Electron cytochemical study of carbogydrate components of surface membrane in cultured glial cells of the snailHelix pomatia

Using a variety of colloidal gold-labelled lectins, the structure and topography of carbohydrate determinants of the surface membrane in different types of cultured glial cells of the snailHelix pomatia have been electron cytochemically investigated. Analysis of lectin binding having different sugar specificities have shown heterogeneity of carbohydrate pools between glial and nerve cells and among different types of glial cells. It was found that satellite glial cells displaying ultrastructural traits of intensive metabolism (type II cells) selectively bindGNA, which is specific for terminal -D-mannose residues, and do not interact (Con A) or slightly interact (LCA) with other mannose-specific lectins.GNA determinants remain during the whole period of cell growth and are absent in satellite type-I glial cells, fibrous glial cells, microglia, and neurons.LTA, PVA, andLABA do not bind to any glial cells.WGA determinants, which are abundant on the neurons, are completely absent onGNA-binding glial cells and single on other types of glial cells. The density ofPNA determinants on microglial cells is the highest, as compared with other types of glial cells or neurons. It is concluded that some lectin determinants (forRCA-1, PNA, LPA) are present on all types of glial cells, while another determinant (GNA) is specific for a certain type of glial cells only and can serve as a marker of these cells. The role of specific carbohydrate determinants for neuron-glia interaction in mature brain is discussed.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 177–189, May–June, 1994.     

Use of lectins to investigate the carbohydrate determinants of cholinergic receptors of various subtypes in molluscan neurons

The chemical nature of the carbohydrate determinants of the plasma membrane that affect the responses of identified neurons of the molluskHelix pomatia, induced by application of acetylcholine (ACh), was studied using D-mannose-specific (ConA), D-galactose-specific (RCA), N-acetyl-D-glucosamino-specific (WGA), and sialo-specific (LPA) lectins. Differences in the change in the Ach-induced chloride and sodium-potassium currents under the action of ConA and in the time and temperature dependences of the effects of ConA for these currents are evidence of the presence of different mechanisms of the influence of carbohydrate determinants containing mannose residues on the functional properties of the cholinergic receptors. On the basis of the time and temperature dependences of the inhibiting effects of WGA and RCA on the ACh-induced currents, it was suggested that endocytosis of the lectin-receptor complexes containing N-acetyl-D-glucosamine residues occurs.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 2, pp. 161–169, March–April, 1992.     

Interactions of endogenous lectin from theHelix pomatia reproductive system (HPA) with the surface of CNS neurons of the snail

The surface of unidentified cultured neurons and electrophysiologically identified units RPa1, RPa2, and LPa3 of the snailHelix pomatia was studied using a colloidal gold-labelled endogenous gonad lectin of this molluse (Helix pomatia agglutinin -HPA), which is specific to -anomer N-acetyl-D-galactosamine, and some other lectins. It was found that only two populations of cultured neurons with a specific ultrastructure typical of peptidergic cells and differing from the ultrastructure of other peptidergic and non-peptidergic neurons possessHPA receptors. These neurons average about 1% of cultured nerve cells. Using a variety of plant lectins withHPA-like specificity (LBA, VVA, SBA) showed that the surface of many peptidergic neurons contains -anomer-like structures, yet it does not bindHPA. These data were supported in the experiments on identified snall neurons with the use ofSBA. The results suggest a putative role of endogenous lectins in regulation of activity of neurons participating in processes of secretion in the gonads.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 17–29, January–February, 1996.     

Effects of applying oxytocin to Helix pomatia neurons. II. Hyperpolarization

The ionic mechanisms of hyperpolarization produced by applying oxytocin (OT) were investigated at the membrane of identifiedHelix pomatia neurons. Two types of neuron were known to exist, in one of which hyperpolarization is produced by a reduction in chloride ions at the membrane and a rise in membrane permeability to potassium ions in the other. In the first of these, response to OT had a reversal potential of –40 mV and decreased when furosemide and tolbutamide were added to the external medium. In the second case, the potential of the reversal of the response to OT was –70 mV. Upon doubling of potassium ion concentration in the external solution it was shifted towards depolarization by 15 mV. It is sugested thatHelix pomatia neurons have different types of OT receptors, some of which, when activated, manifest reduced chloride permeability at the membrane (probably through the cell cyclase system) with a rise in potassium permeability at the membrane in others.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 659–666, September–October, 1988.     

Multiplicity of pacemaker zones in Helix pomatia neurons

Intracellular microelectrode recordings from neurons ofHelix pomatia revealed several local zones of action potential generation both on the soma and on some of the branches of the neurons. Under certain conditions the activity of individual loci of the neuron membrane was synchronized to produce a normal action potential. It is suggested that the somatic membrane of neurons is heterogeneous in structure and consists of separate loci of an electrically excitable membrane, incorporating active and latent pacemaker zones. Neurons ofH. pomatia are characterized by two types of action potential with different triggering mechanisms: one (synaptic) type is generated under the influence of the EPSP, the other (pacemaker) arises through activation of endogenous factors for the neuron (pacemaker potentials). The interaction between synaptic and pacemaker potentials during integrative activity of the neuron is discussed.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 5, No. 1, pp. 88–94, January–February, 1973.     

Effects of arginine-vasopressin and its derivatives on Helix pomatia cerebral neurons

Response to application of and superfusion with solutions containing arginine-vasopressin and its derivatives (VPS), was investigated in identifiedHelix pomatia neurons. Different VPS exerted a similar effect on neurons in all cases. De- and hyperpolarizing as well as modulatory effects were shown. Depolarizing and hyperpolarizing response was accompanied by a rise and fall in steady-state conductance of the cell membrane. Reversal potential of response was determined as in the region of chloride reversal potential. Adding furosemide to the extracellular solution reversibly inhibited response to VPS. It was concluded from this that both de- and hyperpolarizing response took the form of an increase in the amplitude of trans-membrane ionic current induced by injecting cAMP into the neuron under the effects of superfusing the preparation with a VPS-containing VPS solution. Specific VPS receptors, probably associated with the cell cyclic nucleotide system, are thought to exist at the membrane of someHelix pomatia neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 368–373, May–June, 1990.     

Electron cytochemical study of carbohydrate components in cultured nerve and glial cells of the snail Helix pomatia

• 1.1. A variety of colloidal gold-labelled lectins with different sugar specificities to determine whether different nerve and glial cells of the snail Helix pomatia cultured in vitro, can be distinguished by the carbohydrates that they express was screened. The analysis of lectin binding has shown substantial differences in the carbohydrate pattern between nerve and glial cells and between the soma of monoaminergic and peptidergic neurons.
• 2.2. The surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine and N-acetyllactosamine determinants, and does not exhibit neuraminic acid and complex branched N-glycosyl chains. Moreover, N-acetylgalactosamine can be detected on peptidergic neuron membranes only.
• 3.3. N-Acetylglucosamine residues are not present on the surface of the glial cells, and the density of the N-acetyllactosamine and/or terminal β-galactose residues is much higher here than on the surface of the nerve cells.
• 4.4. These results suggest that nerve cells in the snail brain can be distinguished from glial cells by the presence of a cell-surface glycoconjugate containing terminal N-acetyl-d-glucosamine residues, whereas peptidergic neurons can be distinguished from monoaminergic neurons by the presence of a surface glycoconjugate containing terminal α-linked N-acetyl-d-galactosamine residues.

     

Effect of the alkaloid lappaconitine on ionic currents through the somatic membrane ofHelix pomatia neurons

The effect of the alkaloid lappaconitine on passive ion transport through the somatic membrane of identified neurons of the snailHelix pomatia was studied under voltage clamp conditions. In a concentration of 4 mM lappaconitine has a reversible blocking action on the calcium channels of the excitable membrane. To study the effect of the alkaloid on inward sodium currents a solution in which calcium ions were replaced by the equivalent number of magnesium ions was used. Lappaconitine has no appreciable effect on the inward sodium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 469–474, September–October, 1979.     

Action of calcium on the somatic membrane of mollusk giant neurons

Early membrane currents of the isolated neuron soma of the mollusksHelix pomatia,Limnaea stagnalis, andPlanorbis corneus in normal and sodium-free solutions differing in their calcium ion concentration were investigated by the voltage clamp method. The early inward current was shown to continue when the sodium ions in the external solution were replaced by an equivalent number of calcium ions and to be increased with an increase in the concentration of those ions in all neurons of these mollusks investigated. A change in the calcium concentration in the external solution shifted the inactivation curves and also the curves of conductance for the inward current along the potential axis. It is concluded that a system of calcium channels exists in the somatic membrane of neurons in these species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 621–627, November–December, 1973.     

Influence of caffeine on processes of regulation of the intracellular Ca2+ concentration in isolated neurons of the edible snail

Changes in the intracellular concentration of Ca²⁺ ([Ca²⁺]_in) that occur during prolonged depolarization of the plasma membrane were studied in isolated neurons of the edible snailHelix pomatia, using the calcium-sensitive probe Fura-2. The dependence of the amplitude of the calcium response on the value of the depolarization in the presence of 5 mM caffeine, in contrast to that observed in a normal solution, practically disappeared. This fact indicates that caffeine promotes calcium-dependent release of Ca²⁺ from the intracellular depots, which is the determining factor in the increase in [Ca²⁺]_in during depolarization. Processes of reduction of [Ca²⁺]_in to the steady-state levels were described by one exponential function, and in the presence of caffeine they occurred twice as rapidly as in the normal solution. Such an acceleration of the kinetics of the relaxation of [Ca²⁺]_in is evidently associated with an increase in the efficiency of the work of the calcium pump of the intracellular calcium depots, which might lead to a decrease in the steady-state of level of [Ca²⁺]_in even below the level observed for the normal extracellular solution.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 66–73, January–February, 1991.     

Involvement of calcium-binding membrane components in neurophysiological mechanics of habituation in Helix pomatia

Dynamics of binding between calcium and hydrophobic membrane components were investigated in vivo in identified neurons ofHelix pomatia while producing habituation to tactile stimuli using a fluorescent chlortetracycline probe technique. A decline in the concentration of membrane-bound calcium (Ca _b ²⁺ ) and likewise in the intensity of electrophysiological response was found in the "command" neurons of defensive behavior when applying a train of stimuli. An increase in Ca _b ²⁺ was noted in the sensory neuron studied and in the spiracle motoneurons. It proved difficult to produce habituation in these cells fulfilling standard electrophysiological criteria. Hydrophobe-hydrophil transfer of calciumbinding molecules is thought to accompany production of habituation in nerve cells.P. K. Anokhin Institute of Normal Physiology, Academy of Medical Sciences of the USSR, Moscow. I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 5, pp. 605–612, September–October, 1989.     

Ionic mechanisms underlying modulating effects of serotonin on acetylcholine-induced responses in Helix pomatia neurons

The ionic mechanisms underlying modulatory effects of serotonin on acetylcholine-response in identified and nonidentifiedHelix pomatia neurons were investigated using voltage-clamping techniques at the neuronal membrane. External application of 10^–5–10^–4 M serotonin to the membrane of neurons responding to application of acetylcholine depending on Na⁺ depolarization (D_Na response) reduced membrane conductivity during response to acetylcholine without changing reversal potential of acetylcholine-induced current. Acetylcholine (10^–6–10^–4 M) administration took place 1–3 min later. Neurons with response to acetylcholine application dependent on Cl⁺ depolarization (D_Cl response) or hyperpolarization (H_Cl response) behaved similarly. Analogous effects could be produced by external application of theophylline which, together with the latency and residual effect characteristic of serotonin action points to the participation of intracellular processes associated with the cellular cyclase system in the changes produced by serotonin in acetylcholineinduced response. Serotonin brought about a shift in reversal potential and an increase in the acetylcholine-induced current in those neurons where this response was associated with changed permeability at the membrane to certain types of ions. During two-stage acetylcholine-induced response of the D_Na-H_K type, serotonin inhibited the inward current stage. Mechanisms underlying modulatory serotonin action on acetylcholine-induced response in test neurons are discussed in the light of our findings.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 57–64, January–February, 1988.     

Modulatory effects of oxytocin on functional properties of three types of cholinoreceptors in molluskan neurons

It was found that applying 10^–8 M oxytocin (OT) affects the functional properties of three types of cholinoreceptors under conditions of voltage clamping at the membrane of identified ganglia neurons inHelix pomatia. This neuropeptide depressed acetycholine-(ACh-)induced sodium-potassium-calcium current in neuron RB3 without altering reversal potential of ACh-induced current. Two (sub-) types of cholinoreceptors were distinguished on the basis of findings on OT effects on ACh-induced chloride currents; ACh-induced chloride current was reduced by the action of OT on the cholinoreceptors of one of these (neuron F4) and increased in the case of neurons D5 and F86. The effects of applying OT and serotonin were reversible but not cumulative. Injection of OT exerted an action on ACh-induced chloride current independent of that of OT application. Involvement of cyclic adenosine monophosphate in OT-induced bimodal modulation of functional properties of three types of cholinoreceptors was demonstrated.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziology, Vol. 22, No. 1, pp. 87–93, January–February, 1990.     

Selectivity of edta-modified calcium channels to monovalent cations

The characteristics of slow inward sodium currents arising in response to membrane depolarization were studied in experiments on isolated dialyzed neurons of the snailHelix pomatia when the calcium-chelating agent EDTA was added to the calcium-free external solution. Values of the relative permeability of the corresponding ionic channels, determined from the shift of the equilibrium potential, were: P_Na+:P_Li+: +=1.00:0.80:0.55:0.21. The ratio between these values for "fast" sodium channels was 1.00:1.04:0.44:0.19. The induced sodium current was blocked by D-600 and nifedipine, which block calcium channels, more effectively than the calcium current of the same membrane (the corresponding dissociation constants were 10^–5 and 0.8·10^–5 mole/liter for the induced sodium current compared with 2.6·10^–5 and 2.3·10^–5 mole/liter for the calcium current). It is postulated on the basis of these data that the calcium channels have a principal selective filter similar to that of sodium channels, but also an additional binding site for bivalent cations, which prevents entry of monovalent cations into the channel. The addition of calcium-chelating agents to the calcium-free external solution liberates this site and thereby modifies the calcium channel into a sodium channel.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 491–498, September–October, 1982.     

Effects of strontium and barium ions on calcium bindings and transport in nerve cells

Absorption of strontium and barium ions by intracellular organelles after loading the cell with these cations together with their effects on Ca release from the intracellular stores were investigated in neurons isolated fromHelix pomatia using fura-2, a Ca-sensitive fluorescent probe. It was found that strontium ions can successively replace intracellular calcium ions in this response, whereas barium ions are not absorbed by the cell; they block calcium channels of the intracellular stores as well as at the surface membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 820–825, November–December, 1989.     

Effects of quinine on calcium current in mollusk neurons

The effects of quinine on the peak amplitude and the decay of calcium currents (I_Ca) were investigated in nonidentified neurons isolated fromHelix pomatia. A concentration of 1×10^–5–5×10^–4 M quinine was found to produce a reversible dose-dependent deceleration in the decline of I_Ca ("lead" effect) and a reversible, slowly evolving dose-dependent reduction in I_Ca amplitude ("lag" effect). A reduction in amplitude down to half control level is observed at a quinine concentration of 6 ×10^–5 M, while the current-voltage relationship of I_Ca shifts by 5–10 mV towards negative potentials. Results show that quinine successfully blocks calcium channels inHelix pomatia neurons.Institute of Brain Research, All-Union Mental Health Research Center, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 413–417, May–June, 1987.     

Investigations of the ionic mechanisms of bursting activity in Helix pomatia neurons

Steady-state current-voltage characteristics of the membrane and ionic currents arising during changes in membrane potential in bursting neurons ofHelix pomatia were studied by the voltage clamp method. The steady-state current-voltage characteristics of the membrane were shown to have a nonlinear region. Replacement of sodium ions by Tris-HC1 ions in the external solution completely abolishes this nonlinearity. Hyperpolarization of the membrane under voltage clamp conditions leads to the development of an outward current which reaches a maximum and then is inactivated. This current has a reversal potential in the region of the potassium equilibrium potential. Depolarization of the membrane to the threshold value for excitation of uncontrollable regions of the axon hillock causes the appearance of a slow inward current. After reaching a maximum, the inward current falls to zero. A model of generation of waves in a bursting neuron is suggested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 193–202, March–April, 1978.     

Structural features of isolated and perfused molluscan (Helix pomatia) neurons

A qualitative analysis of the ultrastructure of isolated neurons of the molluskHelix pomatia after perfusion was undertaken by scanning and transmission electron-microscopic methods. The results were compared with the electron-microscopic characteristics of the structure of intact neurons isolated both mechanically and by the action of proteolytic enzymes. Intracellular perfusion was shown not to induce significant structural changes in the cytoplasmic matrix. Comparison of the ultrastructural characteristics of isolated intact and perfused neurons showed that the ultrastructure of both the plasma membrane and the membranes of most intracellular organelles remains intact and unaffected by the action of the perfusion fluid. Not only the intracellular organelles, but also the spatial relationships between them remained unchanged in the perfused neurons. Regions of free aqueous phase, in the form of perinuclear edema, were found in the cytoplasmic matrix of the perfused isolated neurons. The conditions for diffusion of substances in perfused isolated cells, which certainly differ from conditions of diffusion in isolated intact neurons, are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 3, pp. 297–302, May–June, 1980.     

Effects of cAMP on calcium currents in mollusk neurons differing in the sensitivity of their calcium conductance to serotonin action

The effects of cAMP and serotonin (5-HT) on calcium current (I_Ca) were investigated inHelix pomatia neurons using voltage clamp and intracellular perfusion techniques. Three types of neuronal response to extracellular application of 5-HT (1–10 µM) were found: reversible blockage of calcium conductance, absence of response, and increase in I_Ca amplitude. Intracellular application of exogenous cAMP was also found to produce an increase in I_Ca in cells stimulated by 5-HT action. Effects of 5-HT and cAMP were non-additive under these circumstances and were potentiated equally by cyclic nucleotide phosphodiesterase inhibitor. Applying cAMP led to no noticeable increase in I_Ca amplitude in cells with calcium conductance unchanged or blocked by 5-HT. Findings would indicate that the stimulating action of 5-HT is mediated by a rise in intracellular level of cAMP. It is postulated that two types of calcium channels differing in their dependence on cAMP metabolism exist; the presence of cAMP-dependent calcium channels at the neuronal membrane fits in with a certain type of 5-HT receptor also present in the cell, moreover. A new approach is suggested for research on isolated neurons, i.e., that of functional identification.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 605–512, October–September, 1990.     

Modulation of endogenous bursting pacemaker activity in a Helix pomatia neuron

Considerable membrane depolarization was shown to arise periodically, at intervals of up to a few minutes, in the PPa1 bursting neuron ofHelix pomatia. Pulses of slow depolarizing current were found by the voltage clamping method. The frequency of the pulses was independent of the holding potential. The equilibrium potential for the slow depolarizing current was about 45 mV. During development of the depolarizing current a region of negative conductivity was observed on the steady-state voltage-current characteristic curve of the membrane. It is suggested that the pulses of slow depolarizing current are associated with the presence of secretory connections between the molluscan neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 6, pp. 606–612, November–December, 1977.