Inactivation of calcium currents in the molluscan neuron somatic membrane

The time course of weakening of inward calcium currents (inactivation) during prolonged (of the order of 1 sec) depolarizing shifts of membrane potential was studied in isolated dialyzed neurons of snailHelix pomatia. This decay of the current recorded in this way can be approximated by two exponential functions with time constants of 20–70 and 250–350 msec, respectively. With an increase in pH of the intracellular solution to 8.5 the fast component of the decay disappeared completely; the kinetics of the slow component in this case was very slightly retarded. It is concluded that the fast component of decay of the recorded current does not reflect a change in the calcium current but is due to parallel activation of the nonspecific outward current; the slow component, however, is true in activation of the calcium current. The rate of inactivation of this current was shown to be determined by its maximal value and not by the level of the depolarizing potential shift and it depends on the conditions of accumulation of calcium ions near the inner surface of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 525–531, September–October, 1982.     

Reversal potential for monosynaptic EPSP in frog motoneurons

Experiments were conducted on brain isolated from the frogRana ridibunda using a current chop technique of transmembrane polarization and discrete measurement of membrane potential by a single microelectrode during intervals between waves of current. It was found that the current-voltage relationship of the motorneuron is non-linear; i.e., membrane resistance decreases considerably in step with increased depolarizing current. After the initial reduction, membrane resistance began to climb back when a more protracted current lasting 1–2 min was applied; consequently membrane potential level shifted towards more positive values of +50 mV and above at current levels of 40–60 nA. It then became possible to bring about complete reversal of monosynaptic EPSP produced in the lumbar motoneurons by stimulation of the brainstem reticular formation or by microelectrode stimulation of the ventrolateral tract descending fibers and to measure reversal potential of these EPSP directly, without resorting to computing or extrapolation. Measurements varied mainly between 0 and –10 mV.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 534–542, July–August, 1986.     

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.     

Changes produced in neuronal excitability by subthreshold depolarization as a possible mechanism of interval selective relationships within the central nervous system

A neuronal process was identified inLymnaea stagnalis nerve cells which may be viewed as one of the mechanisms underlying the interval selectivity previously described in research into the functional relationships between mammalian brain cells. This process takes the form of regularly-occurring changes in excitability resulting in a high probability (of 0.6–1) of neuronal spike response to what had previously been subthreshold depolarizing current pulses following similar subthreshold (conditioning) pulses at intervals specific to each individual neuron. It was found that the cycle of change in neuronal excitability following threshold depolarization did not arise from temporal summation of electrotonic local or postsynaptic neuronal potentials; it was an endogenous (cytoplasmic) process insensitive to transmitter (acetylcholine) application but altering irreversibly under the effects of bombesin, one of the modulator peptides.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad; Institute of Experimental Medicine, Academy of Medical Sciences of the USSR, Leningrad. Translated from Neirofiziologya, Vol. 21, No. 3, pp. 291–299, May–June, 1989.     

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.     

Displacement currents in sodium channels of the ranvier node membrane

The displacement current was recorded in the Ranvier node membrane ofRana ridibunda. This current was shown to be due to conversion of charges from the initial state in which they were when a high negative voltage was present on the membrane into the final state. The dependence of the displacement charge on the membrane potential and state of activation of the sodium channels suggests that the displacement current is connected with activation of the m gates of the sodium channels. Considering the density of the displaced charges, the density of the sodium channels can be estimated to be 5000 channels/µ².A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 410–417, July–August, 1976.     

Possible interaction between synaptic and pacemaker mechanisms of electrical activity in bursting neurons of Helix pomatia

Membrane hyperpolarization induced by short pulses of inward current, by stimulation of the anal nerve, which leads to the appearance of a long IPSP in the neuron, and developing during the appearance of spontaneous IPSPs in the neuron was investigated in neuron RPa1 ofHelix pomatia. Short-term hyperpolarization of the neuron membrane by an inward current (10 msec) led to the development of self-maintained (regenerative) membrane hyperpolarization lasting several seconds. The amplitude and duration of regenerative hyperpolarization increased with an increase in amplitude and duration of the pulse of inward current. The time course of IPSPs arising spontaneously in the neuron and in response to stimulation of the anal nerve was similar to that of regenerative hyperpolarization evoked by a pulse of inward current. It is suggested that regenerative hyperpolarization associated with activation of endogenous mechanisms of regulation of the bursting activity of the neuron may be due not only to short-term membrane hyperpolarization of the test neuron by the electric current, but also to hyperpolarization occurring during IPSP generation.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 67–74, January–February, 1981.     

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.     

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.     

Ionic mechanisms of the transmembrane current evoked by injection of cyclic amp into identified Helix pomatia neurons

Ionic mechanisms of the transmembrane current evoked by injection of cyclic AMP into identified neurons ofHelix pomatia were investigated by the voltage clamp method. Injection of cyclic AMP into neurons RPa3, LPa2, LPa3, and LPl1 was shown to cause the development of a two-component transmembrane (cyclic AMP) current. The current-voltage characteristic curve of the early component is linear in the region from –40 to –90 mV; the reversal potential of the early component, determined by extrapolation, lies between –5 and +20 mV; the current-voltage characteristic curve of the late component also is linear and has a reversal potential between –55 and –60 mV. A decrease in the sodium concentration in the external medium from 100 to 25 mM led to a decrease in amplitude of the cyclic AMP current and to a shift of the reversal potential for the early component by 30–32 mV toward hyperpolarization. It is suggested that the early component of the cyclic AMP current in neurons RPa3, LPa2, LPa3, and LPl1 is associated with an increase in permeability of the neuron membrane chiefly for sodium ions, whereas the late component is correspondingly connected with permeability for potassium ions. Injection of cyclic AMP also caused the appearance of a transmembrane inward current in neuron LPa8, but it was independent of the holding potential and was unaccompanied by any change in membrane permeability. It is suggested that this current may be due to a change in the activity of the electrogenic ion pump.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 526–532, September–October, 1980.     

Role of the inward K rectifier in the repetitive activity at the depolarized level in single ventricular myocytes

The role of the inward K⁺ rectifier in the repetitive activity at depolarized levels was studied in guinea pig single ventricular myocytes by voltage- and current-clamp methods. In action potentials arrested at the plateau by a depolarizing current, small superimposed hyperpolarizing currents caused much larger voltage displacements than at the resting potential and sometimes induced a regenerative repolarization. Around –20 mV, sub- and suprathreshold repetitive inward currents were found. In the same voltage range, small hyperpolarizing currents reversed their polarity. During depolarizing voltage-clamp ramps, around –20 mV there was a sudden decrease in the outward current (I_ns: current underlying the negative slope in the inward K⁺ rectifier steady state I–V relation). During repolarizing ramps, the reincrease in outward current was smaller and slower. During depolarizing and repolarizing current ramps, sudden voltage displacements showed a similar asymmetry. Repetitive I_ns could continue as long as the potential was kept at the level at which they appeared. Depolarizing voltage-clamp steps also caused repetitive I_ns and depolarizing current steps induced repetitive slow responses. Cadmium and verapamil reduced I_ns amplitude during the depolarizing ramp. BRL 34915 (cromakalim), an opener of the ATP-sensitive K⁺ channel, eliminated the negative slope and I_ns, whereas barium increased I_ns frequency (an effect abolished by adding BRL). Depolarization-induced slow responses persisted in an NaCl-Ca-free solution. Thus, the mechanism of repetitive activity at the depolarized level appears to be related to the presence of the negative slope in the inward K⁺ rectifier I–V relation.     

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.     

Effects of applying oxytocin to Helix pomatia neurons. I. Depolarization

Inward current produced by applying oxytocin (OT) to the neuronal soma (OT_I) current) under conditions of voltage-clamping at the cell membrane was investigated inHelix pomatia. Replacing sodium with Tris ions in the external medium produced a considerable decline in OT_I current. A reduction in the external concentration of chlorine ions by replacement with HEPES ions induced an increase in OT_I current and a shift in its current-voltage relationship towards depolarization values. The presence of furosemide in the external solution reversibly inhibited OT_I current. This current likewise declined reversibly following external application of imidazole and tolbutamide but was increased by theophylline action. It was inferred that OT receptors are present on the surface membrane of someHelix neurons which, when activated, lead to increased chlorine permeability — a process apparently mediated via the cyclic nucleotide system.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR. Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 652–659, September–October, 1988.     

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.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

Effect of 2,4,5-trichlorophenol on hyphal membrane potentials in rhythmic mutants of Neurospora crassa

The uncoupler 2,4,5-trichlorophenol (TCP) was used to test for differences in maintaining the hyphal membrane potentials in the wild type and rhythmic mutants of Neurospora crassa. Trichlorophenol (0.1 mmol·1^–1) resulted in a depolarization of 93 mV (wild type) and 144 mV in the mutant clock. A total recovery was achieved in both strains after washing out the uncoupler. The circadian conidiation mutant band was more sensitive than the two other strains and showed two different reaction patterns: one with delayed reaction, total breakdown and without recovery; the other one with nearly immediate reaction, slow but not entirely total decline and partial recovery. These differences are discussed in their relation to the circadian rhythm of conidiation.Abbreviations PD potential difference - TCP trichlorophenol     

Spontaneous and induced changes in the membrane potential and resistance ofAcetabularia mediterranea

Summary The normal resting potential across theAcetabularia mediterranea cell membrane is –170 mV and the resistance is about 0.1 k·cm². The time courses of potential and resistance changes have been studied in connection with several slow dynamic properties of the membrane. These effects include spontaneous and stimulated depolarizing spikes, spontaneous repolarization after prolonged maintenance of a quasistable depolarized condition, and a hyperpolarizing response when current is applied to the cell in this depolarized state. These processes show considerable similarities to each other, which suggests that they might all be explained by changes in permeability to Cl^–. In normal conditions, membrane punch-through occurs with a relatively small hyperpolarizing bias.     

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.     

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.     

Mechanisms of generation of long action potentials in barium solutions

Under voltage clamp conditions ionic currents of neurons of the molluskHelix were studied in solutions containing barium ions. Replacement of the calcium ions in the normal external solution by barium ions led to displacement of the potassium conductivity versus membrane potential curve along the voltage axis toward more positive potentials and also to a decrease in the limiting value of the potassium conductance of the membrane. In sodium- and calcium-free solutions containing barium ions two fractions of the inward current are recorded: quickly (I) and slowly (II) inactivated. The rates of activation of these fractions are comparable. Barium ions are regarded as carriers of both fractions of the inward current. It is postulated that both fractions of the barium current are carried along the calcium channels of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 408–414, July–August, 1977.