Amplitude Histograms Research Articles

Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes

The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0.142 for wild type (p = 0.073). The data obtained indicate that deleting 30 amino acids from the first intracellular loop of CFTR affects both processing and function of the CFTR chloride channel.

Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.

Sr2+ and quantal events at excitatory synapses between mouse hippocampal neurons in culture.

1. Whole-cell recording from pairs of adjacent mouse hippocampal neurons in culture was used to study the quantal properties of action potential-evoked excitatory synaptic transmission and to demonstrate the use of Sr2+ in quantifying those properties. 2. In the presence of extracellular Sr2+, excitatory postsynaptic currents (EPSCs) were followed by an after-discharge of miniature excitatory postsynaptic currents (mEPSCs) lasting 1-2 s and generated by evoked asynchronous release of presynaptic quanta of transmitter. Like the EPSC of which it is thought to be an extension, the after-discharge was modulated by procedures expected to modulate Sr2+ influx into the nerve terminal. The number of mEPSCs in the after-discharge was decreased by increasing extracellular [Mg2+], and increased by increasing extracellular [Sr2+] or increasing the number of action potentials used to evoke the after-discharge. 3. EPSCs recorded in media containing either 1 mM Ca2+ or 6 mM Sr2+ were of similar amplitude. Adding Sr2+ to low-Ca2+ media increased EPSC amplitude, while adding Sr2+ to high-Ca2+ media lowered EPSC amplitude. These results suggest that extracellular Sr2+ is less effective than Ca2+ in supporting quantal release. 4. The levels of extracellular Ca2+, Mg2+ and Sr2+ were adjusted so that most after-discharge mEPSCs were discrete and comparable in numbers to the quantal events that contributed to the corresponding evoked EPSCs. In a series of twenty-five pairs of neurons, the mean amplitude of mEPSCs recorded at -80 mV was 35 +/- 10 pA and the mean coefficient of variation was 0.50 +/- 0.10 (range, 0.26-0.62). The mEPSC amplitude histogram was positively skewed. 5. In ten pairs of neurons, the mean and variance of EPSCs and mEPSCs and quantal content were determined from samples of more than 100 evoked events (in superfusion solutions containing (mM): 0.5 Ca2+, 2 Sr2+ and 10 Mg2+) and mean quantal content was determined from the ratio of amplitudes of the mean EPSC and mEPSC. A binomial quantal analysis produced values of 2-12 for Napp (apparent number of independent synapses) and 0.25-0.75 for Papp (apparent probability of releasing a quantum at one of those synapses). These parameters predicted the number of observed failures. The observed coefficient of variation for quantal content predicted the observed coefficient of variation of the EPSC amplitude when the coefficient of variability of quantal amplitude of after-discharge mEPSCs was taken into account. 6. In six pairs of neurons, where more than 250 evoked events were recorded, the observed amplitude histogram for EPSCs could be approximated by a predicted amplitude distribution generated from the estimated binomial parameters and an empirical function describing the amplitude distribution of after-discharge mEPSCs. 7. The observation that parameters derived from mEPSCs that contribute to the Sr(2+)-generated after-discharge can predict the shape of the EPSC amplitude distribution and a quantal content consistent with the observed failure rate and EPSC amplitude variance, suggests that this subset of mEPSCs has the same properties as the quantal events released around the time of the peak of the corresponding EPSCs. The use of Sr2+ to evoke after-discharges of mEPSCs should allow unambiguous determination of the extent to which modification of synaptic strength is pre- or postsynaptic.

Serotonin-induced changes in the activity of single Ca2+ channels inHelix pomatia neurons

The influence of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) on single Ca2+ channel activity was studied on unidentified neurons of the snailHelix pomatia. Only one type of Ca2+ channels with the unitary conductance of 5 pS was identified using 100 mM Ca2+ in the patch pippette under patch-clamp in a cell-attached configuration. The amplitude histogram showed only one peak with the mean value of 0.5 pA at the testing potential of −30 mV. The distribution of channel open times monotonically declined with the mean time constant of 0.2 msec. The distribution of channel closed times could be fitted by a double-exponential curve with time constants of 1 and 12 msec. The study of the effect of 5-HT on Ca2+ single channel activity showed that 5-HT influenced the channel molecule indirectly, as the transmitter could exert its effect by being added to the bath solution, which did not come into contact with the tested membrane fragment under the micropipette tip. 5-HT prolonged the mean channel open time (up to 0.3 msec) and proportionally decreased both channel closed time constants to 0.4 and 6.0 msec, respectively. A conclusion is made that enhancement of Ca2+ macrocurrent by 5-HT is determined by three factors: (i) changes in kinetics of aiready existing channels, (ii) an increase in the number of active channels of the same type, and (iii) an increase in probability of a channel being open. At the same time, the unitary channel conductance was not affected by the transmitter.

Pressure exposure unmasks differences in release properties between high and low yield excitatory synapses of a single crustacean axon

The cellular mechanisms underlying the effect of high pressure on synaptic transmission at two types of synapses were studied in the opener muscle of the lobster walking leg. Excitatory postsynaptic currents (EPSCs) were recorded using a loose macropatch clamp technique at normal pressure and 3.5, 6.9 MPa helium pressure. Responses of the single excitatory axon could be grouped into two types: low yield (L) synapse exhibiting a small mean EPSC with a considerable number of failures, and high yield (H) synapse having a larger mean EPSC with very few failures. The change in several synaptic transmission parameters indicated that high pressure similarly reduced presynaptic evoked release in both L and H synapses. However, some differences in the kinetics and probability of release could be detected. A major difference was the spontaneous miniature EPSCs (mEPSCs) activity. Many of the mEPSCs, observed only in L synapses, were ‘giant’ (size of 2–5 q). High pressure selectively increased the frequency of the giant mEPSCs in the L synapse but had little effect on their amplitude histogram. High pressure depressed evoked synaptic transmission in both synapses by modulating the presynaptic quantal release parameters, but concomitantly enhanced spontaneous quantal release in L synapses by an unknown mechanism.

Quantal secretion from visualized boutons on rat pelvic ganglion neurones

Synaptic transmission from single preganglionic hypogastfc nerves innervating monopolar pelvic ganglion neurones has been studied with intracellular electrodes to record transmission from all the boutons and with extracellular electrodes placed over boutons visualized with DiOC 2(5) in order to record transmission from selected boutons. Intracellular electrodes revealed spontaneous excitatory postsynaptic potentials (EPSPs) with amplitude histograms that show increasing numbers of large EPSPs as the external calcium ([Ca 2+] o) was increased from 0.15 to 1.0 mM. These histograms were in general well fitted by a Poisson mixture of gamma distributions. Extracellular electrodes placed over visualized boutons revealed evoked excitatory postsynaptic potentials (extracellular EPSPs) with amplitude histograms that were best described by single gamma distributions in most cases in low [Ca 2+] o (less than 0.5 mM). The standard deviation of these gammas was not much larger than that of the electrical noise. In a minority of extracelular recordings the amplitude histogram of evoked extracellular EPSPs was best described by a gamma distribution in which the standard deviation was much greater than that of the noise. Confocal microscopy of boutons orthogradely labelled with dextran-rhodamine showed that about 30% of these formed closely apposing pairs on the surface of the neurones. These observations are discussed in terms of the hypothesis that multiquantal release at boutons occurs as a consequence of the coupled secretion from closely apposed boutons.

Rectifying conductance substates in a large conductance Ca(2+)-activated K+ channel: evidence for a fluctuating barrier mechanism.

In this study, we investigated the mechanism underlying the production of inwardly rectifying subconductance states induced in large conductance Ca(2+)-activated K+ channels (maxi K(Ca) channels) by the small, homologous proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin-I (DTX). Low-resolution bilayer recordings of BPTI-induced substates display excess noise that is well described by a beta-distribution characteristic of a filtered, two-state process. High-resolution patch recordings of maxi K(Ca) channels from vascular smooth muscle cells confirm that the BPTI-induced substate is actually comprised of rapid, voltage-dependent transitions between the open state and a nearly closed state. Patch recordings of DTX-induced substates also exhibit excess noise consistent with a similar two-state fluctuation process that occurs at rates faster than those measured for the BPTI-induced substate. The results indicate that these examples of ligand-induced substates originate by a fluctuating barrier mechanism that is similar to one class of models proposed by Dani, J.A., and J.A. Fox (1991. J. Theor. Biol. 153: 401-423) to explain subconductance behavior of ion channels. To assess the general impact of such rapid fluctuations on the practical measurement of unitary currents by amplitude histograms, we simulated single-channel records for a linear, three-state scheme of C (closed)-O(open)-S(substate). This simulation defines a range of transition rates relative to filter frequency where rapid fluctuations can lead to serious underestimation of actual unitary current levels. On the basis of these experiments and simulations, we conclude that fluctuating barrier processes and open channel noise may play an important physiological role in the modulation of ion permeation.

Description of interacting channel gating using a stochastic Markovian model

Single-channel recordings from membrane patches frequently exhibit multiple conductance levels. In some preparations, the steady-state probabilities of observing these levels do not follow a binomial distribution. This behavior has been reported in sodium channels, potassium channels, acetylcholine receptor channels and gap junction channels. A non-binomial distribution suggests interaction of the channels or the presence of channels with different open probabilities. However, the current trace sometimes exhibits single transitions spanning several levels. Since the probability of simultaneous transitions of independent channels is infinitesimally small, such observations strongly suggest a cooperative gating behavior. We present a Markov model to describe the cooperative gating of channels using only the all-points current amplitude histograms for the probability of observing the various conductance levels. We investigate the steady-state (or equilibrium) properties of a system of N channels and provide a scheme to express all the probabilities in terms of just two parameters. The main feature of our model is that lateral interaction of channels gives rise to cooperative gating. Another useful feature is the introduction of the language of graph theory which can potentially provide a different avenue to study ion channel kinetics. We write down explicit expressions for systems of two, three and four channels and provide a procedure to describe the system of N channels.

Description of interacting channel gating using a stochastic Markovian model.

Single-channel recordings from membrane patches frequently exhibit multiple conductance levels. In some preparations, the steady-state probabilities of observing these levels do not follow a binomial distribution. This behavior has been reported in sodium channels, potassium channels, acetylcholine receptor channels and gap junction channels. A non-binomial distribution suggests interaction of the channels or the presence of channels or the presence of channels with different open probabilities. However, the current trace sometimes exhibits single transitions spanning several levels. Since the probability of simultaneous transitions of independent channels is infinitesimally small, such observations strongly suggest a cooperative gating behavior. We present a Markov model to describe the cooperative gating of channels using only the all-points current amplitude histograms for the probability of observing the various conductance levels. We investigate the steady-state (or equilibrium) properties of a system of N channels and provide a scheme to express all the probabilities in terms of just two parameters. The main feature of our model is that lateral interaction of channels gives rise to cooperative gating. Another useful feature is the introduction of the language of graph theory which can potentially provide a different avenue to study ion channel kinetics. We write down explicit expressions for systems of two, three and four channels and provide a procedure to describe the system of N channels.

The variance of successive peaks in synaptic amplitude histograms: effects of inter-site differences in quantal size.

Variability in the measured amplitude of evoked synaptic events can arise from several factors, including: measurement noise, trial-to-trial variation in the amplitude of the response at a single release site, or variation between different release sites (inter-site variation) in the mean amplitude of the single quantal response. Classic (linear) models of variability include only the first two of these factors, although differences in the number of postsynaptic receptors or the degree of electrotonic attenuation for different release sites could cause substantial inter-site variations in quantal size. In this paper, the effect of inter-site variation on the variance of successive histogram peaks has been determined analytically and verified by computational studies. This effect is minimal at the edges of the histogram and contributes maximally to central peaks. Linear approximations to the variance of successive histogram peaks may therefore result in very poor fits to measured data if substantial inter-site variation in quantal size is involved. Our computational results indicate that for synaptic contacts with high release probabilities and substantial inter-site variation, the variance of histogram peaks will decrease with increasing quantal content.

Competitive interactions between supernumerary and normal sensory neurons in the cockroach are mediated through a change in quantal content and not quantal size.

1. The final steps in synapse formation and stabilization involve the adjustment of strength of connections through competitive interactions between neurons contacting a common target. The mechanisms underlying this competition-driven adjustment of synaptic strength are not well understood. We have studied one aspect of this phenomenon using the cercal sensory system of first instar cockroach nymphs. 2. Quantal analysis of excitatory postsynaptic potentials (EPSPs) recorded at the synapse between the lateral filiform hair sensory neuron (L) and giant interneuron 3 (GI3) was carried out to determine whether the reduction in EPSP amplitude observed in the presence of an additional sensory neuron ("space invader neuron" or SIN) was due to pre- or postsynaptic changes. 3. Mean quantal content, not quantal size, was reduced at the L-GI3 synapse in the presence of a SIN. Fitting binomial distributions to the EPSP amplitude histograms gave estimates of the number of releasable quanta or of synaptic sites (n) and the probability of release at these sites (p). The reduction in quantal content is associated with a decrease in the binomial parameter n and not p, suggesting that there is a decrease in the number of contacts, release sites, or quanta available for release, rather than a change in probability of release.

Analysis of evoked and spontaneous quantal release at high pressure in crustacean excitatory synapses.

The cellular mechanisms underlying the effect of high pressure on synaptic transmission were studied in the opener muscle of the lobster walking leg. Excitatory postsynaptic currents (EPSCs) were recorded using a loose macropatch-clamp technique at normal pressure and 3.5, 6.9 MPa helium pressure. Responses of the single excitatory axon could be grouped into two types: low-yield (L) synapses exhibiting small EPSCs with a considerable number of failures, and high-yield (H) synapses having larger EPSCs with very few failures. High pressure reduced the average EPSC amplitude in all synapses and shifted their amplitude histograms to the left by decreasing the quantal content (m) without changing their quantum current (q). A binomial distribution fit of EPSC amplitudes revealed that high pressure greatly decreased n, the number of available active zones, but the effect on p, the probability of release for each zone, was not consistent. Many of the spontaneous miniature EPSCs (mEPSCs), observed only in L-type synapses, were "giant" (size = 2-5 q). High pressure increased the frequency of the giant mEPSCs but had little effect on their amplitude histogram. High pressure depressed evoked synaptic transmission by modulating the presynaptic quantal release parameters, but concomitantly enhanced spontaneous quantal release by an unknown mechanism.

COMPARISON OF EEG TIME SERIES FROM RAT OLFACTORY SYSTEM WITH MODEL COMPOSED OF NONLINEAR COUPLED OSCILLATORS

The so-called “spontaneous,” background, or “basal” time series of electroencephalographic (EEG) data from rat olfactory cortex are compared with those generated by a model of the ol-factory system, denoted KIII, composed of nonlinear coupled second order ODEs with feedback delay. Each ODE (KO) represents a neural mass having either excitatory or inhibitory output to the ODEs to which it connects. Connected mutually inhibitory or mutually excitatory pairs represent populations of similar neurons interacting to form one structure (KI set). A connected set (KII set) of excitatory and inhibitory KI sets (KIe and KIi) represents the activity of a gross structure in the olfactory system (the olfactory bulb—OB, anterior olfactory nucleus—AON, and prepyriform cortex—PPC or PC). The set of KII sets connected with dispersion-delay feedback pathways (KIII set) form a model of the first central stage of olfactory perceptual processing upon olfactory stimulation, with transition on receptor input from a self-sustained basal state of activity to an induced burst. Both basal and stimulated states are studied using amplitude histograms, power spectra, attractor reconstruction, visual inspection of traces, and correlation. A comparison shows that the two systems are similar, having broad spectra, correlation functions that rapidly go to zero, and gaussian amplitude histograms. Activity is nearly periodic and higher amplitude in the excited state and aperiodic, lower amplitude in the basal state with close to 1/f falloff. Cross spectra between OB and PPC show wide frequency bands of coherent high frequency activity in both the model and rat. The excited state nearly periodic frequencies differ between the KIII model and rat (30–60 Hz in KIII, 50–100 Hz in rat EEG), because the KIII model was developed for the cat and the rabbit, which have lower frequencies than the rat. These benchmarks may be used to evaluate the physiological validity of the output produced by a proposed model, as all components of the system and its connectivity were derived from or evaluated by physiological data.

Conductance and kinetics of single cGMP‐activated channels in salamander rod outer segments.

1. The conductance and kinetics of single 3',5'-cyclic guanosine monophosphate (cGMP)-activated channels of retinal rod outer segments were studied in inside-out membrane patches. The size of the single channel currents was increased by using low concentrations of divalent cations. 2. At saturating cGMP concentration, the current flickered at high frequency. Occasionally, the current was interrupted by closures lasting tens or hundreds of milliseconds. At +50 mV the maximum current during an opening was slightly more than 1 pA, but the open channel level was poorly resolved due to the speed of the gating transitions. 3. Amplitude histograms confirmed the presence of a sublevel of current, roughly a quarter the size of the peak current, at low cGMP concentrations. The fraction of time in the sublevel decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening by the partially liganded channel. 4. Consistent with previous macroscopic current recordings, single channel activation by cGMP had an apparent dissociation constant of 8.6 microM, and a Hill coefficient of 2.8. 5. At saturating cGMP concentrations, the channel was modelled as a two-state system with the following parameters. The open channel conductance was 25 pS. The opening rate constant, beta, was 1.5 x 10(4) s-1 at 0 mV, and had a voltage sensitivity equivalent to the movement of 0.23 electronic charges outward through the membrane electric field. The closing rate constant, alpha, was 2.1 x 10(4) s-1 and was voltage insensitive. Assuming that the open-state chord conductance was voltage independent, the inferred voltage dependence of beta largely accounted for the outward rectification in the steady-state macroscopic current-voltage relation of multichannel patches, at saturating cGMP concentration.

Multimodal action of single Na+ channels in myocardial mouse cells

Unitary Na+ currents of myocardial mouse cells were studied at room temperature in 10 cell-attached patches, each containing one and only one channel. Small-pore patch pipettes (resistance 10-97 M omega when filled with 200% Tyrode's solution) with exceptionally thick walls were used. Observed were both rapidly inactivating (6 patches) and slowly inactivating (3 patches) Na+ currents. In one patch, a slow transition from rather fast to slow inactivation was detected over a time of 0.5 h. A short and a long component of the open-channel life time were recorded at the beginning, but only a short one at the end of the experiment. Concomitantly, the first latency was slowed. Amplitude histograms showed that the electrochemical driving force across the pore of the channel did not change during this time. In three patches, a fast and repetitive switching between different modes of Na+ channel action could be clearly identified by plotting the long-time course of the averaged current per trace. The ensemble-averaged current formed in each mode was different in kinetics and amplitude. Each mode had a characteristic mean open-channel life time and distribution of first latency, but the predominant single-channel current amplitude was unaffected by mode switches. It is concluded that two types of changes in kinetics may happen in a single Na+ channel: fast and reversible switches between different modes, and a slow loss of inactivation.

Signal processing pattern classification techniques to improve knock detection in spark ignition engines

The aim of this paper is to illustrate the contribution of signal processing pattern recognition techniques to the resolution of a physical problem: entire knock detection. Knock is an abnormal combustion of spark ignition in automotive engines. Classical detectors use the energy of engine vibration in order to detect it. Unfortunately, detection becomes erroneous at high speeds when noise level increases. Pattern recognition techniques allow the determination of optimal parameters and methods for knock recognition: cepstral coefficients and amplitude histograms improve knock recognition at 5500 rpm and for other speeds. A comparison between five speeds using the conventional detector and the new detector shows that the latter clearly outperforms the former. The method used here of knock detection can be used in several other applications e.g. gear default detection and axle fissure.

Quantal analysis of presynaptic inhibition, low [Ca2+]0, and high pressure interactions at crustacean excitatory synapses

The cellular mechanisms underlying the effects of high pressure, GABAergic presynaptic inhibition, and low [Ca2+]0 on glutamatergic excitatory synaptic transmission were studied in the opener muscle of the lobster walking leg. Excitatory postsynaptic currents (EPSCs) were recorded with or without prior stimulation of the inhibitor using a loose macropatch clamp technique at atmospheric pressure and at 6.9 MPA helium pressure. High pressure reduced the mean EPSC amplitude and variance, decreased the quantal content (m), but did not affect the quantum current (q). Pressure shifted the median of the amplitude histogram to the left by 1-2 q. Under normal pressure conditions, presynaptic inhibition and low [Ca2+]0 induced similar effects. However, quantal analysis using a binomial frequency distribution model revealed that high pressure and low [Ca2+]0 diminished n (available active zones) and slightly increased p (probability of release), but presynaptic inhibition reduced p and slightly increased n. At high pressure, presynaptic inhibition was reduced, at which time the major contributor to the inhibitory process appeared to be reduction in n and not p. The similarity of the alterations in quantal parameters of release at high pressure, low [Ca2+]0, and in some conditions of presynaptic inhibition is consistent with the hypothesis that pressure reduces Ca2+ inflow into the presynaptic nerve terminals to affect the Ca(2+)-dependent quantal release parameters n and p.

Excitatory synaptic connections onto rat hippocampal inhibitory cells may involve a single transmitter release site.

1. Whole-cell tight-seal records of excitatory postsynaptic currents (EPSCs) were made from inhibitory cells in the CA3 region of thin hippocampal slices. We tested the hypothesis that excitatory synaptic connections made on inhibitory cells involve few transmitter release sites. 2. EPSCs impinging on inhibitory cells had a time to peak of 0.4-3.8 ms and an amplitude of 8-90 pA at a holding potential of -60 mV. They were suppressed by the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovaleric acid (APV). 3. Addition of tetrodotoxin (TTX) and Co2+ to the external solution reduced the frequency of EPSCs from 0.90 to 0.25 s-1 (n = 24 cells). In the majority of cells EPSC amplitude distributions were not significantly changed. 4. Increasing Ca2+ and reducing Mg2+ in the external solution, in order to enhance the probability of transmitter release, did not change EPSC amplitude distributions. In contrast, amplitude histograms for IPSCs recorded from pyramidal cells were shifted to higher mean values in this solution. 5. EPSCs were elicited in inhibitory cells by electrical stimulation via a glass pipette placed near to pyramidal cells in stratum pyramidale. EPSCs elicited by weak stimuli had similar amplitude distributions to excitatory synaptic events recorded in the presence of TTX and Co2+. 6. These findings suggest excitatory synaptic connections made with CA3 inhibitory cells involve few or possibly just one transmitter release site.

The quantal size at retinogeniculate synapses determined from spontaneous and evoked EPSCs in guinea-pig thalamic slices.

1. To determine the quantal size at retinogeniculate synapses, spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded in twelve neurones of the dorsal lateral geniculate nucleus in guinea-pig thalamic slices using the whole-cell patch-clamp technique. We limited our study to the fast non-N-methyl-D-aspartate (NMDA) component of the EPSCs by adding the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid to the perfusion medium. 2. Spontaneous EPSCs occurred at a frequency between 0.5 and 6.6 Hz (mean 2.5 Hz). The modal value of the peak conductance change of spontaneous excitatory events varied between cells from 102 to 179 pS. 3. EPSCs were evoked by electrical stimulation in the optic tract. The peak conductance change of EPSCs evoked by stimulation of a putative single input fibre ranged from 0.6 to 3.4 nS (mean 1.7 nS). 4. To resolve the quantal components of evoked EPSCs the external Ca2+ concentration was reduced and the external Mg2+ concentration increased for four cells. In this condition failures occurred and the amplitude histograms were multimodal with approximately equidistant peaks. 5. These multimodal histograms could be fitted by a sum of Gaussian functions with mean values corresponding to integer multiples of the modal value of the spontaneous EPSCs for the same cell. Thus, the quantal size of evoked EPSCs was the same as the modal value of spontaneous EPSCs. The mean of the apparent quantal conductance change was 138 pS. The estimated number of quanta released by stimulating a putative single input fibre in the control condition ranged from 4 to 27 (mean 13).(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of single cardiac Na channels at 35 degrees C.

Single Na channel currents were recorded in cell-attached patches of mouse ventricular myocytes with an improved patch clamp technique. Using patch pipettes with a pore diameter in the range of 200 nm, seals with a resistance of up to 4 T omega were obtained. Under those conditions, total noise could be reduced to levels as low as 0.590 pA rms at 20 kHz band width. At this band width, properties of single-channel Na currents were studied at 35 degrees C. Six out of a total of 23 patches with teraohm seals contained channel activity and five of these patches contained one and only one active channel. Amplitude histograms excluding transition points showed heterogenous distributions of levels. In one patch, part of the openings was approximately Gaussian distributed at different potentials yielding a slope conductance of 27 pS. The respective peak open probability at -10 mV was 0.26. The mean open time was determined at voltages between -60 and -10 mV by evaluation of the distribution of the event-related gaps in the center of the baseline noise to be approximately 40 microseconds at -60 mV and 50-74 microseconds between -50 and -10 mV. It is concluded that single cardiac Na channels open at 35 degrees C frequently with multiple levels and with open times in the range of several tens of microseconds.