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P Shrager

Publications and source records attributed to P Shrager.

At least 37 records · Page 2Linked to original sources
PubMed1995

Clustering of Na+ channels and node of Ranvier formation in remyelinating axons.

Polyclonal antibodies were raised against a well conserved region of the vertebrate Na+ channel and were affinity purified for use in immunocytochemistry. Focal demyelination of rat sciatic axons was initiated by an intraneural injection of lysolecithin and Na+ channel clustering was followed at several stages of myelin removal and repair. At 1 week post-injection axons contained long, fully demyelinated regions. Na+ channel clusters appeared only at heminodes forming the borders of these zones, and at widely spaced isolated sites that may represent former nodes of Ranvier. Over the next few days proliferating Schwann cells adhered to axons and began to extend processes. Clusters of Na+ channels appeared at the edges of these structures. As the Schwann cells elongated, the clusters seemed to move with them, since they remained at edges and the distance between aggregates increased. Clusters associated with different Schwann cells ultimately approached each other and appeared to fuse. Na+ channels then coalesced further at these sites, forming new nodes of Ranvier in regions that previously were internodal. If Schwann cell proliferation were blocked by mitomycin, no new clusters of Na+ channels appeared within internodes. Under these conditions, heminodal clusters remained visible at 1 week postinjection, but by 2 weeks they were no longer detectable, suggesting that proliferating Schwann cells are required for their maintenance. Clusters at normal nodes of Ranvier remained. It is concluded that Na+ channel aggregation and mobility in demyelinated nerve fibers is controlled by adhering Schwann cells, resulting in the formation of stable new nodes of Ranvier during remyelination.

Animals↗
PubMed1994

Resolving three types of chloride channels in demyelinated Xenopus axons.

Axons from Xenopus sciatic nerve were demyelinated by intraneural injection of lysolecithin rendering the entire internodal axolema accessible to a patch electrode. In this region, three types of anion selective pores were found and characterized at the single-channel level. These included outwardly rectifying, inwardly rectifying, and maxi Cl- channels. The outwardly rectifying Cl- channels (24 pS) are activated by depolarization with a weak voltage dependence of 42 mV per e-fold change in open probability. The inwardly rectifying Cl- channels (27 pS) are insensitive to voltage, but can be blocked by internal application of 100 microM SITS or DIDS. The I-V curves of rectifying channels are S-shaped and can be fitted by a kinetic model with a single free energy barrier. The rectification may be related to the location of this barrier. The maxi Cl- channel (335 pS) is often open at the resting potential, but is inactivated by a large depolarization. The rectification, voltage dependence, and inactivation of these channels may contribute to the regulation of axonal Cl- balance and resting potential.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Axonal coding of action potentials in demyelinated nerve fibers.

Conduction in individual axons of Xenopus has been measured optically in response to short trains of stimuli, following demyelination of the sciatic nerve. In many cases the initial action potential in a burst is absent. Failure may also occur later in the train, resulting in a profound alteration of signal coding by the axon. Integration leading to delayed transmission occurred at the heminode forming the proximal border of the demyelinated zone, as well as at new nodes of Ranvier forming in remyelinating axons. This process appeared to involve a depolarizing afterpotential and seemed to be analogous to the threshold changes involved in superexcitability. Axonal coding was very sensitive to small changes in the stimulus pattern. Neither 1 mM tetraethylammonium ion, which blocks nodal and Ca2+ activated K+ channels, nor 1 mM 4-aminopyridine, which blocks fast internodal K+ channels, prevented loss of the initial spike in a burst. Similarly, neither block of Ca2+ channels by Cd2+ nor lowering of Cl- had a notable effect. Ouabain, on the other hand, had small but possibly significant effects on responses to repetitive stimuli. A computational model was used to test mechanisms involving passive cable properties. Lowering the myelin resistance and the nodal leakage conductance, in accord with recent evidence from intracellular recordings, reproduced many of the results and was accurate with respect to stimulus frequency, temperature and sensitivity to average potential. The coding of action potentials observed here may have clinical consequences in demyelinating diseases such as multiple sclerosis.

Action Potentials↗
PubMed1993

Single channel characterization of multiple types of potassium channels in demyelinated Xenopus axons.

Single channel currents in internodes of demyelinated Xenopus axons were measured with the gigaseal patch-clamp technique. Demyelination induced by injected lysolecithin allows the entire internodal axolemma to be accessible to a patch electrode. Four classes of K+ channels in this region were characterized. A Ca(2+)-activated K+ channel [K(Ca)] with a single channel conductance of 235 pS was found. In the presence of 10(-3), 10(-4), 10(-5), and 10(-6) M intracellular free [Ca2+]i, the half-activation voltages are -24.1, -20.8, 30.2, and 111 mV, and the voltage sensitivities are 18.3, 17.2, 23.7, and 21 mV per e-fold change in open probability, respectively. The half-activation Ca2+ concentration at 40 mV is 10(-5) M and the Hill coefficient of Ca2+ binding is 1.7. The K(Ca) channels were sometimes found in clusters, three to six channels in a patch. A 125 pS ATP-sensitive K+ channel was inhibited by the internal application of 2 mM ATP. Its activation was voltage independent. This channel may be important in the regulation of resting potential. A background K+ channel exhibited outwardly rectifying unitary current (176 pS) in symmetrical 115 mM KCl solutions but the ensemble-averaged I-V curve was ohmic. The voltage dependence is very weak, 220 mV per e-fold change in open probability. The nearly symmetrical macroscopic I-V curve of the background channel suggests a role in maintaining the axonal resting potential. A 28 pS delayed-rectifier K+ channel is found to be blocked internally by 2 mM 4-aminopyridine and by 10 mM tetraethylammonium. The half-activation voltage is -41 mV and the voltage sensitivity is 8 mV per e-fold change in open probability.

4-Aminopyridine↗
PubMed1991

Channel reconstitution in liposomes and planar bilayers with HPLC-purified MIP26 of bovine lens.

The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than +/- 20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60-80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (approximately 120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 to 7 sec. Cs+ did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs+ occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na+ solutions (zero K+). These data indicate that MIP26 channels are not K(+)-selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels.

Animals↗
PubMed1990

Remyelination of nerve fibers in the transected frog sciatic nerve.

This project tests an important aspect of the cellular events controlling the processes of recovery of function and remyelination that follow demyelination in the peripheral nervous system. Frog sciatic nerves have been shown to survive and remain functional for up to 10 days following transection. We have utilized this property in order to dissociate the recovery process from possible control by the neuronal soma. Xenopus sciatic nerves were demyelinated in one branch by an intraneural injection of lysolecithin. The nerve was cut proximally to the injection site either immediately before, or several days after the lysolecithin injection. Recovery of function and remyelination were then followed by electrophysiological, optical, and ultrastructural techniques applied both to whole branches and single fibers. Controls included the cut but uninjected branch, and injected but uncut nerves. The progression of events during both demyelination and recovery in cut axons was indistinguishable from that in uncut fibers. This suggests that this process may be under local control and can be initiated and carried out in the absence of constant communication with the nerve cell body.

Action Potentials↗
PubMed1990

Optical measurement of conduction in single demyelinated axons.

Demyelination was initiated in Xenopus sciatic nerves by an intraneural injection of lysolecithin over a 2-3-mm region. During the next week macrophages and Schwann cells removed all remaining damaged myelin by phagocytosis. Proliferating Schwann cells then began to remyelinate the axons, with the first few lamellae appearing 13 d after surgery. Action potentials were recorded optically through the use of a potential-sensitive dye. Signals could be detected both at normal nodes of Ranvier and within demyelinated segments. Before remyelination, conduction through the lesion occurred in only a small fraction of the fibers. However, in these particular cases we could demonstrate continuous (nonsaltatory) conduction at very low velocities over long (greater than one internode) lengths of demyelinated axons. We have previously found through loose patch clamp experiments that the internodal axolemma contains voltage-dependent Na+ channels at a density approximately 4% of that at the nodes. These channels alone, however, are insufficient for successful conduction past the transition point between myelinated and demyelinated regions. Small improvements in the passive cable properties of the axon, adequate for propagation at this site, can be realized through the close apposition of macrophages and Schwann cells. As the initial lamellae of myelin appear, the probability of success at the transition zone increases rapidly, though the conduction velocity through the demyelinated segment is not appreciably changed. A detailed computational model is used to test the relative roles of the internodal Na+ channels and the new extracellular layer. The results suggest a possible mechanism that may contribute to the spontaneous recovery of function often seen in demyelinating disease.

Action Potentials↗
PubMed1989

Sodium channels in single demyelinated mammalian axons.

The distribution of ionic channels is thought to play an important role in the recovery of function following demyelination. Rat sciatic nerves were demyelinated by lysolecithin and single fibers were examined with patch clamp techniques. Voltage-dependent sodium currents were measured in both internodal and nodal regions. The results suggested that while there is a sharp gradient in channel density at the node of Ranvier, the total number of internodal channels far exceeds the total number of nodal channels. The nodal channels do not diffuse freely following demyelination and the internodal channels may thus serve important functions as conduction is restored.

Animals↗
PubMed1988

Ionic channels and signal conduction in single remyelinating frog nerve fibres.

1. Ionic currents have been measured in single demyelinated and remyelinating frog sciatic nerve fibres by means of the loose patch clamp technique. Axons were demyelinated by a surgical intraneural injection of lysolecithin and recovery was followed for up to 5 months. 2. Removal of myelin debris continued for the first 2 weeks post-injection. Proliferating Schwann cells were then seen within the lesion. As remyelination proceeded new nodes of Ranvier were formed in regions that previously were internodal. Original nodes, marking the transition from old to new myelin, could be identified at all stages. 3. Peak amplitudes of internodal transient inward Na+ currents were constant over the first 2 months and increased by about 60% after 5 months. Internodal currents in remyelinated axons were recorded after a second injection of lysolecithin to remove the thin myelin sheath. 4. Records from paranodal sites neighbouring transition nodes contained transient outward currents that were strongly voltage dependent and seemed to reflect activation of a very high density of Na+ channels just outside the patch. This sharp gradient in channel density at original nodes persisted throughout the period of remyelination studied suggesting that lateral diffusion from these sites is limited. These currents were never seen at internodal sites nor were they found at new nodes of Ranvier. 5. Paranodal inward current amplitudes in new nodes were similar to those in original (transition) nodes. 6. No transient inward Na+ currents were detected in Schwann cells adhering to demyelinated axons or free standing within the area of the lesion. 7. Conduction in single remyelinating fibres was studied by measuring membrane currents that flowed in response to an invading propagating action potential. At 2 weeks post-injection, prior to the formation of myelin, conduction was decremental, but activation of internodal Na+ channels allowed signals to penetrate further into the demyelinated zone than would have been possible by passive spread alone. After an additional 3 weeks, following formation of thin myelin sheaths, conduction was significantly improved and the fractional activation of Na+ channels was increased. 8. The results suggest that Na+ channels at new nodes of Ranvier come neither from original nodes nor from Schwann cells. They may represent a moderate aggregation of existing internodal channels. New nodes seem to possess a gradient of Na+ channel density that is much less steep than that at original nodes. Continuous conduction appears to be limited to short (approximately 0.2 mm) lengths of demyelinated axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1987

Optical recording of action potential propagation in demyelinated frog nerve.

Conduction in focally demyelinated frog nerves has been measured optically using potential-sensitive dyes. Absorption changes were recorded with an array of photodiodes positioned in the image plane of a microscope. Both the amplitude and conduction velocity of the optical signals decreased in the demyelinated region. Conduction was improved after exposure to the potassium channel blocking agent 4-aminopyridine.

Action Potentials↗
PubMed1987

The distribution of sodium and potassium channels in single demyelinated axons of the frog.

1. Frog sciatic nerves were focally demyelinated by intraneural injection of lysolecithin. Fibres were examined 1-8 days later. At early times axons contained zones just a few micrometers long that were covered by myelin debris. These zones lengthened and the debris was cleared over the next several days. 2. The loose patch clamp technique was used to measure ionic currents in the demyelinated axolemma in both internodal and paranodal regions. 3. Transient inward currents that have been identified as Na+ currents on the basis of voltage dependence and sensitivity to tetrodotoxin were recorded at all stages tested. There was no significant increase in internodal peak Na+ current with time over the first 8 days after injection. 4. Peak inward currents varied relatively little with distance along the internode at 1 day post-injection. However, sharp gradients were detected at demyelinated paranodal and nodal membranes. 5. By combining the patch clamp with external stimulation proximal to the patch site it has been possible to record membrane current responses to propagating signals invading the demyelinated zone. With demyelination only in the distal heminode the extent of Na+ channel activation is similar to that predicted for normal nodes of Ranvier by Frankenhaeuser & Huxley (1964). 6. The ratio of K+ conductance to Na+ conductance is much higher in demyelinated internodes than in normal nodes of Ranvier. 7. It is concluded that Na+ channels are likely to be present in normal internodes. The total number of these internodal channels may be about 40 times the total number at nodes. Steep gradients of Na+ channel density remain at nodal regions at least up to 6 days following demyelination.

Action Potentials↗
PubMed1985

Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells.

Cultured Schwann cells from sciatic nerves of newborn rabbits and rats have been examined with patch-clamp techniques. In rabbit cells, single sodium and potassium channels have been detected with single channel conductances of 20 pS and 19 pS, respectively. Single sodium channels have a reversal potential within 15 mV of ENa, are blocked by tetrodotoxin, and have rapid and voltage-independent inactivation kinetics. Single potassium channels show current reversal close to EK and are blocked by 4-aminopyridine. From these results, and from comparisons of single-channel and whole-cell data, we show that these Schwann cells contain voltage-dependent sodium and potassium channels that are similar in most respects to the corresponding channels in mammalian axonal membranes. Cultured rat Schwann cells also have sodium channels, but at a density about 1/10th that of rabbit cells, a result in agreement with saxitoxin binding experiments on axon-free sectioned nerves. Saxitoxin binding to cultured cells suggests that there are up to 25,000 sodium channels in a single rabbit Schwann cell. We speculate that in vivo Schwann cells in myelinated axons might act as a local source for sodium channels at the nodal axolemma.

Animals↗
PubMed1983

The periaxonal space of crayfish giant axons.

The influence of the glial cell layer on effective external ion concentrations has been studied in crayfish giant axons. Excess K ions accumulate in the periaxonal space during outward K+ current flow, but at a rate far below that expected from the total ionic flux and the measured thickness of the space. At the conclusion of outward current flow, the external K+ concentration returns to normal in an exponential fashion, with a time constant of approximately 2 ms. This process is about 25 times faster than is the case in squid axons. K+ repolarization (tail) currents are generally biphasic at potentials below about -40 mV and pass through a maximum before approaching a final asymptotic level. The initial rapid phase may in part reflect depletion of excess K+. After block of inactivation and reversal of the Na+ concentration gradient, we could demonstrate accumulation and washout of excess Na ions in the periaxonal space. Characteristics of these processes appeared similar to those of K+. Crayfish glial cell ultrastructure has been examined both in thin sections and after freeze fracture. Layers of connective tissue and extracellular fluid alternate with thin layers of glial cytoplasm. A membranous tubular lattice, spanning the innermost glial layers, may provide a pathway allowing rapid diffusion of excess ions from the axon surface.

Action Potentials↗
PubMed1981

Block and inactivation of sodium channels in nerve by amino acid derivatives. I. Dependence on voltage and sodium concentration.

The side chain of arginine, n-propylguanidinium (nPG), reversibly decreases peak sodium conductance and increases the speed of sodium current decay, when perfused internally. Effects are voltage dependent and are more pronounced at high depolarizations. Results are also dependent on the sodium concentration gradient. Both the decline in peak conductance and the speeding of inactivation are greater if the sodium concentration gradient is reversed from the normal. The decrease in peak sodium current is too large to be due solely to the faster decay kinetics. The difference is not due to a change in slow inactivation of the channel. Sodium current inactivation has also been studied with a double pulse procedure. Results show that at - 70 mV, nPG leaves sodium channels rapidly (less than 500 microseconds) in normal sodium gradient, but more slowly (greater than 1 ms) in reversed sodium gradient. Several structural analogs of nPG have been tested. Shortening the alkyl chain weakens effects significantly. Arginine itself, which contains extra charged groups, is also less effective. n-Propylammonium is active but with an apparent affinity only one-fifth that of arginine. We conclude that nPG acts within the sodium channel, and has at least two modes of action.

Animals↗
PubMed1981

Block and inactivation of sodium channels in nerve by amino acid derivatives. II. Dependence on temperature and drug concentration.

The interaction of n-propylguanidinium (nPG) with sodium channels has been further characterized. From experiments at varying temperatures, the Q10 for the sodium current decay time constant in the two [Na+] gradients is 2.6-2.9 independent of drug. Testing several nPG concentrations we find that peak sodium current declines sharply with [nPG] at all levels, but the decay time constant approaches an asymptote above 4 mM. No "hooks" in sodium tail currents are seen. If the sodium current is allowed to decay completely before repolarization no tail current is observed. We have developed a kinetic model in which nPG acts at a single site within the sodium channel. Reaction of nPG with its receptor requires two steps. Fitting the temperature data shows that the first step involves diffusion of the drug to the site and close association with it. The second step may include molecular reorganization of the complex. The rate constants for the reaction are all simple exponential functions of voltage. Using them, the model successfully predicts decay time constants and peak currents, and their dependence on potential, [Na+] gradient, temperature, and nPG concentration. The results are consistent with the idea that an arginine residue may be closely associated with inactivation.

Animals↗
PubMed1981

Modification of sodium and potassium channel gating kinetics by ether and halothane.

The effect of ether and halothane on the kinetics of sodium and potassium currents were investigated in the crayfish giant axon. Both general anesthetics produced a reversible, dose-dependent speeding up of sodium current inactivation at all membrane potentials, with no change in the phase of the currents. Double-pulse inactivation experiments with ether also showed faster inactivation, but the rate of recovery from inactivation at negative potentials was not affected. Ether shifted the midpoint of the steady-state fast inactivation curve in the hyperpolarizing direction and made the curve steeper. The activation of potassium currents was faster with ether present, with no change in the voltage dependence of steady-state potassium currents. Ether and halothane are known to perturb the structure of lipid bilayer membranes; the alterations in sodium and potassium channel gating kinetics are consistent with the hypothesis that the rates of the gating processes of the channels can be affected by the state of the lipids surrounding the channels, but a direct effect of ether and halothane on the protein part of the channels cannot be ruled out. Ether did not affect the capacitance of the axon membrane.

Action Potentials↗