Intracellular nonlinear frequency response measurements in the cockroach tactile spine neuron

The threshold of the cockroach tactile neuron increases strongly with depolarization by a process involving at least two time constants. This effect is probably responsible for the rapid and complete adaptation of the neuron's response to step inputs. A technique for intracellular recording and stimulation of the neuron has recently been established and this allows direct observation of the dynamic response of the neuronal encoder. A white noise stimulus was used to modulate the membrane potential of the neuron. The first-order frequency response function between membrane potential and action potential discharge could be explained by a variable threshold model with two time constants. Second-order frequency response functions could be accounted for by a Wiener cascade model. The dynamic nonlinear behavior of the encoder can therefore be explained by a unidirectional threshold which increases linearly and dynamically with membrane potential.     

Spike generation by spinal neurons evoked by sinusoidal depolarization in cats

Dynamic characteristics of transformation of cell membrane depolarization by spinal neurons into spike discharge frequency were investigated in anesthetized cats. Neurons were activated by sinusoidally modulated currents passed through an intracellular microelectrode. Frequency analysis of this transformation for motoneurons was carried out within a modulation frequency range of 0.2–10 Hz. Frequency characteristics were determined with respect to parameters of the first harmonic of the evoked firing rate; the region of values of current fluctuations was chosen on the linear part of the current intensity versus firing rate characteristic curve. Changes in amplitude characteristics did not exceed 5 dB in absolute terms; at the same time the phase lead of the output signal increased with a rise of frequency. At a frequency of 0.2 Hz phase shifts were virtually absent, but at frequencies of 1, 5, and 10 Hz they amounted to 32, 50, and 83° on average respectively. Transformation of membrane depolarization by neurons into spike discharge frequency is characterized by essentially nonlinear properties, due in particular to the absence of a dynamic component of the response to a negative rate of change of depolarizing current. The frequency characteristics of spike activity of neurons of the motor system are discussed from the standpoint of possible correction of dynamic properties of the whole system at the single unit level.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Dnepropetrovsk State University. Translated from Neirofiziologiya, Vol. 14, No. 1, pp. 35–42, January–February, 1982.     

Sensitivity of high-conductance potassium channels in synaptosomal membranes from the rat brain to intracellular pH

High-conductance potassium channels have been studied in inside-out patches excised from proteoliposomes reconstituted from giant liposomes and rat brain synaptosomes. Acid pH in the medium reduced single channel current amplitude and increased the mean open probability and the frequency of channel opening. This was accompanied by a shortening of the open time constant at positive potential and by shortening of the longer closed time constant. The decrease of channel amplitude, the increase of the open probability and the decrease in the longer closed time constant can be explained by neutralization of negative charges of the membrane and by a decrease in the surface membrane potential which mimics membrane depolarization. The shortening of the mean open time is apparently due to a channel blockade by protons. Correspondence to: H. Zemková     

Depletion and accumulation of potassium in the extracellular clefts of cardiac Purkinje fibers during voltage clamp hyperpolarization and depolarization: experiments in sodium-free bathing media

Voltage clamp hyperpolarization and depolarization result in currents consistent with depletion and accumulation of potassium in the extracellular clefts o cardiac Purkinje fibers exposed to sodium-free solutions. Upon hyperpolarization, an inward current that decreased with time (id) was observed. The time course of tail currents could not be explained by a conductance exhibiting voltage-dependent kinetics. The effect of exposure to cesium, changes in bathing media potassium concentration and osmolarity, and the behavior of membrane potential after hyperpolarizing pulses are all consistent with depletion of potassium upon hyperpolarization. A declining outward current was observed upon depolarization. Increasing the bathing media potassium concentration reduced the magnitude of this current. After voltage clamp depolarizations, membrane potential transiently became more positive. These findings suggest that accumulation of potassium occurs upon depolarization. The results indicate that changes in ionic driving force may be easily and rapidly induced. Consequently, conclusions based on the assumption that driving force remains constant during the course of a voltage step may be in error.     

Reopening of Ca2+ channels in mouse cerebellar neurons at resting membrane potentials during recovery from inactivation.

Recordings of single-channel activity from cerebellar granule cells show that a component of Ca2+ entry flows through L-type Ca2+ channels that are closed at negative membrane potentials following a strong depolarization, but then open after a delay. The delayed openings can be explained if membrane depolarization drives Ca2+ channels into an inactivated state and some channels return to rest through the open state after repolarization. Whole-cell recordings show that the charge carried by Ca2+ during the tail increases as inactivation progresses, whereas the current during the voltage step decreases. Voltage-dependent inactivation may be a general mechanism in central neurons for enhancing Ca2+ entry by delaying it until after repolarization, when the driving force for ion entry is large. Modifying the rate and extent of inactivation would have large effects on Ca2+ entry through those channels that recover from inactivation by passing through the open state.     

非洲爪蟾顶盖神经元的抑制性微突触后电流频率和振幅的电压依赖性

采用全细胞记录膜片钳技术,研究非洲爪蟾脑片视顶盖神经元微突触后电流（miniature inhibitory postsynaptic current,mIPSC）频率和振幅对电压依赖关系。观察到以下结果：（1）当通过改变记录电极内的DC电流,将神经元的膜电位从静息电位逐步（每步10mV的增量）去极化或超极化时,mIPSC的频率和,或振幅分别升高或降低。随着膜电位的去极化,mIPSC的频率逐渐升高;当钳位电压升至＋10mV时,mIPSC的频率达到最高值。（2）当神经元去极化时,振幅仅轻微升高。膜电位去极化达到-30mV或-40mV时,mIPSC的振幅最大：进一步去极化,振幅反而下降。另外,在膜电位去极化至-20mV和＋10mV之间时,可记录到大的mIPSC。（3）在无Ca^2＋浸浴液中,mIPSC的频率和振幅也随膜电位的去极化而逐步增高,但频率的增高幅度远不如在生理盐水浸浴中增高幅度明显。（4）当浸浴液中[K＋]0增高时,mIPSC的频率明显降低,而振幅轻微降低。当细胞外[K^＋]。浓度升高超过20mmol／L时,神经元产生明显的缓慢内向或外向膜电流。mIPSC频率和振幅与膜电位存在依赖性的可能机制在文中作了简短的讨论。     

Bioelectric responses of sea urchin eggs inseminated with oyster spermatozoa: a sperm evoked potential without egg activation

Multiple oyster spermatozoa can enter sea urchin eggs with or often without fertilization membrane formation (Osanai and Kyozuka, 1982). In the present work, electrical responses of sea urchin (Temnopleurus hardwicki) eggs inseminated with oyster (Crassostrea gigas) sperm were examined and correlated to the failure of monospermy and egg activation. With diluted sperm, a transient depolarization of the membrane with a constant pattern appeared repeatedly and discretely, and the depolarizations (sperm evoked potentials, SEPs) were not associated with fertilization membrane elevation. With dense sperm, the SEPs occurred consecutively, and sometimes an assembled consecutive depolarization was followed by an activation potential associated with cortical granule discharge. When the membrane potential was artificially held at positive levels, the frequency of SEPs was strongly suppressed but not completely blocked. The present results indicate that an individual heterologous spermatozoon neither produces a depolarization sufficient to block additional sperm entry, nor stimulates egg activation, and that simultaneous entries of multiple heterologous spermatozoa, as possibly reflected by the assembled consecutive depolarizations, induce cortical granule discharge and egg activation.     

Ultrastructural and physiological studies of the contraction of the trunk musculature of Sagitta setosa (chaetognath)

The relationships between structure and function of the contractile apparatus of the trunk musculature in the chaetognath Sagitta setosa, were studied by electron microscopy and mechanical recordings. We also investigated the nature of the neuromediator at the synaptic level. Contraction, relaxation and stretch can be explained on the sliding model basis. The primary filaments are linked to a large Z line, via C filaments. These C filaments vary in length according to the mechanical states of the contractile apparatus. These muscles are directly excitable by electrical current, high K(+) solutions induced depolarization, or exogenous acetylcholine. The characteristics of the unitary contractile response (twitch) and the effects of the frequency of stimulation set up the contractions of Sagitta among the fastest in the animal kingdom. Acetylcholine appears to be the best candidate for the mediator released at the neuromuscular junction. Its effect on the post-junctional membrane seems to require a receptor of the nicotinic type.     

Modulation of endogenous bursting pacemaker activity in a Helix pomatia neuron

Considerable membrane depolarization was shown to arise periodically, at intervals of up to a few minutes, in the PPa1 bursting neuron ofHelix pomatia. Pulses of slow depolarizing current were found by the voltage clamping method. The frequency of the pulses was independent of the holding potential. The equilibrium potential for the slow depolarizing current was about 45 mV. During development of the depolarizing current a region of negative conductivity was observed on the steady-state voltage-current characteristic curve of the membrane. It is suggested that the pulses of slow depolarizing current are associated with the presence of secretory connections between the molluscan neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 6, pp. 606–612, November–December, 1977.     

Transient Responses to Sudden Illumination in Cells of the Eye of Limulus

Responses recorded from visual cells of Limulus (presumably eccentric cells) following abrupt and maintained illumination consist of depolarization with superimposed spikes. Both the depolarization and the frequency of firing are greater at the beginning of the response than later on. Frequency of firing decreases with time also during stimulation with constant currents, but the decay is then less than it is during constant illumination. Early and steady-state responses do not increase in the same proportion following illumination at different intensities. Membrane conductance is higher during the early peak of the response than in steady state. Early and late potential changes appear to tend to the same equilibrium value. The results support the assumptions that: (a) discharge of impulses is the consequence of depolarization of a specialized "pacemaker region" in the axon; (b) depolarization induced by light is the consequence of increase of membrane conductance. The major conductance changes occurring during constant illumination may be due to corresponding changes of the "stimulus" supplied by the photoreceptor or to changes of sensitivity of the eccentric cell's membrane to this stimulus. Some accessory phenomena may be the consequence of regenerative properties of the nerve cell itself.     

Calcium-activated potassium conductance noise in snail neurons

Current fluctuations were measured in small, 3-6 micrometers-diameter patches of soma membrane in bursting neurons of the snail, Helix pomatia. The fluctuations dramatically increased in magnitude with depolarization of the membrane potential under voltage clamp conditions. Two components of conductance noise were identified in the power spectra calculated from the membrane currents. One component had a corner frequency which increased with depolarization. This component was blocked by intracellular injection of TEA and was relatively insensitive to extracellular calcium levels (as long as the total number of effective divalent cations remained constant). It was identified as fluctuations of the voltage-dependent component of delayed outward current. The second component of conductance noise had a corner frequency which decreased with depolarization. It was relatively unaffected by TEA injection and was reversibly blocked by substitution of extracellular calcium with magnesium, cobalt, or nickel. This second component of noise was identified as fluctuations of the calcium-dependent potassium current. The results suggest that the two components of delayed outward current are conducted through physically distinct channels.     

Reliability of spike and burst firing in thalamocortical relay cells

The reliability and precision of the timing of spikes in a spike train is an important aspect of neuronal coding. We investigated reliability in thalamocortical relay (TCR) cells in the acute slice and also in a Morris-Lecar model with several extensions. A frozen Gaussian noise current, superimposed on a DC current, was injected into the TCR cell soma. The neuron responded with spike trains that showed trial-to-trial variability, due to amongst others slow changes in its internal state and the experimental setup. The DC current allowed to bring the neuron in different states, characterized by a well defined membrane voltage (between ?80 and ?50 mV) and by a specific firing regime that on depolarization gradually shifted from a predominantly bursting regime to a tonic spiking regime. The filtered frozen white noise generated a spike pattern output with a broad spike interval distribution. The coincidence factor and the Hunter and Milton measure were used as reliability measures of the output spike train. In the experimental TCR cell as well as the Morris-Lecar model cell the reliability depends on the shape (steepness) of the current input versus spike frequency output curve. The model also allowed to study the contribution of three relevant ionic membrane currents to reliability: a T-type calcium current, a cation selective h-current and a calcium dependent potassium current in order to allow bursting, investigate the consequences of a more complex current-frequency relation and produce realistic firing rates. The reliability of the output of the TCR cell increases with depolarization. In hyperpolarized states bursts are more reliable than single spikes. The analytically derived relations were capable to predict several of the experimentally recorded spike features.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Evidence for existence of two acid groups controlling the conductance of sodium channel

The inhibition of the sodium current in nodal membrane at low pH external solutions was studied under voltage clamp conditions. Analysis of the data for membrane potentials from +10 to +150 mV shows that the inhibition of the Na+ currents at high positive potentials cannot be described by a titration curve of a single acid group. The data can be explained on assumption that the conductance of each sodium channel is controlled by two acid groups: one is located within the pore, the other just near the outer mouth of the pore. The affinity of both groups for H+ is estimated.     

The Use of an Enzyme Electrode in the Analysis of Indole-3-acetic Acid Oxidase Activity in Avena

The rapid reduction in cell electropotentials induced by metabolic inhibitors is strong evidence for an electrogenic ion pump. According to Ohm's law, such a depolarization might be explained by a reduction in electric current, I, with unidirectional transport of a given ion, or an increase in permeability (decrease in resistance). With cells of etiolated seedlings of Pisum sativum L. cv. Alaska and Zea mays cv. Golden Bantam, carbon monoxide inhibition, which occurs only in the dark and is readily reversed by light, allows repeated cycling of depolarization and repolarization; there is no effect on cell membrane resistance. In contrast, cyanide inhibition results in a marked increase in membrane electrical resistance; with cyanide following repeated pulses of current used in measuring cell membrane resistance, the resistance eventually (about 10 minutes) shows an abrupt drop as in the “punch-through” effect reported by H. G. L. Coster (1965. Biophys. J. 5: 669-686).     

Frequency domain analysis of asymmetry current in squid axon membrane.

The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.     

K Permeability of Nitella clavata in the Depolarized State

Membrane current responses to sudden potential changes were recorded in solutions of various [K]_o on 52 internodal cells of Nitella clavata. The membrane current after sudden depolarization had a component sensitive to [K]_o which increased with time from 0.3 to 2.0 s and remained steady thereafter. This late current became zero at values of E and [K]_o which suggests that the current was nearly all carried by K⁺. The potassium conductivity represented by this current increased with depolarization, with a half-maximum value at about -70 mV, and saturation at about -30 to -20 mV. The potassium conductance also increased with increasing [K]_o, but less rapidly than predicted for constant potassium permeability. This failure of the conductance to increase with [K]_o was relatively the same at all membrane potentials and may be explained by a model with a finite number of channels. No attempt was made to model the dependence of g_K on time after depolarization or on membrane potential. However, the finding that the membrane potential did not affect the way in which the permeability depended on [K]_o suggests that the membrane potential change does not affect the affinity of the sites, and that the increase in g_K with time after depolarization is brought about by an increase in the number of channels with such sites.     

Properties of repetitive firing in cat motor cortical neurons to membrane depolarizing currents

Acute experiments were carried out on immobilized cats under superficial pentobarbital (20 mg/kg) anesthesia to investigate the parameters of the rhythmic discharge of neurons in the motor cortex in the area of representation of the forelimb, evoked by passage of steps of depolarizing current through an intracellular microelectrode. The steady-state repetitive firing rate was found to be a linear function of the strength of the current passing through the membrane; no secondary range was discovered. The slope of the "discharge frequency — current" function (f/I=k) was 18±10.7 spikes/sec/nA. The regression line between the slope of the "discharge frequency-current" function and the input resistance (R_in) drawn by the method of least squares had the form k=0.68, R_in=–11.3. Two types of curve of adaptation of the discharge frequency to the stimulating current were found: exponential and undamped oscillations. The curve of latent period of the first action potential in the rhythmic discharge and the length of the first interspike interval as a function of current strength was shown to be a hyperbola, but with different scales along the abscissa. The connection between the properties of the dendritic tree and the parameters of the rhythmic discharge of cortical neurons is discussed.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 8, No. 5, pp. 476–482, September–October, 1976.     

A study of the connection between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron of the snailHelix pomatia

The connection between an interneuron initiating pacemaker activity in the bursting RPa1 neuron and the bursting neuron itself (Pin and Gola, 1983) has been analyzed in the snail Helix pomatia. Prolonged depolarization of the interneuronal membrane produced in it a series of action potentials as well as a parallel initiation or enhancement of bursting activity in the RPa1 neuron. If the discharge in the interneuron was evoked by short current pulses of threshold amplitude, no bursting activity was seen in the RPa1 neuron. However, short stimuli delivered on the background of subthreshold depolarization of the interneuronal membrane produced bursting activity in the RPa1 neuron. Under voltage-clamp conditions a slow inward current could be recorded in the RPa1 neuronal membrane after stimulation of the interneuron with a latency of about 2 sec. Short shifts of the holding potential in the hyperpolarizing direction at the maximum of this current produced a transient outward current. Replacement of extracellular Ca2+ by Mg2+ ions, as well as addition of 1 mM CdCl2 to the external solution, prevented the response to the interneuronal stimulation in the RPa1 neuron. Electron microscopic investigation of the interneuron has shown the abundance of Golgi complexes in its cytoplasm with electron-dense granules in their vicinity. It is concluded that the connection between the interneuron and the bursting neuron is of chemical origin, based on secretion by the former of some substances which activate at least two types of ionic channels in the membrane of the RPa1 neuron.     

The Mechanism of Discharge Pattern Formation in Crayfish Interneurons

Excitatory and inhibitory processes which result in the generation of output impulses were analyzed in single crayfish interneurons by using intracellular recording and membrane polarizing techniques. Individual spikes which are initiated orthodromically in axon branches summate temporally and spatially to generate a main axon spike; temporally dispersed branch spikes often pace repetitive discharge of the main axon. Hyperpolarizing IPSP's sometimes suppress axonal discharge to most of these inputs, but in other cases may interact selectively with some of them. The IPSP's reverse their polarity at a hyperpolarized level of membrane potential; they sometimes exhibit two discrete time courses indicating two different input sources. Outward direct current at the main axon near branches causes repetitive discharges which may last, with optimal current intensities, for 1 to 15 seconds. The relation of discharge frequency to current intensity is linear for an early spike interval, but above 100 to 200 impulses/sec. it begins to show saturation. In one unit the current-frequency curve exhibited two linear portions, suggesting the presence of two spike-generating sites in the axon. Current threshold measurements, using test stimuli of different durations, showed that both accommodation and "early" or "residual" refractoriness contribute to the determination of discharge rate at different frequencies.