The influence of the chloride currents on action potential firing and volume regulation of excitable cells studied by a kinetic model

In excitable cells, the generation of an action potential (AP) is associated with transient changes of the intra- and extracellular concentrations of small ions such as Na⁺, K⁺ and Cl⁻. If these changes cannot be fully reversed between successive APs cumulative changes of trans-membrane ion gradients will occur, impinging on the cell volume and the duration, amplitude and frequency of APs. Previous computational studies focused on effects associated with excitation-induced changes of potassium and sodium. Here we present a model based study on the influence of chloride on the fidelity of AP firing and cellular volume regulation during excitation. Our simulations show that depending on the magnitude of the basal chloride permeability two complementary types of responsiveness and volume variability exist: (i) At high chloride permeability (typical for muscle cells), large excitatory stimuli are required to elicit APs; repetitive stimuli of equal strength result in almost identical spike train patterns (Markovian behavior), however, long excitation may lead to after discharges due to an outward directed current of intracellular chloride ions which accumulate during excitation; cell volume changes are large. (ii) At low chloride permeability (e.g., neurons), small excitatory stimuli are sufficient to elicit APs, repetitive stimuli of equal strength produce spike trains with progressively changing amplitude, frequency and duration (short-term memory effects or non-Markovian behavior); cell volume changes are small. We hypothesize that variation of the basal chloride permeability could be an important mechanism of neuronal cells to adapt their responsiveness to external stimuli during learning and memory processes.     

A study of the crustacean axon repetitive response. 3. A comparison of the effect of veratrine sulfate solution and potassium-rich solutions

The crustacean single nerve fiber gives rise to trains of impulses during a prolonged depolarizing stimulus. It is well known that the alkaloid veratrine itself causes a prolonged depolarization; and consequently it was of interest to investigate the effect of this chemically produced depolarization on repetitive firing in the single axon and compare it with the effect of depolarization by an applied stimulating current or by a potassium-rich solution. It was found that veratrine depolarization, though similar in some respects to a potassium-rich depolarization of depolarizing current effect, was in many respects quite different. (1) At low veratrine concentration, less than 1 Mg%, the negative after potential following a spike action potential was prolonged and augmented. At higher concentrations or after a long period of time, veratrine caused a prolonged steady state depolarization of the membrane, the “veratrine response”. The prolonged plateau depolarization response could be elicited with or without an action potential spike by a short or long duration stimulating pulse, but only if the veratrine depolarization was prevented or offset by an applied conditioning hyperpolarizing inward current. (2) The “veratrine response” resembled the potassium-rich solution response in the plateau-like contour of the depolarization and the very low membrane resistance during this plateau phase. Like the potassium response, it was possible to obtain a typical hyperpolarizing response with an inwardly directed current pulse if applied during the plateau phase. During the negative after potential augmented with veratrine, however, this hyperpolarizing response was not observed. (3) In contrast to the potassium response, however, the “veratrine response” is intimately associated with the sodium concentration in the external medium. The depolarization in millivolts is linearly related to the log of the concentration of external sodium. Moreover, during veratrine action there is a continuous and progressive inactivation of the sodium mechanism which ultimately terminates repetitive firing and abolishes the spike action potential. Then even with conditioning hyperpolarization only the slow response may be elicited in veratrine, occasionally with a spike superimposed if sodium is present, but without repetitive firing. (4) It is concluded that veratrine action is the result of a chemical or metabolic reaction by the alkaloid in the membrane. It is suggested that veratrine may inhibit the sodium extrusion mechanism, or may itself compete for sites in the membrane with calcium and/or sodium. This explains the inhibiting effect of high calcium, the abolition of the “veratrine response” with low temperature and high calcium combined and the progressive inactivation of the sodium system.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Effects of transient receptor potential-like current on the firing pattern of action potentials in the Hodgkin-Huxley neuron during exposure to sinusoidal external voltage

Transient receptor potential vanilloid-1 (TRPV1) channels play a role in several inflammatory and nociceptive processes. Previous work showed that magnetic electrical field-induced antinociceptive [corrected] action is mediated by activation of capsaicin-sensitive sensory afferents. In this study, a modified Hodgkin-Huxley model, in which TRP-like current (ITRP) was incorporated, was implemented to predict the firing behavior of action potentials (APs), as the model neuron was exposed to sinusoidal changes in externally-applied voltage. When model neuron is exposed to low-frequency sinusoidal voltage, increased maximal conductance of ITRP can enhance repetitive bursts of APs accompanied by a shortening of inter-spike interval (ISI) in AP firing. The change in ISIs with number of interval is periodic with the phase-locking. In addition, increased maximal conductance of ITRP can abolish chaotic pattern of AP firing in model neuron during exposure to high-frequency voltage. The ISI pattern is converted from irregular to constant, as maximal conductance of ITRP is increased under such high-frequency voltage. Our simulation results suggest that modulation of TRP-like channels functionally expressed in small-diameter peripheral sensory neurons should be an important mechanism through which it can contribute to the firing pattern of APs.     

Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels

Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.     

Roles of I(f) and intracellular Ca2+ release in spontaneous activity of ventricular cardiomyocytes during murine embryonic development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca²⁺ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca²⁺ imaging, and RT‐PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down‐regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2‐aminoethoxydiphenyl borate (2‐APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR‐ and IP3R‐mediated ICR coexisted in early‐stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na‐Ca exchanger current (I_Na/Ca) mediated ICR‐triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2‐APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)‐mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852–1862, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of a slow potassium permeability on repetitive activity of the frog node of ranvier

Adding a potassium permeability with slow kinetics to the Frankenhaeuser-Huxley equations describing action potential generation at a frog node of Ranvier has a twofold effect on the maintained repetitive firing the model can show. If the contribution of the slow to the total potassium permeability is increased, the maintained discharge frequency for a given stimulating current experiences a decrease. On the other hand, addition of the slow channel narrows the range of currents for which the model can generate repetitive activity. If as little as 6.2% of the total potassium permeability are provided by the slow channels, the Frankenhaeuser-Huxley equations completely lose the ability to show maintained firing. The introduction of the slow potassium current abolishes especially repetitive activity at low values of stimulating current. This effect is so marked that the minimal discharge frequency the model can maintain increases with increasing contribution of the slow channel. Therefore, an important purpose of the slow potassium channel present at the frog nodal membrane could consist of preventing the node of Ranvier from generating consistent firing on its own.     

Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion

Voltage-gated potassium currents (Kv), primarily due to Kv2.1 channels, are activated by glucose-stimulated pancreatic beta cell depolarization, but the exact role (or roles) of this channel in regulating insulin secretion remains uncertain. Here we report that, compared with controls, Kv2.1 null mice have reduced fasting blood glucose levels and elevated serum insulin levels. Glucose tolerance is improved and insulin secretion is enhanced compared to control animals, with similar results in isolated islets in vitro. Isolated Kv2.1(-/-) beta cells have residual Kv currents, which are decreased by 83% at +50 mV compared with control cells. The glucose-induced action potential (AP) duration is increased while the firing frequency is diminished, similar to the effect of specific toxins on control cells but substantially different from the effect of the less specific blocker tetraethylammonium. These results reveal the specific role of Kv2.1 in modulating glucose-stimulated APs of beta cells, exposing additional important currents involved in regulating physiological insulin secretion.     

Effects of ion channel inhibitors on cold- and electrically-induced action potentials in Dionaea muscipula

Glass microelectrodes were inserted into Dionaea muscipula (Venus flytrap) lobes and the action potentials (APs) were recorded in response to a sudden temperature drop or a direct current (DC) application. The effect of potassium channel inhibitor, tetraethylammonium ion, was the lengthening of the depolarization phase of AP. APs were also affected by the anion channel inhibitor, anthracene-9-carboxylic acid, that made them slower and smaller. Neomycin, which disturbs inositol triphosphate-dependent Ca²⁺ release, caused the visible inhibition of AP, too. Ruthenium red, which blocks cyclic ADP-ribose-dependent Ca²⁺ release, totally inhibited DC-triggered APs and induced the decrease in the amplitudes of cold-evoked APs. Lanthanum ions significantly inhibited both cold- and DC-induced membrane potential changes. It was concluded that during excitation Dionaea muscipula relied upon the calcium influxes from both the extra- and intracellular compartments.     

Effect of primycin on some electric properties of the frog skeletal muscle

The effect of primycin, a guanidine-type antibiotic was studied on the electric properties and 42K+ uptake of the frog sartorius and semitendinosus muscle. Both in normal and choline chloride Ringer solution, primycin evoked a concentration and time dependent depolarization of the surface membrane of the muscle. This depolarization was significantly increased by Na ions. Primycin treatment was shown to evoke a dose-dependent decrease of the depolarization induced by 20 mM K+-Ringer. When the muscles were incubated in a Ringer solution containing choline chloride, during an incubation period of 30 min the uptake of 42K+ was decreased to 12% upon the exposure to 5 x 10(-6) mol primycin as compared to the control value. As the primycin-induced depolarization increased, the shape and amplitude of the action potentials elicited by square-wave electric impulses were altered and decreased, respectively. In sodium isaethionate Ringer 1--2 x 10(-6) M primycin induced a slow depolarization resulting in firing potentials. The results suggest that primycin depolarizes the surface membrane exclusively through the blockade of the resting K+ channels, the other phenomena being the results of this depolarizing effect.     

High frequency electrostimulation of excitable cells

Reactions of nerve fibers to high frequency electrical stimulation are examined with three nerve models. Switching on the signal produces a single AP at the threshold current. Stronger currents lead into a region of repetitive firing. The firing rate depends on the current and the fibers more distant from the electrode will have a lower rate. The AP's are not synchronized. In the "House-Urban" cochlear implant a 16 kHz carrier is used for stimulation. It is modulated by electrical signals derived from sound pressure. An analysis of the modulation shows which signals can produce APs synchronized with the source signal.     

The action of substance P on neurons of the myenteric plexus of the guinea-pig small intestine.

Extracellular and intracellular recordings were made in vitro from single neurons of the myenteric plexus of the guinea-pig small intestine. Synthetic substance P was applied to the neurons by means of the perfusing solution or by electrophoresis from micropipettes. Extracellular recording showed that substance P (100 pm-30 nm), applied by perfusion, increased the firing rate of myenteric neurons. Intracellular recording indicated that perfusion with substance P caused a dose-dependent membrane depolarization which was unaffected by hexamethonium, hyoscine, naloxone or baclofen. The depolarization was also evoked by electrophoretic application of substance P. It was associated with an increase in membrane resistance, augmented by membrane depolarization and reduced by membrane hyperpolarization. The relation between the substance P reversal potential and the logarithm of the extracellular potassium concentration was linear with a slope of 54 mV/log10[K+], which indicates that substance P inactivates the resting potassium conductance of the myenteric neurons. This effect on ion conductance is the same as that of an unknown substance that mediates slow synaptic excitations with the myenteric plexus.     

The physostigmine depolarization potentiating effect of salicylate in frog skeletal muscle.

1) The frog's sartorius muscle was depolarized depending on the degree of concentration 2--4 times more intensely by physostigmine salicylate than by physostigmine sulphate. 2) In normal Ringer's solution, 1 mM physostigmine salicylate decreased the sensitivity of the membrane to potassium depolarization by about 90%. Under similar experimental conditions, physostigmine sulphate and Na salicylate, respectively, decrease the sensitivity of the membrane to potassium depolarization by about 30%. 3) The difference manifested in the depolarizing effect of salicylate and other physostigmine salts (chloride, sulphate, phosphate, formiate, acetate, monochloracetate, benzoate and para-oxy-benzoate) is expressed already at 1 mM concentration (about 10-fold), if the muscle had been equilibrated in chloride-free glucuronate or sulphate milieu. 4) The depolarization develops slowly. It takes 30--60 minutes for the new steady state to develop even in the superficial sartorius fibres. If depolarization has reached its maximum on an average 100 mV, the membrane potential remains unchanged for hours. 5) Depolarization ensues at an unchanged degree in the presence of Na-free (choline) Ringer as well as in the presence of 2X10(-8) g/ml tetrodotoxin; therefore, it is not a Na-dependent process. 6) Under the influence of 1 mM physostigmine salicylate the membrane's resistance to the inward potassium current increased about twofold, while the increase was only 15% to the outward potassium current. It is assumed that the salicylate anion is characteristically capable of potentiating the decreasing effect of physostigmine on potassium permeability, though the role of the metabolic effect of salicylate cannot be excluded.     

Noise controlled synchronization in potassium coupled neural models

The paper applies biologically plausible models to investigate how noise input to small ensembles of neurons, coupled via the extracellular potassium concentration, can influence their firing patterns. Using the noise intensity and the volume of the extracellular space as control parameters, we show that potassium induced depolarization underlies the formation of noise-induced patterns such as delayed firing and synchronization. These phenomena are associated with the appearance of new time scales in the distribution of interspike intervals that may be significant for the spatio-temporal oscillations in neuronal ensembles.     

Modeling NaV1.1/SCN1A sodium channel mutations in a microcircuit with realistic ion concentration dynamics suggests differential GABAergic mechanisms leading to hyperexcitability in epilepsy and hemiplegic migraine

Loss of function mutations of SCN1A, the gene coding for the voltage-gated sodium channel Na_V1.1, cause different types of epilepsy, whereas gain of function mutations cause sporadic and familial hemiplegic migraine type 3 (FHM-3). However, it is not clear yet how these opposite effects can induce paroxysmal pathological activities involving neuronal networks’ hyperexcitability that are specific of epilepsy (seizures) or migraine (cortical spreading depolarization, CSD). To better understand differential mechanisms leading to the initiation of these pathological activities, we used a two-neuron conductance-based model of interconnected GABAergic and pyramidal glutamatergic neurons, in which we incorporated ionic concentration dynamics in both neurons. We modeled FHM-3 mutations by increasing the persistent sodium current in the interneuron and epileptogenic mutations by decreasing the sodium conductance in the interneuron. Therefore, we studied both FHM-3 and epileptogenic mutations within the same framework, modifying only two parameters. In our model, the key effect of gain of function FHM-3 mutations is ion fluxes modification at each action potential (in particular the larger activation of voltage-gated potassium channels induced by the Na_V1.1 gain of function), and the resulting CSD-triggering extracellular potassium accumulation, which is not caused only by modifications of firing frequency. Loss of function epileptogenic mutations, on the other hand, increase GABAergic neurons’ susceptibility to depolarization block, without major modifications of firing frequency before it. Our modeling results connect qualitatively to experimental data: potassium accumulation in the case of FHM-3 mutations and facilitated depolarization block of the GABAergic neuron in the case of epileptogenic mutations. Both these effects can lead to pyramidal neuron hyperexcitability, inducing in the migraine condition depolarization block of both the GABAergic and the pyramidal neuron. Overall, our findings suggest different mechanisms of network hyperexcitability for migraine and epileptogenic Na_V1.1 mutations, implying that the modifications of firing frequency may not be the only relevant pathological mechanism.     

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Physiological types of substantia gelatinosa neurons in the rat spinal cord

Electrophysiological properties of neurons in the substantia gelatinosa (SG, or lamina II) were studied in vitro in spinal cord slices from 3-to 5-week-old rats. Based on the type of action potentials (APs) firing in response to long depolarization (0.5 to 0.8 sec), neurons were categorized into three types: tonic (APs were generated over the whole duration of the stimulus, n = 26, or 41.2%), adapting (a few APs occurred only at the beginning of stimulation, n = 8, 12.7%), and delayed-firing neurons, DFNs (APs occurred at the end of stimulation, n = 22, 35.1%); 11% of the cells had intermediate properties. Neurons of each type expressed distinct ion currents that were subthreshold for AP generation (< −40 mV). Tonic and adapting neurons either had no subthreshold currents (n = 21, or 61.3%) or expressed T-type calcium currents (n = 13, or 38.7%). All DFNs had outward A-type potassium currents. Statistical analysis confirmed this classification scheme: neurons of each type were differentially distributed in a 3-D parametric space of the main cellular properties. Distributions of tonic and adapting neurons partially overlapped, while that of DFNs differed significantly from both the above groups. It is suggested that DFNs perform a special function in the processing of sensory information; the functions of tonic and adapting neurons might be rather similar to each other. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 191–198, May–June, 2008.