Osmotically-induced volume changes in isolated cells of a pond snail

• 1.1. To assess whether the stretch-activated (SA) channels in snail cells could contribute to osmoregulation, information is needed about the behaviour of the cells under anisosmotic conditions.
• 2.2. Cells of Lymnaea stagnalis were therefore examined during acute hyposmotic stress (HOS).
• 3.3. Kidney, heart and neuronal cells (monitored photographically) swelled less than expected for strictly semipermeable cells, but exhibited no regulatory volume decrease.
• 4.4. Long-term viability of the cells was not compromised following acute hyposmotic stress.
• 5.5. Quinidine, which blocks SA channels in Lymnaea, intensified stress-induced swelling most markedly in kidney cells.
• 6.6. The data can, however, be explained without invoking recruitment of SA channels.

     

Aluminum enhances the voltage activated sodium currents in the neurons of the pond snail Lymnaea stagnalis L

1. The effects of aluminum on voltage activated sodium currents (VASCs) were investigated by using the conventional two-electrode voltage clamp technique in Lymnaea stagnalis L. neurons. The peak amplitude, kinetics, and voltage-dependence of activation and inactivation of the sodium currents were studied in the presence of 5-500 microM AlCl3, at pH = 7.7. 2. There was a significant concentration-dependent increase in the peak amplitude of sodium currents after Al treatment, ED50 = 67 microM. The threshold concentration of the enhancement was 50 microM. The maximal peak increase of 143% was caused by a 500 microM aluminum. The action of aluminum on VASCs developed slowly, and it is not recovered by washing within 20 min. 3. There was little alteration of the voltage-dependence of the current. It was not a significant effect on the activation- and inactivation time constants of INa, but the steady-state inactivation curve shifted to negative direction on the voltage axis in the presence of Al. 4. The leak currents were not influenced by aluminum up to the highest concentration applied.     

Properties of GABA-induced transmembrane currents in isolated neurons of the rat spinal ganglia

Using techniques of voltage-clamp in the whole-cell configuration and fast local superfusion, we studied the properties of transmembrane ion currents evoked in freshly isolated neurons of the spinal ganglia of rats by application of γ-aminobutyric acid, GABA, in different concentrations. Increases in the GABA concentration and application time resulted in modification of the amplitude and kinetic parameters of the currents. The dependence between the current amplitude and GABA concentration could be adequately described by the Hill equation. The current rise could be fitted by a sum of two exponential curves with different time constants; the time constant of the second exponent changed with an increase in the GABA concentration, while the first exponent was not sensitive to these changes. The current decay also should be fitted by two exponents. The time constant of the first exponent did not change with increases in the GABA concentration or duration of its application; at the same time the second exponent noticeably depended on the time of GABA application. Our experiments demonstrated that the density of GABA-activated ion channels in the membranes of the studied spinal ganglion cells is relatively high; this finding allows us to suppose possible involvement of these channels in regulation of the transmembrane conductivity in these cells.     

Generalized posttetanic changes in the excitatory postsynaptic and acetylcholine-evoked currents in snail neurons

Heteroreceptor posttetanic changes in excitatory postsynaptic currents (EPSC) and inward currents evoked by the local iontophoretic application of acetylcholine (ACh) on the dorsal surface of PLa3 and PRa3 Helix lucorum neurons were studied. The following changes in the currents were revealed over the course of 1-1.5 h after tetanization. The rhythmical ACh application (0.5-1.0 cps, 10-40 s) evokes potentiation of the orthodromic EPSC. The tetanic orthodromic stimulation of one of the nerves (n. intestinalis, n. pallialis dexter, or n. pallialis sinister; 1-5 cps, 1-2 min) causes the potentiation of the ACh current and also heterosynaptic depression of the EPSC. It is concluded that activation of subsynaptic and nonsynaptic neurotransmitter chemoreceptors evokes the development of generalized posttetanic changes in neuronal responses.     

Role of changes in the transmembrane ion currents in pathological states of the body

Modern data of study of transmembrane ions currents (through the sodium-potassium- and calcium-channels) in the mechanism of pathological pain syndromes were reviewed. Tetrodotoxyn-resistant sodium currents, abnormal combinations of different types of sodium-channels during the inflammation, neuropathies (syndromes hyperalgesii and allodonii) and diabetic neuropathy analyzed. The role of pain sensitivity transmitters was demonstrated. Nerve Grow Factor plays the important role in the sodium-channels genes expression. Different types of transmembrane calcium currents participated in hyperalgesium and hypoalgesium analyzed. The summary mechanism of pain suggested as voltage-calcium-channels activation and transsynaptic NMDA receptors excitation that should be conductive the neurons hyperexcitation and force of synaptic transmission.     

Plastic changes in the spontaneous activity of pond snail neurons during rhythmic and associated intracellular electrostimulation

In experiments on spontaneously active neurones of the isolated CNS of pond snail, under contingent stimulation with selective auto-reinforcement of interpulse intervals longer than their mean background value, 70 per cent of neurones were capable of adaptive rearrangements of the initial firing frequency, leading to frequency minimization or maximization of influences applied to them. After control rhythmic stimulation, usually no substantial changes of spontaneous activity were observed. The detected phenomena of initial transformation of the spontaneous impulse activity are considered as a cellular analogue of the instrumental conditioned reflex.     

Effects of intracellular pH and calcium activity on ion currents in internally perfused neurons of the snail Lymnaea stagnalis

The suction pipet method of intracellular dialysis and voltage clamp of cells has proven extremely useful in analysing the electrical properties of cells too small for the application of conventional microelectrode techniques and in larger cells for studying the effects of alterations in the internal ionic composition. Using neurons of the snail Lymnaea stagnalis, we have analysed several problems involved in the latter application of this technique and present several solutions to them. One major problem centers around the degree of control over the ionic composition of the cytoplasm achieved by altering the pipet solution. Using ion-sensitive microelectrodes during internal dialysis, we found that the efficiency of exchange between pipet and cytoplasm was much poorer for highly buffered ions such as H+ and Ca2+, than for K+, for example. Special precautions are described that can help this situation. The second problem involves the study of the effects of low internal pH on ion-channel properties. We summarize evidence for a specific voltage-dependent hydrogen ion channel, current through which becomes prominent at low internal pH. We analyse how the presence of this heretofore unrecognized current can seriously confuse the results of experiments designed to study the effects of low internal pH on other voltage-dependent currents.     

Functional activity of ganglion neurons from the pond snail upon temperature variations

Changes in the functional state of the synthetic apparatus of neurons of the peripharyngeal ring of the mollusk Lymnaea stagnalis upon elevating the temperature of the habitat from 4 to 18-22 degrees C were studied. In the first series of experiments, it was found that the synthetic activity of neurons of the great and small parietal, visceral, and pleural ganglia increases on the average by 70%. In the second series of experiments, changes in the average synthetic activity for all ganglia at 4.0, 6.5, 13.0, 16.8, 20.5, 21.5, 21.9, and 22.0 degrees C were studied. The increase in the synthetic activity of neuron cytoplasm (35% higher than the control values at 4.0 degrees C) was maximal at 13.0 degrees C; then the synthetic activity decreased to reach the level 20% higher than the control. The physiological range of temperature-dependent changes in the functional state of neurons was determined.     

下丘脑神经元电压依赖性钾电流活动在大鼠出生后发育过程中的变化

采用膜片钳细胞贴附式技术,比较研究SD大鼠下丘脑神经元电压依赖性钾通道(voltage-dependent potassium channel,Kv)单通道电流活动的动力学特性,在出生后发育过程中的变化。出生不同天数的大鼠,其下丘脑神经元上Kv通道的电流强度和电导无显著差别(P>0.05),通道电导接近120 pS;单位时间内封接膜片上N个通道的开放概率的总和升高,由第1天的0.19±0.08(n=10)上升到第9天的0.30±0.09(n=10,P<0.05),单通道活动密度增加,由0.14 channel/μm2升高至0.26 channel/μm2。上述结果提示大鼠下丘脑神经元在出生发育过程中,Kv单通道活动的动力学发生显著变化。     

Role of transmembrane ion currents in the mechanism of action of beta-adrenoblockers

The effects of propranolol, oxprenolol, pindolol, labetalol, and atenolol on the isoproterenol-stimulated slow calcium current as well as on spontaneous slow calcium and fast sodium currents were studied in frog trabeculae by the double sucrose bridge technique. Administration of beta-adrenoblockers in low doses (10(-7)-10(-6)M) antagonized the stimulatory effect of isoproterenol on the slow calcium current (pindolol greater than oxprenolol greater than or equal to propranolol = labetalol greater than atenolol). When administered in high doses (2 X 10(-6)-10(-4)M) the beta-adrenoblockers reduced the slow calcium current. Meanwhile, some of them (propranolol, oxprenolol, labetalol) suppressed the fast sodium current.     

Effect of deltamethrin on transient outward currents in identified snail neurons

• 1.1. The effect of a pyrethroid insecticide deltamethrin was investigated on transient outward potassium currents of identified snail (Helix pomatia) neurones LPa1 and RPa3.
• 2.2. In 5 × 10⁻⁵ M concentration the deltamethrin decreased the I_A amplitude and the slope of I–V curve. The activation variable was shifted left along the voltage axis by 10–20 mV, while the inactivation variable remained unchanged.
• 3.3. Time constant of inactivation decreased, and the relaxation of I_A described by one exponential. “Modified” ionic channel fraction was not observed.
• 4.4. It is suggested that deltamethrin acts on I_A channels through a different molecular mechanism to I_Na channels, since not only the gating machinery but the permeability of the channels were influenced.

     

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.     

Ring neuron control of columellar motor neurons during egg-laying behavior in the pond snail

Egg-laying in Lymnaea is characterized by the stereotyped egg-laying behavior (ELB), composed of foot contractions and shell movements. Egg-laying can be induced by a clean water stimulus, that triggers a discharge of the neuroendocrine caudo-dorsal cells (CDCs), which release the ovulation hormone into the blood. A part of the behavior is lost when egg-laying is triggered by hormone injection, indicating that during natural stimulus-induced or spontaneous egg-laying this part (the first phase) may be controlled by neuronal events in the CNS triggered by (a) factor(s) not released to the blood. The authors have identified an unpaired neuron, the ring neuron, that is excited during an in vitro afterdischarge of the CDCs, and which, by its numerous axonal branches in the pedal ganglia, modulates motorneurons of the columellar muscle, which controls shell movements. These motor-neurons, identified as such in reduced preparations by 1 for 1 muscle potentials and elements in the connecting nerve, all receive either excitatory or inhibitory input from the ring neuron, as well as from an unknown neuron which has common input of the ring neuron and the motorneurons. The action of the CDCs on the ring neuron cannot be mimicked by the ovulation hormone, and we therefore conclude that the first part of the ELB is probably caused by a nonhormonal local action of the CDCs on the ring neuron and possibly the common input neuron.     

Octopaminergic modulation of the membrane currents in the central feeding system of the pond snail Lymnaea stagnalis

Octopamine is released by the intrinsic OC interneurons in the paired buccal ganglia and serves both as a neurotransmitter and a neuromodulator in the central feeding network of the pond snail Lymnaea stagnalis. The identified B1 buccal motoneuron receives excitatory inputs from the OC interneurons and is more excitable in the presence of 10 microM octopamine in the bath. This modulatory effect of octopamine on the B1 motoneuron was studied using the two electrode voltage clamp method. In normal physiological saline depolarising voltage steps from the holding potential of -80 mV evoke a transient inward current, presumably carried by Na(+) ions. The peak values of this inward current are increased in the presence of 10 microM octopamine in the bath. In contrast, both the transient (IA) and delayed (IK) outward currents are unaffected by octopamine application. Replacing the normal saline with a Na(+)-free bathing solution containing K(+) channel blockers (50 mM TEACl, 4 mM 4AP) revealed the presence of an additional inward current of the B1 neurons, carried by Ca(2+). Octopamine (10 microM) in the bath decreased the amplitudes of this current. These results suggest that the membrane mechanisms which underlie the modulatory effect of octopamine on the B1 motoneuron include selective changes of the Na(+)- and Ca(2+)-channels.     

Patch and whole cell calcium currents recorded simultaneously in snail neurons

The flow of Ca ions through single Ca channels has been examined. The gigaseal method was used on identifiable snail neurons that were voltage clamped using a two-microelectrode voltage clamp method. Average Ca patch currents and whole cell currents have similar time courses. They are affected similarly by changes in temperature. The differences in amplitude and inactivation between Ba and Ca whole cell currents were present in the patch records. The stationary noise spectra recorded from ensembles of multichannel patches have two components with fast and slow time constants equivalent to two components in the whole cell tail current relaxations. Elementary current amplitudes measured from the variance-mean relationship and from noise spectra gave values comparable to measurements from single channels. The single channel I-V relationship was curvilinear and the maximum slope conductance in 40 mM Cao was 7 pS. The amplitude of unitary currents was unchanged at long times when inactivation had occurred; hence depletion is not involved in this process. Channel density was approximately 3 microns-2 and was the same for Ba and Ca currents. The whole cell asymmetry currents gave very large values for the gating charge per channel. Changes in temperature from 29 to 9 degrees C had only a slight effect on the two Ca tail current tau's at potentials where turn-on of patch and whole cell currents was markedly slowed and the peak amplitudes were reduced by one-third. Single channel recordings were obtained at these two temperatures, and the mean open time and the fast component of the closed times were scarcely affected. Unit amplitudes were reduced by 30% and the slow closed time component was doubled. Therefore, peak currents and the slow closed time component was doubled. Therefore, peak currents were reduced partly as a result of the reduction in unit amplitude, but mainly as a result of a reduction in opening probability, the latter arising from an increase of the long closed times. It is concluded that the behavior of single Ca channels in membrane patches is the same as it is in whole cells. Cooling from 29 to 9 degrees C acts primarily on transitions among closed states and has little effect on the open to closed transition.     

Zinc modulates a-type potassium currents and neuronal excitability in snail neurons

Summary 1. Zinc-induced actions were studied on the A-current and neuronal activity in identified and unidentified nerve cells of the snail,Helix pomatia L., under voltage and current clamp conditions.2. Extracellularly applied Zn²⁺ attenuated the peak amplitude of the A-current in a potential- and dose-dependent way (K _i=1.8 mM at –30 mV,n _H=0.6).3. Attenuation of the A-currents was initiated as Zn²⁺ shifted the potential dependence of both activation and inactivation of the currents toward more positive potential values.4. Zinc concomitantly prolonged the time to peak and decay time constant of the A-currents (K _d=1.7 mM,n _H=1.4) as well.5. Zn²⁺ decreased the resting membrane potential and the spike amplitude and increased the action potential duration and the input resistance of the cells in current clamp experiments.6. A complex action of zinc increased the neuronal excitability, indicating spontaneous and synaptically evoked spike discharges.7. Common and specific zinc binding sites are supposed on vertebrate and invertebrate A-type potassium channel proteins, where binding Zn²⁺ can modulate the gating properties and kinetics of the fast outward potassium currents.     

Potentiation of glutamate-activated currents in isolated hippocampal neurons

"Fast chemical stimulation" was shown to induce potentiation of glutamate-activated currents in neurons isolated from rat hippocampus. A fast application system allowed solution changes up to a rate of 20 Hz. In Mg2(+)-free solution, the response to glutamate application immediately after repetitive stimulation with glutamate plus glycine was increased by 25%-88%, returning to control levels over 10-15 min. Enhancement of glutamate-induced currents was also seen after stimulation with solutions containing aspartate or NMDA plus glycine. Aspartate-induced currents were not potentiated. These and other observations demonstrate that in a purely "postsynaptic" system, short-term potentiation can be induced and is mediated via NMDA receptors whereas the potentiated current is carried via non-NMDA glutamate receptor channels.