Blocking action of Nephila clavata spider toxin on ionic currents activated by glutamate and its agonists in isolated hippocampal neurons

The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10^–4–10^–5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 152–160, March–April, 1989.     

Ionic mechanisms of depolarization responses in Helix pomatia neurons to glutamate application

Reversal potentials of transmembrane ionic currents induced by glutamate were determined in various D neurons ofHelix pomatia. Two types of neurons were found with mean reversal potentials of –10.6±1.2 and –40.0±0.6 mV. Neurons of the first group responded under ordinary conditions to glutamate application by a volley of action potentials. Neurons of the second group did not generate action potentials under the same conditions during glutamate application. With an increase in the dose of mediator the amplitude of D responses in these neurons increased only up to a certain limit, without reaching the critical depolarization level of the cell; a fall in the external chloride ion concentration led to a decrease in their reversal potential. The possible ionic mechanisms of glutamate-dependent depolarization responses of these groups of neurons are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 572–577, November–December, 1982.     

Interaction between sea anemone toxin BgTX8 and ionic channels of neurons isolated from mammals

The action of the toxin BgTX₈ separated from the sea actiniaBunodosoma granolifera on transient tetrodotoxin-sensitive sodium and outward potassium currents of units isolated from rat sensory ganglia was investigated using techniques of voltage clamping at the membrane and intracellular perfusion. It was found that BgTX₈ decelerates the inactivation kinetics but has little effect on activation kinetics of sodium current. At the same time, a 5–10% increase in the amplitude of inward current was often observed at holding potentials of about –100 to –120 mV at the membrane. The dissociation constant of the receptor-toxin equals 4×10^–6 M and is adequately described by Langmuir's isotherm. It was also established that intracellular perfusion of neurons with anemone toxin-containing solution leads to a reduction in the amplitude of sodium current and decelerates its inactivation process. Suppression of outward potassium current was also noted.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Brain Research, Academy of Sciences, Havana, Cuba. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 32–37, January–February, 1988.     

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Effects of allapinine on sodium currents in neurons isolated from the rat trigeminal ganglion and cardiomyocytes

The effects were investigated of allapinine, diterpene alkaloid on ionic currents in voltage-clamped trigeminal neurons and cardiomyocytes isolated from rats. Allapinine application was found to exert an inhibitory effect on inward tetrodotoxin-sensitive sodium currents without changing their voltage dependence. Potential differences between the mechanisms of antiarrhythmic action of diterpene alkaloids and standard antiarrhythmic substances are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Plant Chemistry, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 201–206, March–April, 1990.     

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.     

Effects of low concentrations of tetradotoxin on rat trigeminal ganglion neurons

Voltage clamping and intracellular perfusion methods were used to investigate ionic currents produced by depolarizing shifts of –120 mV from holding potential during experiments on neurons isolated from the trigeminial ganglion of one-month-old rats. It was found that tetradotoxin at low (external) concentrations of 10^–12–10¹⁰ M produced an increase in the amplitude and alternations in the kinetics of inward ionic currents. Similar effects were observed in 8 test cells out of 29. The extent to which the increase in the amplitude of inward ionic currents depended on concentration level could be described by Langmuir's isotherm, with a dissociation constant of the order of 5·10^–12 M. No such tetrodotoxin effects were observed when chloride ions were replaced by a non-penetrating anion in the intracellular solution. It is suggested that tetrodotoxin-sensitive channels exist in the neuronal membrane of the rat trigeminal ganglion, letting through chloride ions during depolarization of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 723–729, November–December, 1986.     

Blocking action of calmidazolium and chlorpromazine on outward potassium current dependent on extra-cellular calcium ions

The effects of the calmodulin antagonists, calmidazolium (R 24571) and chlorpromazine on delayed outward potassium current at the somatic membrane were investigated in non-identified intracellularly perfused neurons isolated fromHelix pomatia. Voltage was clamped at the membrane. Extracellular application of these substances produced effective depression of the outward current. This effect even occurred at test substance concentrations of 10^–9–10^–8 M. Block-ade of delayed outward current was produced mainly as a result of suppressing the potassium current component dependent on intracellular potassium ions (I_k(Ca/in)). The possibility that the receptor for intracellular calcium responsible for modulating this current may be of a calmodulin-like nature is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 356–361, May–June, 1987.     

Action of a nonhydrolyzable guanosine triphosphate analog on modulation of calcium current by dopamine in Lymnaea stagnalis neuron

Dependence of the blocking effect of dopamine on the calcium current on guanosine triphosphate (GTP) was investigated on dialyzed neurons ofLymnaea stagnalis. Against the background of the effect of the nonhydrolyzable GTP analog guanosine-5-0-3-thiotriphosphate (GTPS) (10^–4 M) marked residual blocking of the calcium current was observed during rinsing out of dopamine, and this was potentiated in the case of repeated application of the hormone. It is suggested that the receptor-mediated effect of GTPS on the calcium current is effected through activation of phospholipase C.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 43–48, January–February, 1991.     

Taurine-activated currents in isolated neurons of the rat cerebellum

Taurine-activated currents were investigated in rat cerebellar neurons using techniques of voltage clamping at the membrane and intracellular perfusion. Activation of both chloride and calcium conductance at the membrane were produced by applying taurine to the membrane surface. The dose-response curve for taurine-activated current is in the 1×10^–4–1×10^–1 M concentration region. The dissociation constant of the taurine-receptor complex equals 2×10^–3 M. Activation of taurine-induced currents is a cooperative process: Hill's coefficient –2. It was found that bicuculline and strychnine exert a blocking action on taurine-activated currents, while pentobarbital and oxazepam potentiate taurine action.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 6, November–December, 1990, pp. 780–786.     

Effects of glutamic acid diethylester on synaptic transmission in the skate ampullae of Lorenzini

The effects on synaptic transmission of glutamic acid diethylester (GDEE), a glutamate receptor blocker, were investigated by recording spike activity from single nerve fibers in the electroreceptor cells of the skate (Raja clavata) ampullae of Lorenzini. It was found that adding GDEE to the bathing medium led to a concentration-dependent reduction in or complete blockade of background and evoked receptor activity; 10^–6 M GDEE was the minimum effective concentration. It was also shown that GDEE reversibly blocked postsynaptic response produced by excitatory amino acids: L-glutamate (L-GLU) and L-asparate (L-ASP). Findings suggest the involvement of L-GLU or a related substance in synaptic transmission in the ampullae of Lorenzini.I. P. Pavlov Institute of Physiology of the Academy of Science of the USSR, Leningrad, USSR. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 323–327, May–June, 1987.     

Characteristics of chloride-dependent effects of acetylcholine and neutral aminoacids on neurons of the snail (Achatina)

The response was investigated of neurons composing the cerebral ganglia inAchatina fulica (the Giant African snail) to application of acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly). Chloride-dependent currents induced by these transmitters in 1 1/2-month old siblings were inhibited by dibutyryl-cAMP and strychnine. Inhibition of ACh response produced 10^–8 M GABA was mimicked by application of dibutyryl-cAMP and isobutylmethylxanthine. Complete cross-desensitization was characteristic of both GABA- and Bly-induced response, but this effect did not occur when ACh and GABA (or Gly) were applied. A conclusion was reached on the basis of the pharmacological relationship between GABA- and Gly-induced response that these amino acids act on a single receptor — channel complex in the neurons of infant snails, whereas ACh-, GABA-, and Gly-induced chloride currents were not so related in cells of 4 year-oldAchatina.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 35–43, January–February, 1991.     

Model investigation of firing mechanisms of mammalian neurons

A model of electrical activity of mammalian neurons, based on properties of the corresponding ionic components of the transmembrane current, is suggested. Its analysis showed that two types of firing are possible for these neurons. First, the firing pattern is determined by fast sodium and delayed potassium currents, and the period of oscillations is 74±27 msec. Second, the firing pattern is determined by calcium and Ca-activated potassium currents, and the mean duration of the interspike interval is 850±40 msec. A comprehensive examination of all ionic currents enabled two mechanisms of lengthening of the interspike interval (adaptation) of the discharge to be predicted. One is due to the fast potassium current, the other to accumulation of Ca⁺⁺ ions inside the cell and the development of a Ca-activated potassium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 445–451, July–August, 1984.     

Extrasynaptic action of vasopressin,oxytocin, and vasotocin neuropeptides on electrically operated ionic channels at the mollusk neuronal membrane

Techniques of intracellular dialysis and neuronal perfusion in the visceral ganglion ofLymnaea stagnalis used during voltage-clamping at the neuronal membrane helped to ascertain that a concentration of 1×10^–16–1×10^–6 M neuroactive peptides (vasopressin, oxytocin, and vasotocin) alter the amplitude of electrically-operated transmembrane ionic currents considerably without affecting the kinetics of current activation and inactivation and surface potential at the membrane. The experimental conditions applying made it possible to record incoming sodium and calcium currents separated from each other as well as outward delayed and transient potassium currents. It was found that electrically-operated cerebral currents could either increase or decline in amplitude under the effects of peptides applied at different concentrations to the membrane of the same unit. Receptors of the peptides investigated in this study are thought to be located within the structure of electrically-operated channels at the neuronal membrane.A. I. Gertsen Teaching Institute, Leningrad. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 526–533, July–August, 1990.     

Single potassium channel of anomalous (inward) rectification in mollusk neurons

Currents passing through individual potassium channels with anomalous (inward) rectification were recorded at the neuronal membrane ofPlanorbarius corneus using the patch clamp technique. These currents could be detected, whether in "right side out" or "inside out" configurations in the presence of 50 mM potassium ions or one of the potassium channel blockers: tetraethylammonium (TEA), barium, or cesium (2–20 mM) on the external side of the membrane. Inward currents were observed in individual channels at potentials more negative than level of potassium equilibrium potential (E_k); conductance of these measured 81±12 pS (n=11). At more positive potentials than E_k, conductance fell to zero. Potassium channels with anomalous (inward) rectification inPlanorbarius corneus resemble equivalent channels in other cells in their kinetics: time scale of the open state may be described by a single exponential function. This would imply that the ionic channel has a single open state. Time scale of the closed state was biexponential, thus indicating the possible existence of two kinetically different nonconducting states of the potassium channel with anomalous (inward) rectification at the neuronal membrane ofPlanorbarius corneus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 31–38, January–February, 1989.     

Interaction between pentobarbital and GABA-activated ionic channels in rat cerebellar neurons

Using techniques of voltage clamping at the membrane, intracellular perfusion, and concentration clamping, GABA- and barbiturate-activated currents were investigated in single neurons isolated from the rat cerebellum. The dissociation constant for interaction between GABA and GABA receptors was measured at 3±0.8 × 10^–5 M. The presence of pentobarbital in the bathing solution exerts a potentiating effect on GABA-induced conductance in isolated neurons, shifting the dose-response curve for GABA towards lower concentration values without increasing peak chloride conductance. The concentrations at which GABA effects are potentiated range between 10^–6–10^–4 M. High concentrations of pentobarbital inhibit GABA-activated conductance; at concentrations in excess of 5 × 10^–4 M, it also brings about activation of chloride conductance, depressed by bicuculline and picrotoxin, in the absence of GABA. A short-term increase in membrane conductance is produced by rapid pentobarbital washout.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 93–98, January–February, 1990.     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

Response induced in prog motoneurons by glutamate application

It was shown during experiments on isolated frog brain (fromRana ridibunda) that response to microelectrophoretically injected glutamate on to various points on the somatodendritic motoneuronal membrane (GLU response) displayed the same properties as EPSP induced in the same motoneuron by activation of three different synaptic inputs. Techniques of transmembrane polarization and current chop by means of a single microelectrode were used in this research. Mean levels of reversal potentials of GLU response and EPSP occurring as a result of stimulating the reticular formation, dorsal root, and microstimulation of presynaptic elements at the point of glutamate application equaled –16.9 ± 1.7 (n=13), –6.8 ± 1.7 (n=13), –9.8 ± 1.8 (n=6), and –15.1 ± 1.4 mV (n=13), respectively. Summation of GLU response and EPSP were quasilinear. Changes (upwards) in conductance associated with GLU response did not exceed 10%. Findings would indicate that glutamate, acting on the postsynaptic membrane receptors, induces depolarization and may serve as transmitter in all three inputs investigated.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 776–785, November–December, 1988.     

Naloxone-dependent depression by morphine of responses of snail neurons to serotonin

Morphine, added to the extracellular solution in a concentration of 1·10^–5 M, quickly and reversibly weakens the depolarizing and hyperpolarizing responses of neurons of the snailHelix lucorum evoked by 1·10^–6 M serotonin. The inhibitory effect of morphine is completely abolished by the addition of naloxone (1·10^–5 M), suggesting that opiate receptors are involved in the process. Interaction between morphine and serotonin is noncompetitive in type, as is shown by the character of the dose-effect curves recorded during the action of serotonin before and after morphine application.Institute of Psychiatry, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 6, pp. 589–593, November–December, 1981.     

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.