多通道神经元锋电位检测和分类的新方法

大脑神经元胞外单细胞动作电位(即锋电位)的检测和分类是提取神经元脉冲序列、研究神经系统信息处理机制的关键．为了提高锋电位的检出率和分类的正确性,设计了一种处理多通道锋电位记录信号的算法,用于分析微电极阵列记录的大鼠海马神经元锋电位信号,电极阵列上的测量点排列紧密,4个通道可以同时记录到来自相同神经元的信号．该算法首先利用一种多通道阈值检测法检出四通道记录信号中的锋电位,然后利用一种基于复合锋电位的主成分特征参数分类法将锋电位分类．仿真数据和实验记录信号的检验结果表明：与相应的单通道算法相比,该算法的锋电位检出率和分类的正确性显著提高,并且可以增加单次实验测得的神经元数目．因此,该算法为实现神经元锋电位的自动检测提供了一种简单有效的新 方法．     

Neuromuscular glutamatergic and GABAergic channels

Our laboratory has worked extensively on glutamatergic and GABA-ergic channels, predominantly in crayfish, but also in locust,Drosophila and recentlyAscaris. Channel currents were recorded in the different modes of the patch-clamp technique (Hamillet al., 1981). The opening kinetics of the channels were derived from open and closed time histograms obtained from single channel recordings. From these, channel conductances could also be evaluated. The most relevant data were obtained by very rapidly rising and falling pulses (time of change about 0.1 ms) of agonists applied to outside-out patches containing the respective channels (Frankeet al., 1987). From such recordings we constructed dose-response curves for peak and steady-state currents, for the rise times of the currents and for the time constants of desensitization. In double-pulse experiments we measured recovery from desensitization and predesensitization due to low agonist concentrations. For most of the channel types, we succeeded in constructing a reaction scheme which in computer simulations mimicked channel behaviour to a good approximation.     

在体膜片钳技术的新进展

在体膜片钳是指在整体动物上直接对其中枢神经元进行全细胞膜片钳记录的技术,在生理学和药理学研究中具有良好的应用前景.常规采用的是盲法记录,最近出现的可视法记录,采用双光子靶向膜片钳（two-photon targeted patching,TPTP）技术,通过基因操作在动物脑内目标神经元中构建特异表达的荧光标志,可以做到对特定神经元亚群的靶向研究.对这两种方法的原理和操作进行了简单的介绍.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

Mathematical model and numerical simulation of the cell growth in scaffolds

A scaffold is a three-dimensional matrix that provides a structural base to fill tissue lesion and provides cells with a suitable environment for proliferation and differentiation. Cell-seeded scaffolds can be implanted immediately or be cultured in vitro for a period of time before implantation. To obtain uniform cell growth throughout the entire volume of the scaffolds, an optimal strategy on cell seeding into scaffolds is important. We propose an efficient and accurate numerical scheme for a mathematical model to predict the growth and distribution of cells in scaffolds. The proposed numerical algorithm is a hybrid method which uses both finite difference approximations and analytic closed-form solutions. The effects of each parameter in the mathematical model are numerically investigated. Moreover, we propose an optimization algorithm which finds the best set of model parameters that minimize a discrete l ₂ error between numerical and experimental data. Using the mathematical model and its efficient and accurate numerical simulations, we could interpret experimental results and identify dominating mechanisms.     

The patch clamp is more useful than anyone had expected

The patch clamp was developed into its present form by Neher and Sakmann for recording the currents through individual ionic channels in excitable membranes. Recent advances now allow the patch-clamp technique to be used to record from cell-free membrane patches and from entire small cells. The technique has been used extensively for recordings from single channels that have provided new insights into their functioning; it is also being applied to a surprising variety of other physiological problems.     

Heterogeneity of Volume-sensitive Chloride Channels in Basolateral Membranes of A6 Epithelial Cells in Culture

A new technique allowing single-channel patch-clamp recordings from basolateral membranes of A6 renal epithelial cells in culture was developed. Using this technique we studied the chloride channels activated in these basolateral membranes during hypo-osmotic stress. Four different types of channel were identified and classified according to their current/voltage (I/V) relationships as observed in the on-cell configuration of the patch-clamp technique. Three of these channels had linear I/V relationships with unitary conductances of 12, 30 and 42 pS. The fourth type had an outwardly rectifying I/V curve with inward and outward conductances of 16 and 57 pS respectively. The kinetic properties of each class of channel were studied and kinetic models developed for two of them: the 42 pS channel and the outward rectifier. These models permitted the study of the evolution of the kinetic parameters during hypo-osmotic shock and revealed two different kinetic schemes of channel activation. The results of experiments made on the basolateral membranes were also compared with those of a set of analogous patch-clamp experiments carried out on isolated A6 cells. In these latter, the frequency of successful observations of active channels in a patch was 13%, whereas it was 31% for basolateral membranes. Also, of the four types of channel observed in basolateral membranes, two were never found in isolated cells, only the 12 pS channel and the outward rectifier were present in these isolated cells. Received: 17 April 1996/Revised: 26 June 1996     

Ion channels: does each subunit do something on its own?

The advent of the patch-clamp technique 25 years ago revolutionized the study of ion channels. This method also made it possible to measure the kinetic behavior of single protein molecules. The low-noise recordings of ionic currents through single channels, coupled with other cutting-edge technologies, have revealed a rich complexity of functional states that are not readily explained by simple allosteric protein models such as the popular concerted model and the sequential model. Although these models can each account for elements of ion channel function, we propose that variations or extensions of the lesser-known general allosteric model provide a more promising framework for explaining the intricate behaviors of ion channels.     

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter''s experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.     

Erratum: High speed sCMOS‐based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

In the article by M.B. Sikkel et al. (doi: 10.1002/jbio.201500193), published in J. Biophotonics 9 , 311–323 (2016), an error occurred in the computer code that was used to generate Figure 3. This erratum is published to correct Figure 3, the calculated value of t_geom and the experimentally determined value of t_optics in the text of the article.     

A model for the development of genetic translation

Several models have been advanced, both in this journal and others, for the development of the genetic code and translation apparatus. Eigen in particular has put forward a detailed model based on the hypercycle. This paper uses some of these previous ideas to develop a new model of the code and translation in which the pairs AU and GC play complementary roles, and in whichtRNAs develop from a molecule withtwo loops which stacks in repetitive patterns without the need for a messenger RNA. Thus a bridge is provided between random, (or autocatalytic) polymerization, and coded translation. In addition, alternative postulates to several of Eigen's ideas are tested by computer simulation.     

ALGORITHM FOR APPLICATION OF FOURIER ANALYSIS FOR BIORHYTHMIC BASELINES OF PHARMACODYNAMIC INDIRECT RESPONSE MODELS

The change of an indirect pharmacological response R(t) can be described by a periodic time-dependent production rate k_in (t) and a first-order loss constant k_out. If k_in(t) follows some biological rhythm (e.g., circadian), then the response R(t) also displays a periodic behavior. A new approach for describing the input function in indirect response models with biorhythmic baselines of physiologic substances is introduced. The present approach uses the baseline (placebo) response R_b(t) to recover the equation for k_in(t). Fourier analysis provides an approximate equation for R_b(t) that consists of terms (usually two or three) of the Fourier series (harmonics) that contribute most to the overall sum. The model differential equation is solved backward for k_in(t), yielding the equation involving R_b(t). A computer program was developed to perform the square L²-norm approximation technique. Fourier analysis was also performed based on nonlinear regression. Cortisol suppression after inhalation of fluticasone propionate (FP) was modeled based on the inhibition of the secretion rate k_in(t) using ADAPT II. The pharmacodynamic parameters k_out and IC₅₀ were estimated from the model equation with k_in(t) derived by the new approach. The proposed method of describing the input function needs no assumption about the behavior of k_in(t), is as efficient as methods used previously, and is more flexible in describing the baseline data than the nonlinear regression method. (Chronobiology International, 17(1), 77–93, 2000)     

Thermal behaviors of elastin-like polypeptides (ELPs) according to their physical properties and environmental conditions

Elastin-like polypeptides (ELPs) have a distinctive thermal property, transition temperature (T_t), which leads to phase transition. This thermal property depends on the molecular weight (MW) of ELP, ELP concentration, composition of the amino acids constituting ELPs, and ionic strength of the aqueous solution. In order to investigate the effects of ELP length, ionic strength and existence of fusion protein, ELP genes of three different sizes were cloned using the recursive directional ligation (RDL) method and expressed in Escherichia coli. Following purification, thermal behaviors of ELPs were monitored using a spectrophotometer with temperature scanning. The results of our study indicated that T_t shifted to low in accordance with ELP length or increased ionic strength. Additionally, it was observed that T_t was affected by the physical properties of the protein fused with ELPs.     

Preferential KAT1-KAT2 Heteromerization Determines Inward K+ Current Properties in Arabidopsis Guard Cells

Guard cells adjust their volume by changing their ion content due to intense fluxes that, for K⁺, are believed to flow through inward or outward Shaker channels. Because Shaker channels can be homo- or heterotetramers and Arabidopsis guard cells express at least five genes encoding inward Shaker subunits, including the two major ones, KAT1 and KAT2, the molecular identity of inward Shaker channels operating therein is not yet completely elucidated. Here, we first addressed the properties of KAT1-KAT2 heteromers by expressing KAT1-KAT2 tandems in Xenopus oocytes. Then, computer analyses of the data suggested that coexpression of free KAT1 and KAT2 subunits resulted mainly in heteromeric channels made of two subunits of each type due to some preferential association of KAT1-KAT2 heterodimers at the first step of channel assembly. This was further supported by the analysis of KAT2 effect on KAT1 targeting in tobacco cells. Finally, patch-clamp recordings of native inward channels in wild-type and mutant genotypes strongly suggested that this preferential heteromerization occurs in planta and that Arabidopsis guard cell inward Shaker channels are mainly heteromers of KAT1 and KAT2 subunits.     

Comparing short protein substructures by a method based on backbone torsion angles

An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes Δt, the root mean square difference in ? and ψ torsion angles over a small number of amino acids (typically 3–5). Using this algorithm, large number of protein substrates comparisons were feasible. The parameter Δt was sensitive to variations in local protein conformation, and it correlates with Δr, the root mean square deviation in atomic coordinates. Values for Δt were obtained that define similarity thresholds, which determine whether two substructure are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of Δt due to measurement noise fromcomparisons of independently refined coordinates of the same protein. A sample distribution of Δt from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons large numbers of nonmatching comparisons. Unlike methods based on Cα atoms alone, Δt was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologus proteins by Δt showed that the active site torsion angles are highly conserved. The Δt method was applied to the α-chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.     

Activation of Shaker Potassium Channels : I. Characterization of Voltage-dependent Transitions

The conformational changes associated with activation gating in Shaker potassium channels are functionally characterized in patch-clamp recordings made from Xenopus laevis oocytes expressing Shaker channels with fast inactivation removed. Estimates of the forward and backward rates for transitions are obtained by fitting exponentials to macroscopic ionic and gating current relaxations at voltage extremes, where we assume that transitions are unidirectional. The assignment of different rates is facilitated by using voltage protocols that incorporate prepulses to preload channels into different distributions of states, yielding test currents that reflect different subsets of transitions. These data yield direct estimates of the rate constants and partial charges associated with three forward and three backward transitions, as well as estimates of the partial charges associated with other transitions. The partial charges correspond to an average charge movement of 0.5 e₀ during each transition in the activation process. This value implies that activation gating involves a large number of transitions to account for the total gating charge displacement of 13 e₀. The characterization of the gating transitions here forms the basis for constraining a detailed gating model to be described in a subsequent paper of this series.     

Imaging single-channel calcium microdomains

The Ca²⁺ microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca²⁺ signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca²⁺ imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of ‘optical patch-clamp recording’ is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca²⁺ ions.     

Integral equation methods for computing likelihoods and their derivatives in the stochastic integrate-and-fire model

We recently introduced likelihood-based methods for fitting stochastic integrate-and-fire models to spike train data. The key component of this method involves the likelihood that the model will emit a spike at a given time t. Computing this likelihood is equivalent to computing a Markov first passage time density (the probability that the model voltage crosses threshold for the first time at time t). Here we detail an improved method for computing this likelihood, based on solving a certain integral equation. This integral equation method has several advantages over the techniques discussed in our previous work: in particular, the new method has fewer free parameters and is easily differentiable (for gradient computations). The new method is also easily adaptable for the case in which the model conductance, not just the input current, is time-varying. Finally, we describe how to incorporate large deviations approximations to very small likelihoods. Action Editor: Barry J. Richmond     

Rapid kinetic analysis of multichannel records by a simultaneous fit to all dwell-time histograms

A method is presented for rapidly extracting single-channel transition rate constants from patch-clamp recordings containing signals from several channels. The procedure is based on a simultaneous fit of the observed dwell-time distributions for all conductance levels, using a maximum likelihood approach. This algorithm allows estimation of single-channel rate constants in cases where more advanced methods may be impractical because of their extremely long computational time. A correction is included for the limited time resolution of the recording system, according to theory developed by Roux and Sauvé (Biophys. J. 48:149-158, 1985), by accounting for the impact of undetected transitions on the dwell-time distributions, and by introducing an improved practical implementation of a fixed dead time for the case of more than one channel. This feature allows application of the method to noisy data, after filtering. A computer program implementing the method is tested successfully on a variety of simulated multichannel current traces.     

Human Adipose Cells Have Voltage-dependent Potassium Currents

The whole-cell patch-clamp method was used to study the membrane electrical properties of human adipocyte cells obtained by differentiating from precursors of human abdominal and mammary tissues. All differentiated cells exhibited outward currents with sigmoidal activation kinetics. The outward currents showed activation thresholds between –20 to –30 mV and slow inactivation. The ionic channels underlying the macroscopic current were highly selective for K⁺. Their selectivity was for typical K⁺ channels with relative permeabilities of K⁺>NH ₄ ⁺ >Cs⁺>Na⁺. No evidence of any other type of voltage-gated channel was found. The potassium currents (I _KV) were blocked reversibly by tetraethylammonium and barium. The IC ₅₀ value and Hill coefficient of tetraethylammonium inhibition of I _KV were 0.56 mM and 1.17 respectively. These results demonstrate that human adipose cells have voltage-dependent potassium currents.