for fast signal processing, yet it is little understood. We have investigated intracellular Ca(2+) transients induced by a single action potential (AP) in Retzius neurons in situ of isolated ganglia of the leech Hirudo medicinalis using confocal laser scanning microscopy in the cell body, in different axonal branches, and in dendrites. In the cell body, a single AP induced a Ca(2+) transient in submembrane regions, while in central regions no fluorescence change was detected. Burst activity evoked a much larger Ca(2+) influx, which elicited Ca(2+) signals in central somatic regions, including the cell nucleus. A single AP induced a Ca(2+) transient in distal branches of the axon and in dendrites that was significantly larger than in the proximal axon and in the cell body (p <.05), and the recovery of the Ca(2+) transient was significantly faster in axonal branches than in dendrites (p <.01). The AP-induced Ca(2+) transient was inhibited by Co(2+) (2 mM). The P/Q-type Ca(2+) channel blocker omega-agatoxin TK (500 nM) and the L-type Ca(2+) channel blocker nifedipine (20 microM) had no effect on the Ca(2+) transient, whereas the L-type Ca(2+) channel blocker methoxyverapamil (D600, 0.5-1 mM) irreversibly reduced the Ca(2+) transient by 37% in axons and by 42% in dendrites. Depletion of intracellular Ca(2+) stores following inhibition of endoplasmic Ca(2+)-ATPases by cyclopiazonic acid (10 microM) decreased the AP-induced Ca(2+) transient in the dendrites by 21% (p <.01), but not in axons, and increased the Ca(2+) recovery time constant (tau) in the axonal branches by 129% (p <.01), but not in dendrites. The results indicate that an AP evokes a voltage-gated Ca(2+) influx into all subcompartments of the Retzius neuron, where it produces a Ca(2+) signal of different size and/or kinetics. This may contribute to the modulation of electrical excitation and propagation of APs, and to different modes of synaptic and nonsynaptic processes.

Action Potentials↗
PubMed2004

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons.

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.

Analysis of Variance↗
PubMed1976

Magnesium-resistant excitatory synaptic potentials in the leech Retzius cell.

Postsynaptic potentials (PSPs) recorded from leech Retzius cells in response to stimulation of interganglionic connective could not be reversed by soma depolarization or abolished by 40 mM Mg ion, nor could input resistance changes be detected during them. Alteration of external Cl and K over a tenfold range provided no clear evidence that the PSPs involved a conductance change to either ion. The method of extrapolation yielded an apparent PSP equilibrium potential of about -20 mV. The steep portion of the relationship between Retzius cell action potential amplitude and membrane potential extrapolated to an apparent reversal potential of -13 mV. It is likely that the connective-to-Retzius cell PSPs were principally electrical events. Their apparent reversal potentials could have been in the range associated with chemical synapses because they traversed an electrical synapse with a variable coupling resistance, or because the polarizing currents, passing "backwards" across electrical synapses, changed the amplitude of the presynaptic action potentials.

Action Potentials↗
PubMed1976

The identification of two inhibitory cells in each segmental ganglion of the leech and studies on the ionic mechanism of the inhibitory junctional potentials produced by these cells.

The present study identifies a pair of inhibitory cells that are located on each anterolateral margin of a leech segmental ganglion. These cells, which we label as cells 119, are electrically interconnected. These cells give rise to inhibitory junctional potentials (ijp's) in contralteral longitudinal body wall muscle cells. The latencies of the ijp's following spikes in cell 119 are variable. The ijp's are caused by transient increases in premeability to the Cl- ion. Previous studies demonstrated that 5-HT causes a hyperpolarization of body wall muscle cells by increasing the permeability of muscle membrane to the Cl- ion. Accordingly, 5-HT was searched for in the 119 cell bodies, but autoradiography, fine structure, and gas chromatography-mass spectrometry gave no indication that 5-HT was present in these cells. However, the variable latencies of the ijp's may indicate that there is a neuron interposed between cell 119 and the muscle cells. If this is the case, then the interposed neuron should be analyzed for 5-HT. Further experiments to locate the terminals of cells 119 and the cell bodies of the presumed interposed neurons are thus desirable.

Action Potentials↗
PubMed1980

The projections of neurosecretory cells in the brain of the North-American medicinal leech, Macrobdella decora, using intracellular injection of horseradish peroxidase.

The projections of four anatomically distinct groups of putative neurosecretory cells found within the supra-oesophageal ganglion of the leech Macrobdella decora were studied by intracellular injection of horseradish peroxidase. All four groups have their own characteristic branching pattern while sharing the common feature of possessing primary branches that project into the dorsal commissure. Numerous secondary processes extend from these primary branches to terminate within the neural lamella, as well as within the neuropile. Electron microscopy of the regions into which these secondary processes project reveals numerous neurosecretory terminals. The data suggests that the midregion of the dorsal commissure constitutes a neurohemal complex. These observations strengthen the argument that the four groups of identified cells are indeed neurosecretory.

Animals↗
PubMed1986

Differential time course of the response to axotomy induced by cut or crush in the leech AP cell.

The time course of the reaction to axotomy in the leech AP cell was determined by measuring the duration of the spontaneous spikes at different times after the operation. The axotomy performed by section of the segmental roots containing the AP axon induced an increase of the spike duration, which persisted over 30 days. A different time course was found when the axotomy was performed by nerve crush: the changes in duration of the spontaneous spikes, which occurred during the early 2 weeks, were significantly reduced afterwards. Dye staining of some cells axotomized by crushing revealed that the reversion of the changes, which had been set up by axotomy, was in some cases concomitant with the reconnection between proximal and distal axon stumps. The section of a single axonal branch was never sufficient to affect the membrane properties of the AP cells. It is concluded that the changes observed in axotomized AP cells are not produced by simple axonal injury and that the maintainance of normal properties in the somatic membrane requires the presence of at least part of the distal axon arborization.

Animals↗