A study of the crustacean axon repetitive response. 3. A comparison of the effect of veratrine sulfate solution and potassium-rich solutions

The crustacean single nerve fiber gives rise to trains of impulses during a prolonged depolarizing stimulus. It is well known that the alkaloid veratrine itself causes a prolonged depolarization; and consequently it was of interest to investigate the effect of this chemically produced depolarization on repetitive firing in the single axon and compare it with the effect of depolarization by an applied stimulating current or by a potassium-rich solution. It was found that veratrine depolarization, though similar in some respects to a potassium-rich depolarization of depolarizing current effect, was in many respects quite different. (1) At low veratrine concentration, less than 1 Mg%, the negative after potential following a spike action potential was prolonged and augmented. At higher concentrations or after a long period of time, veratrine caused a prolonged steady state depolarization of the membrane, the “veratrine response”. The prolonged plateau depolarization response could be elicited with or without an action potential spike by a short or long duration stimulating pulse, but only if the veratrine depolarization was prevented or offset by an applied conditioning hyperpolarizing inward current. (2) The “veratrine response” resembled the potassium-rich solution response in the plateau-like contour of the depolarization and the very low membrane resistance during this plateau phase. Like the potassium response, it was possible to obtain a typical hyperpolarizing response with an inwardly directed current pulse if applied during the plateau phase. During the negative after potential augmented with veratrine, however, this hyperpolarizing response was not observed. (3) In contrast to the potassium response, however, the “veratrine response” is intimately associated with the sodium concentration in the external medium. The depolarization in millivolts is linearly related to the log of the concentration of external sodium. Moreover, during veratrine action there is a continuous and progressive inactivation of the sodium mechanism which ultimately terminates repetitive firing and abolishes the spike action potential. Then even with conditioning hyperpolarization only the slow response may be elicited in veratrine, occasionally with a spike superimposed if sodium is present, but without repetitive firing. (4) It is concluded that veratrine action is the result of a chemical or metabolic reaction by the alkaloid in the membrane. It is suggested that veratrine may inhibit the sodium extrusion mechanism, or may itself compete for sites in the membrane with calcium and/or sodium. This explains the inhibiting effect of high calcium, the abolition of the “veratrine response” with low temperature and high calcium combined and the progressive inactivation of the sodium system.     

Electrophysiological responses to bradykinin and microinjected inositol polyphosphates in neuroblastoma cells. Possible role of inositol 1,3,4-trisphosphate in altering membrane potential

Addition of bradykinin to mouse N1E-115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several-fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pretreatment of the cells with a Ca2+-ionophore abolishes the hormone-induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.     

On the effect of the ionophore X-537A on neuromuscular transmission in the rat

In the rat phrenic nerve-diaphragm muscle preparation, X-537A at 6×10^?6 to 3×10^?5 M (1) depolarized muscle fibre membranes, (2) caused an occasional transient increase in and ultimate block of spontaneous transmitter release, (3) did not increase the amplitude of the end-plate potential (epp) but abruptly blocked stimulus-evoked transmitter release, and (4) produced an increase in the occurrence of “giant” miniature epp's (mepp's). The possibility is discussed that the sporadically raised mepp frequency was due to an ionophore-induced depolarization of nerve terminals. The increased occurrence of “giant” mepp's apparently reflected a X-537A-induced spontaneous multiquantal release of acetylcholine. This was not dependent on extracellular calcium but appeared to be of presynaptic origin.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

Blue Light Pulse-Induced Transient Changes of Electric Potential and Turgor Pressure in the Motor Cells of Phaseolus vulgaris L.

Blue light induces both depolarization of membrane potentialin the motor cell and turgor movement in the laminar pulvinusof bean plant. This paper describes the changes of electricpotential and turgor pressure induced in Phaseolus vulgarisL. by blue light pulses. A transient depolarization of membranepotential as large as 40 mV was induced by a short pulse of15 s blue light in motor cells of the laminar pulvinus. Thischange was not an action potential because of the absence ofa refractory period and threshold. The magnitudes of the responsewere dependent on the fluence of light. The response was long-lived,indicating that continuous input of light energy is not requiredfor a sustained response. The potential change was always followedby a transient turgor movement of the pulvinus. A molecular mechanism similar to a model postulated for theblue light response of stomata may operate in the motor cell.However, the direction of the electrical response to blue light(depolarization) in the motor cell was the opposite of thatin the guard cell (hyperpolarization). Turgor change of themotor cell by blue light was also opposite in direction (decrease). (Received February 19, 1988; Accepted June 28, 1988)     

A pulse of blue light induces a transient increase in activity of apoplastic K+ in laminar pulvinus of Phaseolus vulgaris L

A pulse of blue light induced both a transient increase in activity of apoplastic K+ and membrane depolarization in laminar pulvinus of Phaseolus vulgaris L. This shows that blue-light-induced net efflux of K+ from motor cells is closely related to membrane depolarization.     

Does a homogeneous population of elementary movement detectors activate the landing response of blowflies,Calliphora erythrocephala?

The landing response of tethered flying blowflies, Calliphora erythrocephala, was elicited by moving periodic gratings, and by stripes moving apart. The influence of binocular interactions on the landing response was investigated by comparing the responses of intact (“binocular”) animals to the response of flies which had one eye covered with black paint (“monocular” flies) effectively eliminating the input from this eye. Directions of motion eliciting a maximal response (preference direction) were determined in intact animals, and in “molecular” flies for different regions of the visual field. Preference directions determined in “monocular” flies follow the orientation of Z-axes (Fig. 4). Preference directions determined in intact animals and in “monocular” flies differ in the binocular eye region: in intact animals, the preference directions corresponds to vertical directions of motion; whereas the preference direction determined for the same area in “monocular” flies are inclined obliquely against the vertical plane. Sex-specific differences were found for the ventral binocular eye region in which the shift of preference directions is more pronounced in male than in female flies. The experimental data support the hypothesis that elementary movement detectors are aligned along the Z-axes of the eye, and that preference directions deviating from the orientation of elementary movement detectors are caused by binocular interactions.     

Increased chloride conductance as the proximate cause of hydrogen ion concentration effects in Aplysia neurons

A fall in extracellular pH increased membrane conductance of the giant cell in the abdominal ganglion of Aplysia californica. Chloride conductance was trebled whereas potassium conductance was increased by 50%. Half the giant cells were hyperpolarized (2–8 mv) and half were depolarized (3–10 mv) by lowering the pH. The hyperpolarizing response always became a depolarizing response in half-chloride solutions. When internal chloride was increased electrophoretically, the hyperpolarization was either decreased or changed to depolarization. The depolarizing response was reduced or became a hyperpolarizing response after soaking the cell in 10.0 mM chloride, artificial seawater solution for 1 hr. Depolarization was unaffected when either external sodium, calcium, or magnesium was omitted. A glass micropipette having an organic liquid chloride ion exchanger in its tip was used to measure intracellular chloride activity in 14 giant cells; 7 had values of 27.7 ± 1.8 mM (SEM) and 7 others 40.7 ± 1.5 mM. Three of the first group were hyperpolarized when pH was lowered and three of the second group were depolarized. In all six cells, these changes of membrane potential were in the direction of the chloride equilibrium potential. Intracellular potassium activity was measured by means of a potassium ion exchanger microelectrode.     

Cold-sensitive responses in the Paramecium membrane

A transient depolarization was recorded in response to the cooling of a deciliated Paramecium. The amplitude of the depolarization was almost proportional to the cooling rate. Therefore, the cells are sensitive to the rate of temperature change. The input resistance of the membrane transiently increased during the cooling. When constant current was applied to shift the resting membrane potential to a negative or positive level, the initial depolarization in response to cooling decreased, and the following hyperpolarization during cooling reversed to a gradual depolarization during a positive shift. The potential at which the reversal occurred was independent of K+ concentration and was slightly dependent on Ca2+ concentration (10 mV/log[Ca2+]o). The amplitude of the initial depolarization decreased with the increase in K+ and was not affected by Ca2+. These results are discussed in terms of changes in membrane conductances in response to cooling.     

Action of Certain Tropine Esters on Voltage-Clamped Lobster Axon

Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClA), which differs from TPTA only by the substitution of a p-Cl for a p-CH₃ group on the benzene ring, had a negligible effect on axonal excitability.     

Two classes of visual motion sensitive interneurons differ in direction and velocity dependency of in vivo calcium dynamics

Neurons exploit both membrane biophysics and biochemical pathways of the cytoplasm for dendritic integration of synaptic input. Here we quantify the tuning discrepancy of electrical and chemical response properties in two kinds of neurons using in vivo visual stimulation. Dendritic calcium concentration changes and membrane potential of visual interneurons of the fly were measured in response to visual motion stimuli. Two classes of tangential cells of the lobula plate were compared, HS-cells and CH-cells. Both neuronal classes are known to receive retinotopic input with similar properties, yet they differ in morphology, physiology, and computational context. Velocity tuning and directional selectivity of the electrical and calcium responses were investigated. In both cell classes, motion-induced calcium accumulation did not follow the early transient of the membrane potential. Rather, the amplitude of the calcium signal seemed to be related to the late component of the depolarization, where it was close to a steady state. Electrical and calcium responses differed with respect to their velocity tuning in CH-cells, but not in HS-cells. Furthermore, velocity tuning of the calcium response, but not of the electrical response differed between neuronal classes. While null-direction motion caused hyperpolarization in both classes, this led to a calcium decrement in CH-cells, but had no effect on the calcium signal in HS-cells, not even when calcium levels had been raised by a preceding excitatory motion stimulus. Finally, the voltage-[Ca2+]i-relationship for motion-induced, transient potential changes was steeper and less rectifying in CH-cells than in HS-cells. These results represent an example of dendritic information processing in vivo, where two neuronal classes respond to identical stimuli with a similar electrical response, but differing calcium response. This highlights the capacity of neurons to segregate two response components.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

An interplexiform cell in the goldfish retina: light-evked response pattern and intracellular staining with horseradish peroxidase

Summary The light-evoked response pattern and morphology of one interplexiform cell were studied in the goldfish retina by intracellular recording and staining. The membrane potential of the cell spontaneously oscillated in the dark. In response to a brief light stimulus, the membrane potential initially gave a slow transient depolarization. During maintained light, the oscillations showed a tendency to be suppressed; the response of the cell to the offset of the stimulus was not so prominent. The perikaryon of the interplexiform cell was positioned at the proximal boundary of the inner nuclear layer. The cell had two broad layers of dendrites; one was diffuse in the inner plexiform layer, the other was more sparse in the outer plexiform layer. The morphological and electrophysiological characteristics of the cell are discussed in relation to dopaminergic interplexiform cells and the light-evoked release pattern of dopamine in the teleost retina.     

Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels

Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.     

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.     

ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels

During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (DeltaG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (DeltaG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl- from guard cells and a rapid but confined reduction in stomatal aperture.     

Voltage-gated ion conductances corresponding to regenerative positive and negative spikes in the dinoflagellateNoctiluca miliaris

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellateNoctiluca miliaris were examined under voltage-clamp conditions.Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of –80 mV to a potential level more positive than –50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na⁺ concentration was lowered. The transient inward current is assumed to be responsible for the Na⁺-dependent positive spike in non-clamped specimens ofNoctiluca.Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of –20 mV to a potential level more negative than –30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl^– concentration was lowered. The transient outward current is responsible for the Cl^–-dependent negative spike in non-clamped specimens ofNoctiluca.Abbreviations ASW artificial seawater - TRP tentacle regulating potentials - TTX tetrodotoxin     

The Effects of Mechanical Stimulation on Some Electrical Properties of Axons

Rapid, short duration mechanical compression of lobster giant axons by a crystal-driven stylus produces a depolarization and an increase in membrane conductance which develop immediately with compression but take several seconds to recover. The conductance increase occurs even when the depolarization is prevented electrically. If sodium is removed from the external medium or if procaine is added to it, compression produces almost no depolarization. Small bundles of myelinated frog fibers are depolarized by rapid compression but recover very rapidly (milliseconds); "off" responses are occasionally seen. The results are discussed in terms of the mechanoelectric transducer behavior of an axon membrane.     

Membrane potential responses of paramecium caudatum to external Na+

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.     

Distribution of chemoreceptors to quinine on the cell surface of Paramecium caudatum

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni²⁺-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.Abbreviation CNR caudatum non reversal