Amplification of neuromuscular transmission by postjunctional folds

Previously, suggestions have been made that postjunctional folds at the vertebrate motor end plate might, in some way, serve to enhance neuromuscular transmission. This suggestion was examined quantitatively using a model junction with geometry similar to that seen in mammalian 'fast twitch' muscles. It was found that the depolarization produced at the top of an interfold by a quantum of acetylcholine is significantly greater than that produced in the absence of folds because of the series resistance of the interfold myoplasm. As a result, voltage-sensitive sodium channels in the postsynaptic membrane are activated more readily. In the model, activation of as few as four interfolds by eight quanta is sufficient for excitation to spread to the remainder of the muscle. With no folds, 19 quanta are required.     

Connecting the protein structure universe by using sparse recurring fragments

The quest to order and classify protein structures has lead to various classification schemes, focusing mostly on hierarchical relationships between structural domains. At the coarsest classification level, such schemes typically identify hundreds of types of fundamental units called folds. As a result, we picture protein structure space as a collection of isolated fold islands. It is obvious, however, that many protein folds share structural and functional commonalities. Locating those commonalities is important for our understanding of protein structure, function, and evolution. Here, we present an alternative view of the protein fold space, based on an interfold similarity measure that is related to the frequency of fragments shared between folds. In this view, protein structures form a complicated, crossconnected network with very interesting topology. We show that interfold similarity based on sequence/structure fragments correlates well with similarities of functions between protein populations in different folds.     

Extracellular action potentials of skeletal muscle fibre affected by 4-aminopyridine: a model study

A new specially designed analytical function approximating the intracellular action potentials (ICAPs) for calculation of the extracellular potentials (ECAPs) at various radial and axial distances from the active fibre is proposed. 4-Aminopyridine (4-AP) was used to obtain ICAPs with a prolonged repolarization phase in order to investigate the influence of changes in ICAP shape on the ECAPs. From the experimentally recorded ICAPs before and after treatment of frog skeletal muscle fibres with 4-AP, approximated by the new function, the ECAPs were calculated applying the linesource model in a finite fibre. Using this function allowed calculation of the ECAPs at distances not accessible for the experimental recordings. The total ionic current (I _i) during the action potential was calculated using the cable equation. Our results showed that the ratio of the first positive to the negative phases of the ECAPs of treated fibres increased at large radial distances (3000 m and more) and the terminal positive phase was asymmetric with an abrupt initial deflection followed by a slow inverse deflection. The calculated ECAPs at various axial distances from the fibre end (cylindrical and conical part) and at radial distances from the fibre membrane ranging from 0 to 5000 m, corresponded in shape to the experimentally recorded potentials of untreated and 4-AP-treated muscle fibres.     

The effect of vanadate on the electrogenic Na+/K+ pump, intracellular Na+ concentration and electrophysiological characteristics of mouse skeletal muscle fibre

The present study reports a discrepancy between the effects of vanadate on the membrane Na+-K+-ATPase and the Na+/K+ pump of the skeletal muscle. Vanadate in concentration 4 X 10(-6) mol/l which is necessary to block the enzyme Na+-K+-ATPase activity of membrane fractions failed to inhibit the electrogenic Na+/K+ pump of intact muscle cells. The effect of vanadate on the electrophysiological parameters of the muscle fibre membrane required much higher vanadate levels, but again, Na+/K+ pump was still active. Vanadate in concentrations 4 X 10(-4) and 4 X 10(-5) mol/l depolarized the membrane potential and decreased the membrane resistance [apparently in consequence of enhanced passive membrane permeability for Na+ ions]. Action potentials and the electrical excitability of the muscle fibre membrane were reduced by these vanadate concentrations.     

Spatial organization of intestinal microbiota in the mouse ascending colon

Complex microbial populations are organized in relation to their environment. In the intestine, the inner lining (mucosa) is a potential focal point for such organization. The proximal murine colon contains mucosal folds that are known to be associated with morphologically distinct microbes. To identify these microbes, we used the technique of laser capture microdissection (LCM) to sample microbes associated with these folds (interfold region) and within the central lumen (digesta region). Using 16S rRNA gene tag pyrosequencing, we found that microbes in the interfold region were highly enriched for the phylum Firmicutes and, more specifically, for the families Lachnospiraceae and Ruminococcaceae. Other families such as Bacteroidaceae, Enterococcaceae and Lactobacillaceae were all enriched in the digesta region. This high-resolution system to capture and examine spatial organization of intestinal microbes should facilitate microbial analysis in other mouse models, furthering our understanding of host–microbial interactions.     

A new electrical method for the determination of the cell membrane area in plant cells

The membrane are of giant algal cells of Valonia utricularis was determined electrically by using the charge-pulse technique. The membrane was charged to low voltages between 2 and 20 mV by injecting charge pulses of defined amplitude and very short duration (about 100 ns). The injected charge was calculated by measuring the current increment via a potential drop across a 10 resistance in the outer circuit and by considering the preselected charging time. The initial voltage across the membrane was calculated by extrapolation to time zero (=end of the charge pulse). From the values of the injected charge and the voltage built up initially across the membrane, the capacitance of the membrane could be calculated. Assuming that the specific capacity of the two membranes, tonoplast and plasmalemma, arranged in series was 0.5 F cm^-2, the membrane area could be derived from the membrane capacity. The electrically determined membrane area agrees with the geometrically determined one to within 10%.     

Contributions to Physiology of the Antenna-Heart in Periplaneta americana (L.) (Blattodea: Blattidae)

An accessory pulsatile organ of an open circulatory system in insects supplying the antennae with haemolymph was investigated. The rhythm of this so-called antenna-heart is generated by a myogenic automatism and can be neuronally influenced via the nervus cardioantennalis.The action potentials of the muscle fibres show typical pre-depolarization and mostly no overshoot. A specific membrane resistance (R(m)) of about 660Omegacm(-2) was calculated for the fibres. Some electrical coupling between the muscle fibres is presumed for synchronization of any myogenically triggered heart beat which could actually be proved experimentally by current injection in the antenna-heart. However, intercalated disks or gap junctions could not be found. Nevertheless, a good coupling factor (U(2)/U(1)) between all fibres was demonstrated by parallel recordings and can be well described by a conductance model according to fibre topology.     

Sensory characteristics of the P afferent neurone of the crab thoracic-coxal muscle receptor organ

Intracellular recordings were made from the P fibre, the smallest of the three afferent neurones innervating the thoracic-coxal muscle receptor organ of the crab (Carcinus maenas). While the two larger afferents are nonspiking, the response of the P fibre to a trapezoidal change in receptor muscle length consists of a single action potential signalling the onset of stretch superimposed on a graded amplitude receptor potential. The P fibre is sensitive to the velocity of the applied stretch, but is insensitive to static joint position, stretch amplitude and the velocity of the release phase. The presence and amplitude of the action potential depends on the initial length of the receptor muscle, the tension caused by efferent activation of the receptor muscle prior to receptor stretch, and on the velocity of stretch. Length constant (1.9 mm) and specific membrane resistance (76 K · cm²) values obtained for the P fibre, together with its small diameter (7 m) suggest that this neurone is less well adapted to conveying passive signals to the thoracic ganglion than are the S and T fibres. It is likely that the P fibre complements the length sensitivity of the S fibre and the tension and velocity sensitivity of the T fibre by signalling the onset of receptor stretch via single action potentials.Abbreviations TCMRO thoracic-coxal muscle receptor organ - TTX tetrodotoxin     

Electrotonic spread of current in monolayer cultures of neonatal rat heart cells

Summary The passive electrical properties of neonatal rat heart cells grown in monolayer cultures were determined. Hyperpolarizing current pulses were injected through one microelectrode via an active bridge circuit. Membrane voltage displacements caused by the injected current pulses were measured at various distances from the first with a second microelectrode. Using a modified least-squares method the experimental results were fitted to a Bessel function, which is the steady-state solution of the differential equation describing the relation between membrane voltage caused by current injection and interelectrode distance in a very large and very thin plane cell. Best fit was obtained with a space constant of 360 m and an internal resistivity of 500 cm. From these figures, specific membrane resistance was calculated to be 1,300 cm², assuming all current to leave through the upper surface of the monolayer.The time constant of the membrane was measured from the time course of the current-induced membrane voltage displacements. From its value of 1.7 msec a membrane capacity of 1.3 F/cm² was calculated.From these results and some literature data on nexus distribution (A. W. Spira,J. Ultrastruct. Res. 34:409, 1971) specific nexus resistance was calculated to range between 0.25 and 1.25 cm², depending on the amount of folding of the intercalated discs. The results suggest that spread of activation in monolayer cultures of heart cells by means of local circuit currents is very likely.     

Measurement of the electrical resistance of plasmodesmata and membranes of corn suspension-culture cells

Electrophysiological investigations of intercellular communication and membrane resistance in higher plants have been hampered by the difficulty in measuring these quantities independently. Uncertainty about the position of an electrode inserted into vacuolate tissue has further complicated such measurement. To overcome these problems sister cell pairs of a Zea mays L. Black Mexican Sweet suspension culture were used and dye was injected from the current-injecting electrode to determine the location of the electrode tip in each experiment. Of the impalements, 72% were cytoplasmic. The presence of plasmodesmata was fully incorporated into the electriccircuit model for the cell, and the resistance of the membrane of the current-injected cell was calculated, separate from the plasmodesmata resistance. This avoided some of the confusion resulting from work on multicellular tissue in which the position of the electrode and the extent of intercellular coupling is not determined. Using this technique, plasma-membrane resistivity was measured as 0.65 ·m², the resistivity of the tonoplast and plasma membrane in series was 1.35 ·m², and the resistance of a single plasmodesma was calculated to be 53 ± 11 G.Abbreviations BMS Black Mexican Sweet - PD potential difference - R_j resistance of the plasmodesmata in the junction between cells - R_m resistance of the plasma membrane of the current-injected cell - R_t resistance of the tonoplast - V₁, V₂ membrane PDs of sister cells This work was funded by an Australian Research Council grant to R.L.O. We are grateful to Dr. Maret Vesk (Electron Microscope Unit, The University of Sydney) for assistance with the preparation of EM sections, and to Dr. Richard Brettell (C.S.I.R.O. Division of Plant Industry) for assistance with the BMS culture.     

A scanning and transmission electron microscopic study of the membranes of chicken egg.

Questions regarding the structure of the inner and outer shell membranes of the chicken egg were addressed in this study by correlating observations from light microscopy and scanning and transmission electron microscopy. The egg membrane had a limiting membrane, which measured .9 to .15 microns in thickness and appeared to be a continuous and an impervious layer, but the shell membrane did not. Under the SEM, each membrane was seen to be made up of several fibre layers. In the tear preparations viewed under the SEM two layers were observed in the egg membranes and three to five layers in the shell membrane, with an apparent plane of cleavage between each layer. Each fibre was made up of a central core and an outer mantle layers. The central core was perforated by channels which measured .08 to 1.11 microns in diameter and ran longitudinally along the length of the fibre. Between the mantle layer and the fibre core was a gap or cleft measuring between .03 to .07 microns. The diameter of the fibres of the inner layer of the egg membrane ranged between .08 to .64 microns, whereas those of the outer layer of the same membrane ranged from .05 to 1.11 microns. Fibres in the shell membrane ranged from .11 to 4.14 microns diameter.     

Electrotonic conduction of excitation and inhibition along the dendritic fiber with the uniformly distributed synaptic conductance

The existence of an optimum radius of fibre is shown, when the physical parameters of the intracellular medium and the nonexcitable dendritic membrane are known and the length and load resistance are fixed. This provides the maximum potential for the proximal end of the fibre if synaptic conductance is distributed uniformly along the fibre. A formula for calculating the soma potential of the whole neuron is proposed. The optimal ratio l/lambda is 0.9193 ... if the volume of the fibre and synaptic conductivity are fixed.     

Conduction along myelinated and demyelinated nerve fibres during the recovery cycle: Model investigations

The membrane excitability changes as well as the underlying mechanisms of these changes in a normal and in a systematically paranodally demyelinated nerve fibre have been investigated by paired stimulation during the first 30 ms of the recovery cycle. The ionic current kinetics determining the observed changes in the action potential parameters are presented also. The simulation of the conduction in the normal fibre is based on the Frankenhaeuser and Huxley (1964) and Goldman and Albus (1968) equations, while in the case of a demyelinated fibre according to the same equations modified by Stephanova (1988a). It has been shown for the demyelinated membrane that increased demyelination increases both the threshold current for the second potential as well as the absolute refractory period. With increasing interpulse interval, the subnormality of the membrane excitability is followed by supernormality in the case of the demyelinated membrane. For the recovery cycle of 30 ms under consideration no supernormality of the normal membrane excitability is obtained. With interpulse interval from 8.8 to 10.9 ms, the highest degree of demyelination (l=30 m) is accompanied by a refractory period of transmission. The membrane properties of the normal and demyelinated fibres recover 20 ms after the first pulse. For short interpulse intervals, the amplitude of the second action potential is decreased, and a slower propagation velocity is obtained. The most sensitive phenomenon is the excitability of the demyelinated membrane, which remains unrecovered 30 ms after the first pulses has been applied.     

Local development of action potentials in slow muscle fibres after complete or partial denervation.

Pyriformis muscles of Rana temporaria were completely or partially denervated by cutting the sciatic nerve or some of the small nerve branches entering the muscle. One stimulating and one to three recording microelectrodes were inserted along the fibres in order to compare the electrical activity at these points. In an early period following denervation action potentials of variable size and shape could be observed; these action potentials were often composed of two, sometimes of three or four, components. The size of individual components depended on the position of the recording microelectrode. Individual components could occasionally be triggered separately by adjusting the strength of the stimulating current pulse; propagation of these "all or none" responses was absent. In other fibres one component of the action potential could trigger another one several millimetres apart, thus indicating propagation. Conduction velocities were approximately 0.4 m/s. In partially denervated slow fibres, endplate potentials were confined to one lateral segment of the fibres, while the action potential occupied the denervated part of the membrane. The amplitudes of endplate and action potentials varied inversely with distance. Rough estimates of the length constant of the slow fibre membrane were calculated from the spatial decay of action potentials, endplate potentials and hyperpolarizing electrotonic potentials; mean values obtained were 2.5, 4.8 and 7.7 mm respectively. The results suggest that following denervation Na channels are built into discrete areas of the slow fibre membrane and that this process depends on the amount of denervation in individual fibres.     

Passive electrical constants of skeletal muscular fibres studied in different muscles of normal and thiamine deficient rats (author's transl)]

10 Some effects of thiamine deficiency were studied in three skeletal rat muscles, having different proportions of "fast" and "slow" fibres: extensor longus digiti IV (a nearly pure fast muscle), soleus (having a predominant population of slow fibres) and diaphragm muscle (mixed fibre population). 20 Cross section area of fibres (fig. 2) is reduced in thiamine deficient animals, mostly for fast fibres having a glycolytic metabolism, the histochemical profile of which tends to become similar to that of slow fibres, in which oxydative metabolism is predominant, as shown by a marked increase in succinodehydrogenase activity. 30 Measurements of resting potential E, of membranes time constant tau and of fibre input resistance R were performed in normal and thiamine deficient muscles (table I). R and tau were obtained from square pulse analysis, using a double shifted sampling method permitting the use of a single microelectrode. E is not greatly affected by thiamine deficiency. tau changes appear not to be significant, except for fast fibres from extensor longus muscle, where tau is slightly reduced. R is increased in thiamine deficient animals (fig. 3). 40 Changes in R and tau do not exactly follow the predictions of cable theory, if one assumes that a purely dimensional factor is involved. Thus, the view that thiamine deficiency does not change basic passive electrical constants of fibres (membrane specific resistance and capacity, myoplasm resistivity) can be considered only as a first approximation. 50 R and tau values obtained in normal muscles are larger than data taken from other studies. The reasons for this discrepancy are discussed. It is suggested that diet differences may play a role.     

Membrane Resistance of Human Red Cells

A method has been devised to measure the specific membrane resistance of single human red cells. The cells were sucked into a 3 to 5 micron diameter pore in the end of a glass tube. By passing a small current through the cells, the total cell resistance was measured. The dimensions of the cell were measured optically and the specific membrane resistance was then calculated. Leakage of current between the cell and the walls of the pore was minimized by filling this region with isotonic sucrose. The measured specific membrane resistance values of four human red cells were 6.3, 6.32, 10.0, and 19.7 ohm-cm².     

Effect of formaldehyde and glutaraldehyde on electrical properties of cardiac Purkinje fibers

The effects of formaldehyde, glutaraldehyde, 1-fluoro-2,4-dinitrobenzene, and 1,5-difluoro-2,4-dinitrobenzene on the electrophysiological properties of cardiac Purkinje fibers were studied. At concentrations of 2.5 mM the aldehydes produced a transient hyperpolarization, lengthening of the plateau of the action potential, and an increase in action potential overshoot and upstroke velocity. If exposure to aldehyde was continued, the fiber failed to repolarize after an action potential and the membrane potential stabilized at about -30 mv. If exposure was terminated before this, recovery was usually complete. At the time the fibers were hyperpolarized the input resistance was increased without much change in length constant, leading to an increase in both calculated membrane resistance and calculated core resistance. Although it was anticipated that an effect of the aldehydes on the membrane was to increase fixed negative charge, it was difficult to explain all the electrophysiological changes on this basis. The major effects of the fluorobenzene compounds were not the same; they produced a shortening of the action potential and a rapid loss of excitability.     

Mathematical analysis of the changes in the parameters of the action potentials,membrane and ionic currents of frog muscle fibre during the recovery cycle

The method of mathematical modelling was used to study the excitability changes of the membrane of a frog skeletal muscle fibre and the parameters of the action potentials, membrane and ionic currents during the first 30 ms of the recovery cycle.The threshold current for a fibre at rest was found to be 0.32 A and the durations of the absolute and relative refractory periods were respectively 4 ms and 5.2 ms. With increasing interpulse interval, the subnormality of the membrane excitability is followed by supernomality. Under the same condition the supernormality in the velocity recovery cycle is not obtained.In the recovery cycle, the shape (polarity, sequence and number of phases) of the action potentials, of the membrane and ionic currents and their conductances, are unchanged. Only the time and amplitude parameters of the quantities listed above are known to vary. With increasing the interpulse interval, the amplitudes of the quantities increase and their durations are shortened attaining the values of the corresponding quantities of the initial action potential.The membrane properties are recovered 30 ms after application of the initial pulse, but the supernormality of the excitability is still preserved.     

The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation

Summary Intracellular potentials were measured, using a piezoelectric electromechanical transducer to impale Ehrlich ascites tumor cells with capillary microelectrodes. In sodium Ringer's, the potential immediately after the penetration was –24±7 mV, and decayed to a stable value of about –8 mV within a few msec. The peak potentials disappeared in potassium Ringer's and reappeared immediately after resuspension in sodium. Ringer's, whereas the stable potentials were only slightly influenced by the change of medium. The peak potential is in good agreement with the Nernst potential for chloride. This is also the case when cell sodium and potassium have been changed by addition of ouabain. It is concluded that the peak potentials represent the membrane potential of the unperturbed cell, and that chloride is in electrochemical equilibrium across the cell membrane.The membrane potential of about –11 mV previously reported corresponds to the stable potential in this study, and is considered as a junction potential between damaged cells and their environment. Similar potential differences were recorded between a homogenate of cells and Ringer's.The apparent membrane resistance of Ehrlich cells was about 70 cm². This is two orders of magnitude less than the value calculated from³⁶Cl fluxes, and may, in part, represent a leak in the cell membrane.For comparison, the influence of an eventual leak on measurements in red cells and mitochondria is discussed.     

Effect of chlordimeform on nerve-muscle system of the larva of the waxmoth, Galleria mellonella

The action of chlordimeform on the nerve-muscle preparation of the larvae of the waxmoth Galleria mellonella has been studied by means of microelectrodes. Exitatory junction potential evoked by nerve stimulation is reversibly suppressed by 2 × 10^?3 M chlordimeform, and spike-like component is abolished. The resting membrane potential of the muscle fibre and the action potential from the nerve terminal are not affected at 5 × 10^?3 M chlordimeform. The depolarizing membrane response caused by outward current and the effective membrane resistance are not appreciably affected. It appears that chlordimeform exerts its blocking action on the neuromuscular junction rather than the conductance mechanism of muscle fibre membrane.