Action potentials of isolated single muscle fibers recorded by potential-sensitive dyes

Light transmission changes upon massive stimulation of single muscle fibers of Xenopus were studied with the potential-sensitive nonpermeant dyes, merocyanine rhodanine (WW375) and merocyanine oxazolone (NK2367). Upon stimulation an absorption change (wave a) occurred, which probably represents the sum of action potentials in the transverse tubules and surface membrane. In WW375-stained fibers wave a is a decrease in transmission over the range of 630 to 730 nm (with NK2367, over the range of 590 to 700 nm) but becomes an increase outside this range, thus showing a triphasic spectral pattern. This spectrum differs from that of the squid axon, in which depolarization produces only an increase in transmission over the whole range of wavelengths (Ross et al. 1977. J. Membr. Biol. 33:141-183). When wave a was measured at the edge of the fiber to obtain more signal from the surface membrane, the spectrum did not seem to differ markedly from that obtained from the entire width of the fiber. Thus, the difference in the spectrum between the squid axon and the vertebrate muscle cannot be attributed to the presence of the tubular system.     

Mechanism of response of potential-sensitive dyes studied by time-resolved fluorescence

The mechanism of response of two potential-sensitive dyes, diOC₂(5) (3,3′-diethyloxadicarbocyanine iodide) and oxonol V (bis-[3-phenyl-5-oxoisoxazol-4-yl]pentamethine oxonol), were studied by using steady-state and time-resolved fluorescence techniques. The lipid concentration dependence of the Δψ (membrane potential)-induced change in total fluorescence intensity was quite different for these two dyes. Time-resolved fluorescence measurements showed that the fluorescence decay of these dyes in membranes could be resolved into at least three exponentials. Δψ-induced changes in the levels of these three populations were also measured under a variety of conditions. In the case of diOC₂(5) an inside negative Δψ increased the levels of the bound forms. This shows that diOC₂(5) responds to Δψ mainly by an “on-off” mechanism whereby Δψ perturbs the membrane-water partition coefficient of the dye. The Δψ-induced changes approached zero when the dye was totally membrane bound. In contrast, the Δψ-induced response of oxonol V increased with increased membrane binding. An inside negative Δψ decreased the level of the bound form with a longer lifetime. This shows that the mechanism of response of oxonol V is a Δψ-induced shift in the equilibrium between bound forms of the dye.     

Energetic aspects of propagation of action potential

On the basis of the electrical analogue of an action potential suggested previously, the distribution of electrical field outside and inside the membrane was obtained. The energy released upon the propagation of a single action potential along the nerve fibre was calculated. A comparison of the results with the available data showed the correctness of the calculations performed.     

Presynaptic nicotinic cholinoreceptors modulate velocity of the action potential propagation along the motor nerve endings at a high-frequency synaptic activity

Experiments on frog neuromuscular junctions have demonstrated that asynchrony of the acetylcholine quantal release forming the multi-quantal evoked response at high-frequency synaptic activity is caused, in particular, by a decrease in velocity of the action potential propagation along the non-myelinated nerve endings, which is mediated by activation of the α7 and α4β4 nicotinic cholinoreceptors.     

On the theory of action potential propagation in plant cells

The distribution of an electric field in plant cells and zooblasts has been investigated during propagation of the action potential. The behavior of ions in the cytoplasm and in the extracellular fluid has been described with the equations of electric charge motion in electrolytes. It has been shown that the action potential causes an electric potential change not only in the depth of the cytoplasm but also in the extracellular area far from the lipid bilayer. The biomembrane resistance has been expressed by physical parameters of a cell, such as ionic diffusion coefficient in fluid, Debye-Hückel radius, dielectric constant etc. The presence of breaks in the action potential diagrams has been explained as a result of insufficient resolving power of the measuring devices at the instant the sodium ion channels of the bilayer open.     

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.     

Simulation of action potential propagation in complex terminal arborizations.

Action potential propagation in complex terminal arborizations was simulated using SPICE, a general purpose circuit simulation program. The Hodgkin-Huxley equations were used to simulate excitable membrane compartments. Conduction failure was common at branch points and regularly spaced boutons en passant. More complex arborizations had proportionally more inactive synapses than less complex arborizations. At lower temperature the safety factor for impulse propagation increased, reducing the number of silent synapses in a particular arborization. Small structural differences as well as minute changes in the discharge frequency of the action potential resulted in very different activation patterns of the arborization and terminal boutons. The results suggest that the structural diversity of terminal arborizations allows a wide range of presynaptic information processing. The results from this simulation study are discussed in the context of experimental results on the modulation of synaptic transmission.     

Depth-resolved measurement of transient structural changes during action potential propagation

We report noncontact optical measurement of fast transient structural changes in the crustacean nerve during action potential propagation without the need for exogenous chemicals or reflection coatings. The technique, spectral domain optical coherence tomography, provides real-time cross-sectional images of the nerve with micron-scale resolution to select a specific region for functional assessment and interferometric phase sensitivity for subnanometer-scale motion detection. Noncontact optical measurements demonstrate nanometer-scale transient movement on a 1-ms timescale associated with action potential propagation in crayfish and lobster nerves.     

Repolarization of the action potential enabled by Na<Superscript>+</Superscript> channel deactivation in PSpice simulation of cardiac muscle propagation

Background  

In previous studies on propagation of simulated action potentials (APs) in cardiac muscle using PSpice modeling, we reported that a second black-box (BB) could not be inserted into the K⁺ leg of the basic membrane unit because that caused the PSpice program to become very unstable. Therefore, only the rising phase of the APs could be simulated. This restriction was acceptable since only the mechanism of transmission of excitation from one cell to the next was being investigated.     

Stochastic simulations on the reliability of action potential propagation in thin axons

It is generally assumed that axons use action potentials (APs) to transmit information fast and reliably to synapses. Yet, the reliability of transmission along fibers below 0.5 μm diameter, such as cortical and cerebellar axons, is unknown. Using detailed models of rodent cortical and squid axons and stochastic simulations, we show how conduction along such thin axons is affected by the probabilistic nature of voltage-gated ion channels (channel noise). We identify four distinct effects that corrupt propagating spike trains in thin axons: spikes were added, deleted, jittered, or split into groups depending upon the temporal pattern of spikes. Additional APs may appear spontaneously; however, APs in general seldom fail (<1%). Spike timing is jittered on the order of milliseconds over distances of millimeters, as conduction velocity fluctuates in two ways. First, variability in the number of Na channels opening in the early rising phase of the AP cause propagation speed to fluctuate gradually. Second, a novel mode of AP propagation (stochastic microsaltatory conduction), where the AP leaps ahead toward spontaneously formed clusters of open Na channels, produces random discrete jumps in spike time reliability. The combined effect of these two mechanisms depends on the pattern of spikes. Our results show that axonal variability is a general problem and should be taken into account when considering both neural coding and the reliability of synaptic transmission in densely connected cortical networks, where small synapses are typically innervated by thin axons. In contrast we find that thicker axons above 0.5 μm diameter are reliable.     

Influence of anisotropic conduction properties in the propagation of the cardiac action potential

Anisotropy, the property of being directionally dependent, is ubiquitous in nature. Propagation of the electrical impulse in cardiac tissue is anisotropic, a property that is determined by molecular, cellular, and histological determinants. The properties and spatial arrangement of connexin molecules, the cell size and geometry, and the fiber orientation and arrangement are examples of structural determinants of anisotropy. Anisotropy is not a static property but is subject to dynamic functional regulation, mediated by modulation of gap junctional conductance. Tissue repolarization is also anisotropic. The relevance of anisotropy extends beyond normal propagation and has important implications in pathological states, as a potential substrate for abnormal rhythms and reentry.     

Spatiotemporal properties of the action potential propagation in the mouse visual cortical slice analyzed by calcium imaging

The calcium ion (Ca(2+)) is an important messenger for signal transduction, and the intracellular Ca(2+) concentration ([Ca(2+)](i)) changes in response to an excitation of the cell. To reveal the spatiotemporal properties of the propagation of an excitatory signal with action potentials in the primary visual cortical circuit, we conducted a Ca(2+) imaging study on slices of the mouse visual cortex. Electrical stimulation of layer 4 evoked [Ca(2+)](i) transients around the stimulus electrode. Subsequently, the high [Ca(2+)](i) region mainly propagated perpendicular to the cortical layer (vertical propagation), with horizontal propagation being restricted. When the excitatory synaptic transmission was blocked, only weak and concentric [Ca(2+)](i) transients were observed. When the action potential was blocked, the [Ca(2+)](i) transients disappeared almost completely. These results suggested that the action potential contributed to the induction of the [Ca(2+)](i) transients, and that excitatory synaptic connections were involved in the propagation of the high [Ca(2+)](i) region in the primary visual cortical circuit. To elucidate the involvement of inhibitory synaptic connections in signal propagation in the primary visual cortex, the GABA(A) receptor inhibitor bicuculline was applied. In this case, the evoked signal propagated from layer 4 to the entire field of view, and the prolonged [Ca(2+)](i) transients were observed compared with the control condition. Our results suggest that excitatory neurons are widely connected to each other over the entire primary visual cortex with recurrent synapses, and inhibitory neurons play a fundamental role in the organization of functional sub-networks by restricting the propagation of excitation signals.     

Fluorescent styryl dyes applied as fast optical probes of cardiac action potential

Several styryl dyes were tested as fast optical probes of membrane action potentials in mammalian heart muscle tissue. After staining, atrial specimens were superfused in physiological salt solution, and fluorescence was excited by an argon ion laser. Excitation spot size on the surface of the preparation was 60 m in diameter. Dyes RH 160, RH 237, and RH 421 performed excellently as fast fluorescent probes of cardiac membrane potential. Fractional fluorescence changes, F/F, due to the action potential were in the range 2 to 6% at 514.5 nm excitation. Rise times of the action potential onset detected with each of the dyes were less than 0.5 ms, which is as fast or even faster than microelectrode measurements (atria of the rat). Thus membrane potential changes could be monitored with high resolution in both time and space. Emission spectra from heart muscle preparations stained with these dyes were shifted to shorter wavelengths by 70 nm and more as compared to spectra of the dyes in ethanol solution. The fluorescence spectrum of RH 160 at resting potential and the spectrum recorded during the plateau phases of the action potential were measured and showed no difference within the spectral resolution. As can be concluded from measurements of fluorescence changes at different excitation wavelengths, electrochromism cannot be the only mechanism causing the potential response.     

Computer simulation of action potential propagation in septated nerve fibers.

The nonlinear, core-conductor model of action potential propagation down axisymmetric nerve fibers is adapted for an implicit, numerical simulation by computer solution of the differential equations. The calculation allows a septum to be inserted in the model fiber; the thin, passive septum is characterized by series resistance Rsz and shunt resistance Rss to the grounded bath. If Rsz is too large or Rss too small, the signal fails to propagate through the septum. Plots of the action potential profiles for various axial positions are obtained and show distortions due to the presence of the septum. A simple linear model, developed from these simulations, relates propagation delay through the septum and the preseptal risetime to Rsz and Rss. This model agrees with the simulations for a wide range of parameters and allows estimation of Rsz and Rss from measured propagation delays at the septum. Plots of the axial current as a function of both time and position demonstrate how the presence of the septum can cause prominent local reversals of the current. This result, not previously described, suggests that extracellular magnetic measurements of cellular action currents could be useful in the biophysical study of septated fibers.     

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.