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 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 extracellular potassium ions on outward potassium current dependent on extracellular calcium ions

Changes in outward potassium current occurring in response to changes in the concentration of potassium ions in the extracellular medium were investigated in unidentified neurons isolated fromHelix pomatia using an intracellular perfusion technique. It was found that introducing potassium ions (5–10 mM) into the extracellular solution produces a reversible increase in the component of outward potassium current which is dependent on extracellular calcium ions. Increased amplitude of this component occurs as a result of attenuated inactivation of the current under the action of extracellular potassium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 351–356, May–June, 1987.     

Restoration of voltage-dependent antibrain antibody-inhibited calcium current by cyclic adenosine monophosphate

A dual-microelectrode voltage clamp technique was used for recording voltage-dependent calcium current (I_ca) in unidentified neurons isolated fromHelix pomatia. Neither intracellular injection of cyclic adenosine monophosphate (cAMP; 10 nA, 5 min) nor intracellular application of dibutyril-cAMP (dcAMP; 1 mM, 10–20 min) induced a change in normal I_ca or produce a reversible 10–20% reduction in amplitude. Adding S-100 protein fraction antibodies to the external medium led to the onset of calcium-dependent inactivation of I_ca, bringing amplitude of I_ca down to 15±12% of its initial level. Either cAMP or dcAMP then restored inhibited I_ca to 50±11% of its original level. It was found that the effects of cAMP on I_ca of intact neurons depend on level of cytoplasmic Ca²⁺.Institute for Brain Research, All-Union Center for Mental Health Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 247–252, March–April, 1989.     

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.     

Action of calcium ions on channels of inward and outward currents in the molluscan neuron membrane

Changes in the characteristics of activity of sodium, calcium, and potassium channels in the surface membrane during variation of the calcium ion concentration in the extracellular and intracellular medium were investigated by the voltage clamp method during intracellular dialysis of isolated neurons of the mollusksLimnea stagnalis andHelix pomatia. Besides their direct role in passage of the current through the membrane, calcium ions were shown to have two actions, differing in their mechanism, on the functional properties of this membrane. The first was caused by the electrostatic action of calcium ions on the outer surface of the membrane and was manifested as a shift of the potential-dependent characteristics of the ion transport channels along the potential axis; the second is determined by closer interaction of calcium ions with the specific structures of the channels. During the action of calcium-chelating agents EGTA and EDTA on the inner side of the membrane the conductivity of the potassium channels is substantially reduced. With an increase in the intracellular free calcium concentration the conductivity is partially restored. The action of EGTA and EDTA on the outer side of the membrane causes a substantial decrease in the ion selectivity of the calcium channels and changes the kinetics of the portal mechanism. These changes are easily abolished by rinsing off the chelating agents or by returning calcium ions to the external medium. A specific blocking action of an increase in the intracellular free calcium concentration on conductivity of the calcium channels was found.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 69–77, January–February, 1977.     

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.     

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.     

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.     

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.     

Control of Cl− influx inChara by internal pH

Summary The possible regulation of Cl^– influx inChara by the cytoplasmic Cl^– concentration and cytoplasmic pH was investigated using both intact and intracellularly perfused cells. In perfused cells Cl^– influx was sensitive to changes in the internal Cl^– concentration but only when the concentration was less than 1mm.In intact cells the metabolic inhibitors, CCCP, DCMU, and oligomycin which inhibit Cl^– influx also reduced the cytoplasmic pH. A correlation between ATP concentration and cytoplasmic pH was shown to apply when the ATP concentration was lowered using these inhibitors. The possible relationships between ATP status, cytoplasmic pH, and Cl^– influx are discussed.     

Neurochemical mechanisms of burst activity regulation in isolated snail endogenous oscillators: Role of monoamines in opoid peptides

The electrical activity of three identified bursting neurons (including one "plateau-generating" neuron) is studied in the brain ofHelix lucorum under conditions of complete isolation. A method for isolating the identified neurons is worked out (mechanical extraction by means of microelectrodes). All three neurons are shown to be endogenous oscillators. In the isolated state in the absence of any stimulation they exhibit spontaneous changes in activity: from an inactive state to nongrouped regular activity, from regular activity to burst activity, and vice versa. It is also found that the burst activity of all three isolated neurons can be regulated (initiated, discontinued, parameters altered) using the same neurochemical agents: serotonin, opioid enkephalins. Burst activity can be generated by the combined action of dopamine and enkephalin and discontinued just with dopamine. The possible significance of the described neurochemical mechanism of regulating burst activity in neurons that are endogenous oscillators is discussed with regard to the generation of the rhythm of various cyclic functions.N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 472–480, July–August, 1991.     

Stimulation of neuronal calcium conductance by parathyroid hormone

The effects of 10^–10–10^–5 M parathyroid hormone (PTH) on voltage-dependent potassium channels at theHelix pomatia neuronal membrane were investigated in voltage-clamped experiments using intracellular perfusion techniques. The hormone was found to produce a 2-stage effect on calcium current (I_Ca). The initial, brief stage of PTH action consisted of a minor (7–10%) increase in I_Ca and was partially reversible. This was followed by the second (slow) stage, developing for 60–70 min, whereupon level of I_Ca doubled. This hormonal action was not easily reversed and did not occur unless the intracellular solution contained ATP or the hormone was applied after perfusing the cell. Introducing 10 mM EDTA into the perfusate induced a considerable decline in PTH effects. Adding concentrations of 100 and 60 µM of exogenous cAMP and cGMP, respectively, did not imitate the action of this hormone. The first-mentioned effect is thought to be produced by indirect PTH action on channel protein or structures closely associated with the channel and the second by metabolic processes, possibly the phosphoinositide pathway of signal transmission.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Medical Institute, Erevan. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 373–380, May–June, 1990.     

Serotonin-induced changes in the action of functionally distinct neurons in Helix pomatia

The effects of serotonin on the amplitude of summated EPSP in interneurons and on the duration of action potentials in sensory neurons, interneurons, and motoneurons involved in avoidance behavior were investigated in functionally distinct neurons isolated from theHelix pomatia nervous system. The duration of action potentials in sensory neurons was found to increase under the effects of serotonin (and this could underly the rise in EPSP amplitude), although that of interneuron and motoneuron spikes did not change. The functional significance of selective neuronal response to a rise in serotonin concentration is discussed, together with the mechanics underlying such effects.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 316–322, May–June, 1987.     

Effect of pH on properties of the cholinergic receptor membrane of Limnaea stagnalis neurons

The effect of pH on responses of completely isolated neurons ofLimnaea stagnalis during perfusion and microapplication of acetylcholine was investigated by a microelectrode voltage clamp method. A decrease in pH led to a decrease in the sensitivity of the cholinergic receptor membrane. At pH 5.8–6.0 responses to acetylcholine disappeared completely. Increasing the pH to 10.6 did not affect the response to this agent. The ionization constant of the group responsible for the change in sensitivity averaged 6.7. The effect of pH was shown to be unconnected with its influence on the ion channels of the cholinergic receptor membrane and it is evidently due to protonation of the functionally important group in the combining site of the cholinergic receptor.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 8, No. 6, pp. 640–644, November–December, 1976.     

Electric reactions of the auditory cortex areas in bats to ultrasonic stimuli with various fill frequencies

The overall electric reactions and action potentials of single neurons in the auditory cortex were investigated for Vespertilionidae (Myotis oxygnathus) and Rhinolophidae (Rhinolophus ferrum equinum) narcotized with Hexenal. In the Vespertilionidae the greatest sensitivity to ultrasound is manifest at frequencies from 10 to 50 kHz, and in the Rhinolophidae for the ranges from 10 to 40 and from 82 to 84 kHz. The shapes of the response areas of single neurons in both types of bats are similar except for neurons discovered in Rhinolophidae that have three response areas with characteristic frequencies in the ranges 27–28, 40–42, and 80–84kHz. Narrow response areas with characteristic frequencies in the range from 70 to 90kHz appear on a considerable proportion of the neurons in the Rhinolophidae, but not the Vespertilionidae. Low thresholds are recorded to the stimulus cutoff in the range from 76 to 86 kHz.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 3, No. 5, pp. 526–532, September–October, 1971.     

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.     

The isolated perfused rabbit ovary — A model for studies of ovarian function

Summary In the present investigation the ultrastructure of isolated rabbit ovaries, perfused with different media for various time periods, was studied. The steroid hormone production by the perfused ovary was also determined. Perfusion with Medium 199 results in prominent interstitial ovarian oedema which increases with perfusion time. Even after the addition of 6–10 % Dextran T40, oedema appears in the interstitial tissue of the ovary. Perfusion solutions with osmotically active colloid particles of large molecular size (Dextran T70; average molecular weight 70,000 and bovine serum albumin), cause less distortion in the ovarian structure, and ultrastructurally the ovarian tissues appear essentially the same as in the control ovaries.The results indicate that the perfused rabbit ovary, under strictly controlled conditions, can be used as an experimental model for studies of various aspects of ovarian function, including follicular rupture.     

Mechanisms of the inhibitory effect of cyclic adenosine monophosphate on calcium current in intact mollusk neurons

Inhibitory effects of cyclic adenosine monophosphate (cAMP) on calcium current (I_Ca) were investigated in experiments on unidentified neurons isolated fromHelix pomatia by means of voltage clamping techniques using two microelectrodes. Intracellular level of cAMP was raised by intracellular injection of this substance or by extracellular application of dibutyryl-cAMP or isobutylmethyl-xanthine. A set of neurons showing inhibitory effects of cAMP on I_Ca was used. Effects on barium current (I_Ba) of an equal extent were also revealed. Injection of cGMP through a double-barreled microelectrode into these neurons produced an increase in amplitude of I_Ca. Intracellular application of phorbol ester had no effect on this current, however. Intracellular injection of EGTA led to enhancement of I_Ca amplitude, but the inhibitory effect of cAMP persisted following the action of EGTA. Tolbutamide and H-8 (but to a lesser extent) inhibited I_Ca. The inhibitory effects of tolbutamide and dibutyryl cAMP were not found to be cumulative in six out of twelve instances. These findings would imply that the inhibitory action of cAMP on I_Ca is unassociated with activation of cAMP-dependent protein kinase, cGMP-dependent protein kinase or protein kinase C; nor does it depend on level of intracellular Ca²⁺. The possibility of direct interaction between cAMP and channel-forming protein is considered.Institute of Brain Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 54–61, January–February, 1990.     

Characteristics of electrical responses by the medial geniculate bodies in Vespertilionidae and Rhinolophidae to ultrasonic stimuli with different frequencies

The total electrical responses and action potentials of the neurons in the medial geniculate bodies in Vespertilionidae and Rhinolophidae were investigated. Maximum sensitivity to ultrasonic stimuli was recorded inMyotis oxygnathus (Vespertilionidae) in the range 10–40 kc/sec and 65–80 kc/sec, and in Rhinolophidae in the ranges 10–70 kc/sec and 81–86 kc/sec. Low thresholds were observed inMyotis oxygnathus for the frequencies covered by their echo-location cries, whereas the thresholds recorded in Rhinolophidae in the 80 kc/sec band (the principal frequency of their echo location cries) were 15–30 dB higher than those for adjacent frequencies. Minimum thresholds of off-responses were observed inMyotis oxygnathus in the range 50–60 kc/sec, and in Rhinolophidae in the range 78–80 kc/sec. The regions of neuronal response in both species of bat were generally similar in form to those of responses recorded in the medial geniculate bodies of other mammals. Some of the neurons in Rhinolophidae with a characteristic frequency of about 80 kc/sec were also sensitive to stimuli with one-half and one-third of the principal frequency. In Rhinolophidae the greatest selectivity for frequencies was possessed by neurons that responded within the range from 80 to 90 kc/sec.A. A. Zhadanov Leningrad State University. Translated from Neirofiziologiya, Vol. 3, No. 2, pp. 138–144, March–April, 1971.