Effects of laser-induced hyperthermia treatment on ionic permeability of myelinated nerve

Summary The effect of laser-induced hyperthermia on the ionic permeability of nerve membranes was studied using the nodes of Ranvier in amphibian myelinated nerve as a model. To effect a photothermal modification of nerve membrane functions, con trolled laser irradiation consisting of a 5-sec thermal pulse was applied to the nodal membrane, increasing the temperature to a maximum of 48–58°C at the node. Major electrophysiological changes observed in the nodal membrane following laser-induced hyperthermia were a differential reduction of the sodium and potassium permeability, an increase in the leakage current, and a negative shift on the potential axis of the steady-state Na inactivation. There was no significant change in the kinetics of ion channel activation and inactivation for treatments below 56°C. The results suggest that a primary photothermal damage mecha nism at temperatures below 56°C could be a reduction in the number of active Na channels in the node, rather than a change in individual channel kinetics, or in the properties of the lipid bilayer of intervening nerve membrane. A differential heat sensi tivity between the noninactivated and the inactivated Na channels is also suggested. For the treatments of 56°C and above, a signifi cant increase of membrane leakage current suggests an irrevers ible thermal damage to the lipid bilayer. This work was supported by the ONR/SDIO N00014-86-K-0188 Medical Free-Electron-Laser Program and the Columbus-Cabrini Foundation.     

More on myelinated nerve model analysis

An error in a previous analysis of a simple model for myelinated nerve propagation is pointed out and rectified. The model previously chosen does not lend itself to analysis because it has a region ofinfinite negative conductivity which leads to an instability in steady propagation. A model which assumes afinite negative conductivity is examined in detail and a more general results is derived.     

Effects of some potassium channel blockers on the ionic currents in myelinated nerve

The effects of some potassium channel blockers on the ionic currents and on the so-called K(+)-depolarization in intact myelinated nerve fibres were studied. 4-AP, and in particular, Flaxedil, proved to be selective K(+)-current blockers. However, TEA, a crown ether (DCH18C6), a longchained triethylammonium compound (C10-TriEA), capsaicin, and the extract from the medicinal herb Ruta graveolens proved not to be selective K(+)-current blockers; they all block Na(+)-currents as well, although to a lesser extent. The sodium inactivation curve did not change under TEA and Flaxedil but was shifted on the potential axis in negative direction by DCH18C6, 4-AP, capsaicin and the Ruta extract whereas C10-TriEA caused a shift of both sodium inactivation and activation parameters in positive direction. Regarding to the kinetics of the persisting K(+)-current fraction, two different kinds of blockade were found: 1. Unchanged K(+)-kinetic which is typical for the effects of TEA, 4-AP, Flaxedil, and C10-TriEA. 2. Clearly changed K(+)-kinetic, characterized by K(+)-transients; which is typical for the effects of capsaicin and in particular, for those of DCH18C6 and of the Ruta extract. The possibly different modes of action of both groups of blockers are discussed in terms of current models for the action of potassium channel blockers.     

Effects of some chemical reagents on sodium current inactivation in myelinated nerve fibers of the frog.

The effect of several chemical reagents on the sodium current was studied in voltage-clamped single nerve fibers of the frog. The oxidants halazone and hypochlorous acid drastically inhibited inactivation. Their effect was similar to that of chloramine T (Wang, 1984a). The curve relating the steady-state inactivation parameter h infinity to the conditioning potential E became nonmonotonic after treatment with the oxidants, i.e., dh infinity/dE greater than 0 for E greater than -20 mV. By contrast, the oxidants periodate, iodate, and hydrogen peroxide (applied for the same time, but at higher concentrations) merely produced a parallel shift of the h infinity(E) curve to more negative values of membrane potential. Diethylpyrocarbonate, a reagent that preferentially modifies histidine groups, had one marked effect: a strong shift of the h infinity(E) curve to more negative values of membrane potential. Almost no effect was observed after application of the tyrosine-reactive reagent N-acetylimidazole. Similarly, the arginine-reactive reagent glyoxal had only minor effects on the Na permeability. The results suggest that methionine is not critically involved in the kinetics of Na current inactivation. Similarly, an essential tyrosine or arginine residue seems to be unavailable to chemical reagents from outside on the frog node of Ranvier. Deduced from the reactivities of (some of) the reagents used, modification of membrane lipids is a tentative explanation for the effects observed on inactivation kinetics.     

Extracellular currents and potentials of the active myelinated nerve fiber.

This paper is concerned with the accurate and rapid calculation of extracellular potentials and currents from an active myelinated nerve fiber in a volume conductor, under conditions of normal and abnormal conduction. The neuroelectric source for the problem is characterized mathematically by using a modified version of the distributed parameter model of L. Goldman and J. S. Albus (1968, Biophys. J., 8:596-607) for the myelinated nerve fiber. Solution of the partial differential equation associated with the model provides a waveform for the spatial distribution of the transmembrane potential V(z). This model-generated waveform is then used as input to a second model that is based on the principles of electromagnetic field theory, and allows one to calculate easily the spatial distribution for the potential everywhere in the surrounding volume conductor for the nerve fiber. In addition, the field theoretic model may be used to calculate the total longitudinal current in the extracellular medium (I0L(z)) and the transmembrane current per unit length (im(z)); both of these quantities are defined in connection with the well-known core conductor model and associated cable equations in electrophysiology. These potential and current quantities may also be calculated as functions of time and as such, are useful in interpreting measured I0L(t) and im(t) data waveforms. An analysis of the accuracy of conventionally used measurement techniques to determine I0L(t) and im(t) is performed, particularly with regard to the effect of electrode separation distance and size of the volume conductor on these measurements. Also, a simulation of paranodal demyelination at a single node of Ranvier is made and its effects on potential and current waveforms as well as on the conduction process are determined. In particular, our field theoretic model is used to predict the temporal waveshape of the field potentials from the active, non-uniformly conducting nerve fiber in a finite volume conductor.     

Effects of reagents modifying carboxyl groups on the gating current of the myelinated nerve fiber

Summary K⁺ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K⁺-selective channels ranging from 15 to 211 pS in symmetrical 140mm KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140mm KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H⁺ ions (pH=6.8); a 211 pS channel that was Ca²⁺-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917±128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4±8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140mm KCl solutions. Thus, there appears to be a correlation between the presence of functional K⁺ channels and insulin secretion.     

Effects of nitric oxide on protein-lipid interactions in the membranes of the myelinated nerve fiber

The influence of nitric oxide (NO) on the myelinated nerve fiber and the impact of modification of SHgroups of axon and myelin membrane proteins on the amplitude and propagation velocity of action potential (AP), amount of the membrane-bound calcium (Ca _mb ²⁺ , viscosity of the axon membrane, and saturation factor of phospholipid fatty acids (Sf) of myelin have been investigated. We established that the decrease in the number of extracellular SH-groups in membrane proteins induced by p-chloromercuribenzoate (pCMB, 10^?4 M), led to a decrease in the AP amplitude and a reversible desorption of Ca _mb ²⁺ but did not affect the axolemma viscosity and Sf. Nitric oxide (NO) caused a decrease in the AP amplitude and propagation velocity, an increase in the axolemma viscosity and a decrease in Sf of myelin; it also induced a reversible desorption of Ca _mb ²⁺ . Pretreatment of the nerve fiber with pCMB weakened the NO-induced desorption of Pretreatment of the nerve fiber with K⁺-channel blocker tetraethylammonium (10^?2 M) completely abolished the NO-induced change in the amount of Ca _mb ²⁺ . We suppose that NO-mediated changes in axolemma viscosity, Sf of myelin and desorption of Ca _mb ²⁺ affect protein-lipid interactions in axolemma and myelin, which in their turn influence the propagation of AP.     

Effects of deuterium oxide on the rate and dissociation constants for saxitoxin and tetrodotoxin action. Voltage-clamp studies on frog myelinated nerve

The actions of tetrodotoxin (TTX) and saxitoxin (STX) in normal water and in deuterium oxide (D2O) have been studied in frog myelinated nerve. Substitution of D2O for H2O in normal Ringer's solution has no effect on the potency of TTX in blocking action potentials but increases the potency of STX by approximately 50%. Under voltage clamp, the steady-state inhibition of sodium currents by 1 nM STX is doubled in D2O as a result of a halving of the rate of dissociation of STX from the sodium channel; the rate of block by STX is not measurably changed by D2O. Neither steady-state inhibition nor the on- or off-rate constants of TTX are changed by D2O substitution. The isotopic effects on STX binding are observed less than 10 min after the toxin has been added to D2O, thus eliminating the possibility that slow-exchange (t 1/2 greater than 10 h) hydrogen-binding sites on STX are involved. The results are consistent with a hypothesis that attributes receptor-toxin stabilization to isotopic changes of hydrogen bonding; this interpretation suggests that hydrogen bonds contribute more to the binding of STX than to that of TTX at the sodium channel.     

Ultraviolet-induced alterations of the sodium inactivation in myelinated nerve fibres.

Ultraviolet radiation irreversibly reduces the sodium permeability in nerve membranes and, in addition, induces a change of the potential dependence of the kinetic parameters of sodium inactivation in the node of Ranvier. This second ultraviolet effect shifts the kinetic parameters of sodium inactivation h infinity (V), alpha h (V), and beta h (V) to more negative potentials (no changes of the slopes of the curves). The amount of the displacement delta V along the potential axis is equal for the three parameters and depends on the ultraviolet dose. It is about delta V = --10 mV after an irradiation dose of 0.7 Ws/cm2 at 280 nm. Both ultraviolet-induced effects depend on membrane potential and on the wavelength of the applied radiation. But while the potential shift is enhanced at more negative holding potentials, the ultraviolet blocking is diminished and vice versa. Further, the ultraviolet-induced potential shift is greater at 260 nm than at 280 nm, whereas a maximum sensitivity of ultraviolet blocking is found at 280 nm. Therefore, the two radiation effects are the result of two separate photoreactions. For explanation of the radiation-induced potential shift it is assumed that ultraviolet radiation decreases the density of negative charges at the inner surface of the nodal membrane. From this hypothesis a value for the inner surface potential psii was derived. --19 mV less than or equal to psii less than or equal to --14 mV.     

Conditioning hyperpolarization-induced delays in the potassium channels of myelinated nerve.

Hyperpolarizing conditioning pulses delay the onset of potassium channel current in voltage-clamped myelinated nerve fibers. Both the development of and recovery from this conditioning are approximately exponential functions of time: the time constants are functions of the conditioning voltage. The delay is larger and develops faster for more hyperpolarized conditioning pulses. The magnitude of the delay (but not the rate of development or recovery) depends upon the test potential-small test depolarizations produce larger delays than large depolarizations. The currents with and without the conditioning pulse cannot be made to superimpose by a simple time translation.     

Slow actions of hyperpolarization on sodium channels in the membrane of myelinated nerve.

The mean sodium current, I, and the variance of sodium current fluctuations, var, were measured in myelinated nerve during a depolarization to V = 40 mV applied from the resting potential (VH = 0) or from a hyperpolarizing holding potential VH = -28 mV. From I and var the relative variations in the number N and the conductance gamma of sodium channels following changes of the holding potential were calculated. Hyperpolarizing the membrane from VH = 0 to -28 mV increased N by a factor of 3.7, whereas gamma decreased by a factor of 0.53. These actions of holding potential on sodium channels develop slowly since 500 ms prepulses to 0 or -28 mV do not alter the values of N and gamma.     

A distributed-parameter model of the myelinated nerve fiber

This paper presents a new model for the characterization of electrical activity in the nodal, paranodal and internodal regions of isolated amphibian and mammalian myelinated nerve fibers. It differs from previous models in the following ways: (1) in its ability to incorporate detailed anatomical and electrophysiological data; (2) in its approach to the myelinated nerve fiber as a multi-axial cable; and (3) in the numerical algorithm used to obtain distributed model equation solutions for potential and current. The morphometric properties are taken from detailed electron microscopic anatomical studies (Berthold & Rydmark, 1983a, Experientia 39, 964-976). The internodal axolemma is characterized as an excitable membrane and model-generated nodal and internodal membrane action potentials are presented. A system of describing equations for the equivalent network model is derived, based on the application of Kirchoff's Current Law, which take the form of multiple cross-coupled parabolic partial differential equations. An implicit numerical integration method is developed and the numerical solution implemented on a parallel processor. Non-uniform spatial step sizes are used, enabling detailed representation of the nodal region while minimizing the number of total segments necessary to represent the overall fiber. Conduction velocities of 20.2 m sec-1 at 20 degrees C for a 15 microns diameter amphibian fiber and 57.6 m sec-1 at 37 degrees C for a 17.5 microns diameter mammalian fiber are achieved, which agrees qualitatively with published experimental data at similar temperatures (Huxley & St?mpfli, 1949, J. Physiol., Lond. 108, 315-339; Rasminsky, 1973, Arch, Neurol. 28, 287-292). The simulation results demonstrate the ability of this model to produce detailed representations of the transaxonal, transmyelin and transfiber potentials and currents, as well as the longitudinal extra-axonal, periaxonal and intra-axonal currents. Also indicated is the potential contribution of the paranodal axolemma to nodal activity as well as the presence of significant longitudinal currents in the periaxonal space adjacent to the node of Ranvier.     

Properties of internally perfused, voltage-clamped, isolated nerve cell bodies

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.