Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors.

The time course of most quantal currents recorded with a small diameter electrode placed over visualized varicosities of sympathetic nerve terminals that secrete ATP was determined: these had a time to reach 90% of peak of 1.3-1.8 ms and a time constant of decay of 12-18 ms; they were unaffected by blocking ectoenzymes or the uptake of adenosine. Monte Carlo methods were used to analyze the stochastic interaction between ATP, released in a packet from a varicosity, and the underlying patch of purinoceptors, to reconstitute the time course of the quantal current. This leads to certain restrictions on the possible number of ATP molecules in a quantum (about 1000) and the density of purinoceptors at the junctions (about 1000 microns-1), given the known geometry of the junction and the kinetics of ATP action. The observed quantal current has a relatively small variability (coefficient of variation < 0.1), and this stochastic property is reproduced for a given quantum of ATP. Potentiation effects (of about 12%) occur if two quanta are released from the same varicosity because the receptor patch is not saturated even by the release of two quanta. The simulations show that quantal currents have a characteristically distinct shape for varicosities with different junctional cleft widths (50-200 nm). Finally, incorporation of an ectoenzyme with the known kinetics of ATPase into the junctional cleft allows for a quantal current of the observed time course, provided the number of ATP molecules in a quantum is increased over the number in the absence of the ATPase.     

Quantal potential fields around individual active zones of amphibian motor-nerve terminals

The release of a quantum from a nerve terminal is accompanied by the flow of extracellular current, which creates a field around the site of transmitter action. We provide a solution for the extent of this field for the case of a quantum released from a site on an amphibian motor-nerve terminal branch onto the receptor patch of a muscle fiber and compare this with measurements of the field using three extracellular electrodes. Numerical solution of the equations for the quantal potential field in cylindrical coordinates show that the density of the field at the peak of the quantal current gives rise to a peak extracellular potential, which declines approximately as the inverse of the distance from the source at distances greater than about 4 microm from the source along the length of the fiber. The peak extracellular potential declines to 20% of its initial value in a distance of about 6 microm, both along the length of the fiber and in the circumferential direction around the fiber. Simultaneous recordings of quantal potential fields, made with three electrodes placed in a line at right angles to an FM1-43 visualized branch, gave determinations of the field strengths in accord with the numerical solutions. In addition, the three electrodes were placed so as to straddle the visualized release sites of a branch. The positions of these sites were correctly predicted on the basis of the theory and independently ascertained by FM1-43 staining of the sites. It is concluded that quantal potential fields at the neuromuscular junction that can be measured with available recording techniques are restricted to regions within about 10 microm of the release site.     

Latency of transmitter release at crayfish motor nerve endings examined by intracellular depolarization

Latency of release of individual quanta of transmitter was studied at neuromuscular junctions of a crayfish (Procambarus clarkii). Postsynaptic quantal currents were recorded at individual motor nerve endings with a macropatch electrode while the subterminal axon branch was depolarized by current passed through an intracellular microelectrode. For depolarizing currents of moderate size, the latency of transmitter release did not change when the duration of the depolarizing current was altered. Previous studies in which a contrary result was obtained may have been compromised by artefacts or by the sampling methods employed. The present results do not support the hypothesis of a depolarization-induced "repressor" of quantal release.     

Quantal glutamate release is essential for reliable neuronal encodings in cerebral networks

Background

The neurons and synapses work coordinately to program the brain codes of controlling cognition and behaviors. Spike patterns at the presynaptic neurons regulate synaptic transmission. The quantitative regulations of synapse dynamics in spike encoding at the postsynaptic neurons remain unclear.

Methodology/Principal Findings

With dual whole-cell recordings at synapse-paired cells in mouse cortical slices, we have investigated the regulation of synapse dynamics to neuronal spike encoding at cerebral circuits assembled by pyramidal neurons and GABAergic ones. Our studies at unitary synapses show that postsynaptic responses are constant over time, such as glutamate receptor-channel currents at GABAergic neurons and glutamate transport currents at astrocytes, indicating quantal glutamate release. In terms of its physiological impact, our results demonstrate that the signals integrated from quantal glutamatergic synapses drive spike encoding at GABAergic neurons reliably, which in turn precisely set spike encoding at pyramidal neurons through feedback inhibition.

Conclusion/Significance

Our studies provide the evidences for the quantal glutamate release to drive the spike encodings precisely in cortical circuits, which may be essential for programming the reliable codes in the brain to manage well-organized behaviors.     

A model for analysis of the anesthetic response

When a dose d of an anesthetic is administered intravenously, either sleep will be observed for a period of time or no sleep will occur. We developed a general approach for formulating a model for this response which reduces to a quantal response model if the duration of sleep is ignored. Several specific models illustrate the general formulation. In each case the underlying dose response relationship that corresponds to the quantal response problem is the logistic. To include the duration of sleep in the model we introduced a concept of an "active dose" which is a function of the administered dose and time. We use a set of data from a study of the response of guinea pigs to ketamine for our numerical examples.     

Monte Carlo simulation of miniature endplate current generation in the vertebrate neuromuscular junction.

A Monte Carlo method for modeling the neuromuscular junction is described in which the three-dimensional structure of the synapse can be specified. Complexities can be introduced into the acetylcholine kinetic model used with only a small increase in computing time. The Monte Carlo technique is shown to be superior to differential equation modeling methods (although less accurate) if a three-dimensional representation of synaptic geometry is desired. The conceptual development of the model is presented and the accuracy estimated. The consequences of manipulations such as varying the spacing of secondary synaptic folds or that between the release of multiple quantal packets of acetylcholine, are also presented. Increasing the spacing between folds increases peak current. Decreased spacing of adjacent quantal release sites increases the potentiation of peak current.     

Quantal analysis of long-term posttetanic changes in minimal postsynaptic potentials of hippocampal slices in vitro

Minimal excitatory postsynaptic potentials (EPSP) were investigated in 13 neurons under single or double-pulse near-threshold microstimulation of the radial layer (Schaffer's collaterals) and stratum oriens in surviving hippocampal slices (area CA1) in guinea pigs. The amplitude of 23 EPSP (9 units; 12 pathways) rose after tetanization of Schaffer's collaterals over a 5–55 min period, taken as long-term potentiation (LTP). Statistical analysis conducted using four methods of quantal hypothesis based on a binomial approximation revealed an increase in mean quantal content (m) during LTP. The rise in quantal size was only statistically significant when using data obtained from a section of these methods (mainly for stretches of over 15 min following tetanization) and shows no correlation with intensity of LTP. The pronounced rise in m demonstrated using different methods matches data from experiments on intact animals and indicates a presynaptic location of the mechanisms underlying protracted persistence of residual tetanization lasting some tens of minutes.Institute for Brain Research, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Max-Planck Institute of Biophysical Chemistry, Göttingen, Germany. Zoological Institute, Jagiellonian University, Cracow, Poland. Translated from Neirofiziologiya, Vol. 22, No. 6, pp. 752–761, November–December, 1990.     

Applicability of the coefficient of variation method for analyzing synaptic plasticity.

The classical coefficient of variation method for "quantal" analysis of synaptic responses allows unambiguous identification of pre- and postsynaptic loci underlying synaptic plasticity only when extensive simplifying restrictions are made. They include invariance of quantal parameters and the assumption that a single afferent produces the evoked potentials or currents. More general theoretical formulations and simulations demonstrate that the standard criteria do not always provide useful guidelines because when the other sources of physiological variance are included, putative pre- and postsynaptic domains may overlap. For example, data typically interpreted as indicating modifications at both sites can be due to a mechanism localized to only one of the two, if parameter variances are taken into consideration in the case of a single input cell, or if there are multiple inputs and the stimulus does not activate all of them reliably. With this perspective, other physiologically realistic hypotheses relevant to the expression of synaptic plasticity, such as that during long-term potentiation, can be envisioned.     

Changes in the kinetics of quanta secretion-effective mechanism of synaptic transmission modulation

It is widely accepted that the leading presynaptic mechanisms underlying the synaptic plasticity involve changes of the number of neurotransmitter quanta released by one nerve pulse (the quantal content of postsynaptic response) and of the size of a single quantum. In addition, the existence of one more effective though previously ignored mechanism of modulation of synaptic plasticity was suggested related to the change in the time course (kinetics) of secretion of single neurotransmitter quanta forming the multiquantal response. This article reviews current data (including the authors' own results) on the kinetics of evoked neurotransmitter quanta secretion from motor nerve endings in peripheral synapses, mechanisms of their modulation and methods of quantitative analysis.     

F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling

A short comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect, the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating endoplasmic/sarcoplasmic reticulum-derived vesicles equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive channel-receptors for Ca-release. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of actin filaments. Cellular sites of F-actin-based Ca storage are microvilli and the submembrane cytoskeleton. Several specific features of Ca signaling such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release (CICR), which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling.     

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.     

Probabilistic secretion of quanta from nerve terminals at synaptic sites on muscle cells: non-uniformity, autoinhibition and the binomial hypothesis

A model of the secretion of a quantum at a release site is proposed in which, following the influx of calcium ions, synaptic vesicles are made available for release by the activation of kappa phosphorylation steps with rate alpha. At any time during this process the vesicles may become unavailable for secretion at rate gamma. On completion of the kappa phosphorylation steps the vesicles participate in the formation of a fusion pore with the terminal membrane to give exocytosis at rate delta. Changes in alpha, delta and kappa are shown to produce characteristic changes in the number and timecourse of quantal secretions following a nerve impulse, which are similar to those observed following drug treatments that are thought to act selectively on each of these processes. The number of quanta secreted from nerve terminals that consist of many release sites does not fluctuate much during a low frequency train of impulses: the variance is small compared with the mean level, so secretion follows binomial rather than Poisson statistics. A theory is derived that shows that variations in the probability of secretion amongst these release sites of any particular kind fails to reduce the variance of the total secretion from the terminal; Poisson rather than binomial statistics then still apply. The theory shows that an interaction between release sites is required to reduce this variance and such an effect is provided if secretion at a site inhibits secretion at nearby sites. Simulations show that incorporating this process of autoinhibition into the model reproduces the experimental observations on the effects of calcium ions on the binomial parameters p and n as well as on the relative constancy of p during facilitation and depression of quantal secretion. Methods for estimating the timecourse of changes in the probability of secretion at release sites following an impulse, by using either the time of occurrence of first, second, third or later quantal latencies, are given. These procedures show that current methods for estimating the time-dependent probability changes are inadequate for detecting interaction between release sites, such as autoinhibition, unless this is relatively large. Therefore, estimates from third quantal latencies are used.     

Endurance exercise modulates neuromuscular junction of C57BL/6NNia aging mice

Fahim, Mohamed A. Endurance exercise modulatesneuromuscular junction of C57BL/6NNia aging mice. J. Appl. Physiol. 83(1): 59-66, 1997.The effect ofage and endurance exercise on the physiology and morphology ofneuromuscular junctions (NMJ) of gluteus maximus muscle was studied inC57BL/6NNia mice. Mice were exercised, starting at 7 or 25 mo of age,at 28 m/min for 60 min/day, 5 days/wk for 12 wk, on a rodent treadmill.Intracellular recordings of spontaneous miniature endplate potentials(MEPP) and the quantal content of endplate potentials (EPP) wererecorded from NMJ of 10- and 28-mo-old control and exercised mice.Endurance exercise resulted in significant increases in MEPP amplitudes (23%), quantal content, and safety margin, and a significant decrease in MEPP frequency of young mice, with no change in resting membrane potential or membrane capacitance. Three months of endurance exercise resulted in an increase in MEPP frequency (41%) and decreases in MEPPamplitudes (15%), quantal content, and safety margin of old mice.Endurance exercise resulted in significantly larger nerve terminals(24%) in young animals, suggesting functional adaptation. Nerveterminals in exercised 28-mo-old mice were smaller than in thecorresponding control mice, an indication that exercise minimizedage-related nerve terminal elaboration. It is concluded that thedifferent physiological responses of young and old gluteus maximusmuscles to endurance exercise parallel their morphological responses.This suggests that the mouse NMJ undergoes a process of physiologicaland morphological remodeling during aging, and such plasticity could bemodulated differently by endurance exercise.

     

Calcium modulation of the release kinetics of the acetylcholine quanta generating multiquantal postsynaptic response

The effects of calcium on the quantal content of nerve-evoked endplate currents (EPC) and on the temporal parameters of quantal release were studied in the frog neuromuscular synapse using the method of "subtractions". It was shown that under physiological conditions quanta generating multiquantal postsynaptic responses were released nonsynchronously because of a considerable variability of latencies of the uniquantal responses forming multiquantal EPC. Different calcium dependences for EPCs quantal content and time course of the quantal release were revealed. The average quantal content grew exponentially with the increase in calcium concentration from 0.4 to 1.8 mmol/L, whereas the release synchronicity reached the maximum at 1 mmol/L calcium. It was suggested that the changes in the synchronicity of the evoked release were one of the mechanisms of the synaptic plasticity.     

Extracting Information from the Power Spectrum of Synaptic Noise

In cortical neurons, synaptic "noise" is caused by the nearly random release of thousands of synapses. Few methods are presently available to analyze synaptic noise and deduce properties of the underlying synaptic inputs. We focus here on the power spectral density (PSD) of several models of synaptic noise. We examine different classes of analytically solvable kinetic models for synaptic currents, such as the "delta kinetic models," which use Dirac delta functions to represent the activation of the ion channel. We first show that, for this class of kinetic models, one can obtain an analytic expression for the PSD of the total synaptic conductance and derive equivalent stochastic models with only a few variables. This yields a method for constraining models of synaptic currents by analyzing voltage-clamp recordings of synaptic noise. Second, we show that a similar approach can be followed for the PSD of the the membrane potential (Vm) through an effective-leak approximation. Third, we show that this approach is also valid for inputs distributed in dendrites. In this case, the frequency scaling of the Vm PSD is preserved, suggesting that this approach may be applied to intracellular recordings of real neurons. In conclusion, using simple mathematical tools, we show that Vm recordings can be used to constrain kinetic models of synaptic currents, as well as to estimate equivalent stochastic models. This approach, therefore, provides a direct link between intracellular recordings in vivo and the design of models consistent with the dynamics and spectral structure of synaptic noise.     

Light-sensitive voltage responses in the neurons of the cerebral ganglion of Ciona savignyi (Chordata: Ascidiacea)

Light-responsive behaviors such as siphon contraction (1), phototropism (2), and gamete release (3, 4) have been described in several ascidian species. The pigmented spots around the siphon openers (5), the epithelial cells of the sperm duct (6, 7), and the cerebral ganglion (8) have been suggested to be the photoreceptor candidates underlying these behaviors. However, these arguments have not yet been settled because no direct electrophysiological recordings of light-induced receptor potentials have been reported. In this study, we focused on the cerebral ganglion and performed intracellular recordings from the neurons in the ventral side of the cerebral ganglion in an isolated in vitro preparation of the neural complex in Ciona savignyi. We found that 24% (n = 115) of the recorded neurons showed various types of voltage responses to light stimuli. Almost all (27/28) of the recorded voltage responses were "on" responses that included hyperpolarizing and depolarizing responses and could be categorized into five types, except for a complex response recorded in one cell; the remaining one (1/28) was a depolarizing "off" response. This is the first report of electrophysiological recordings of light-sensitive voltage responses from ascidian cerebral ganglion neurons.     

Regulation of ryanodine receptor opening by lumenal Ca(2+) underlies quantal Ca(2+) release in PC12 cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.     

Quantifying Spike Train Oscillations: Biases,Distortions and Solutions

Estimation of the power spectrum is a common method for identifying oscillatory changes in neuronal activity. However, the stochastic nature of neuronal activity leads to severe biases in the estimation of these oscillations in single unit spike trains. Different biological and experimental factors cause the spike train to differentially reflect its underlying oscillatory rate function. We analyzed the effect of factors, such as the mean firing rate and the recording duration, on the detectability of oscillations and their significance, and tested these theoretical results on experimental data recorded in Parkinsonian non-human primates. The effect of these factors is dramatic, such that in some conditions, the detection of existing oscillations is impossible. Moreover, these biases impede the comparison of oscillations across brain regions, neuronal types, behavioral states and separate recordings with different underlying parameters, and lead inevitably to a gross misinterpretation of experimental results. We introduce a novel objective measure, the "modulation index", which overcomes these biases, and enables reliable detection of oscillations from spike trains and a direct estimation of the oscillation magnitude. The modulation index detects a high percentage of oscillations over a wide range of parameters, compared to classical spectral analysis methods, and enables an unbiased comparison between spike trains recorded from different neurons and using different experimental protocols.     

Invasive recordings from the human brain: clinical insights and beyond

Although non-invasive methods such as functional magnetic resonance imaging, electroencephalograms and magnetoencephalograms provide most of the current data about the human brain, their resolution is insufficient to show physiological processes at the cellular level. Clinical approaches sometimes allow invasive recordings to be taken from the human brain, mainly in patients with epilepsy or with movement disorders, and such recordings can sample neural activity at spatial scales ranging from single cells to distributed cell assemblies. In addition to their clinical relevance, these recordings can provide unique insights into brain functions such as movement control, perception, memory, language and even consciousness.