Experimental study of the conducted action potential in cardiac Purkinje strands

Conduction velocity is a complex physiological process that integrates the active and passive properties of the excitable cell. The relations between these properties in determining the conduction velocity are not intuitively obvious, and models have been used frequently to illustrate important relationships. To study the relationships of important parameters and to evaluate commonly used models, we changed conduction velocity experimentally in sheep cardiac Purkinje strands by reducing extracellular Na systematically. Cable analyses were also performed to obtain passive membrane and cable properties. Resting membrane resistance and capacitance did not change, nor did core resistance. Active properties measured in addition to conduction velocity included maximal upstroke velocity, action potential height, time constant of the foot, peak inward current, and upstroke power. With reduction in extracellular Na, all of these parameters of the action potential changed nonlinearly and not in direct proportion to the change in conduction velocity. The only simple relation found was a linear relationship between maximal upstroke velocity and peak inward current, normalized by the capacity of the foot. Models based on the cable equation and the wave equation offer a basis for quantitative analysis of conduction, and these data can be used to test the models.     

Electrophysiology of identified neurosecretory and non-neurosecretory cells in the cockroach pars intercerebralis

Two cell types can be distinguished with intracellular recording from the pars intercerebralis of the American cockroach (Periplaneta americana). The first type, which corresponds morphologically to the medial neurosecretory cell, always had spontaneously occurring, overshooting action potentials. These action potentials are probably endogenously produced. Tetrodotoxin experiments revealed that sodium is the dominant ion of the action potential. The action potentials are followed by a relatively long after-hyperpolarization. The input resistance of these cells ranged from 120 to 390 M omega. A mathematical model, based on cellular morphology and response to current pulses, revealed a membrane time constant of about 100 msec and an axonal:somatic conductance ratio of approximately 13. Area-specific membrane resistance was estimated at 33 k omega cm2. These cells also often had reversible and spontaneous inhibitory postsynaptic potentials. The second cell type, which is non-neurosecretory, never produced spontaneous action potentials and rarely had synaptic potentials. Action potentials could be evoked by current injection into the cell body or by extracellular stimulation of their axons in the posteroventral portion of the the protocerebrum. These action potentials also depend on sodium ions. Their input resistance ranged from 16 to 35 M omega. They had a membrane time constant of approximately 15 msec and an axonal:somatic conductance ratio of about 9. Their area specific membrane resistance was estimated at 14 k omega cm2.     

低Ca~(2 )对豚鼠心室乳头肌电缆特性的影响

应用常规细胞内微电极技术和一支细胞外玻璃吸引电极,观察低Ca~(2 )对豚鼠心室乳头肌电缆特性及动作电位传导速度的影响。实验结果表明,1/6正常Ca~(2 )浓度(0.3mmol/L)溶液灌流时,与正常对照相比,空间常数减小23.9％(p<0.01),细胞内纵向电阻增大37.1％(p<0.01),动作电位传导速度减小16.1％(p<0.01)。单纯缺氧时,与正常对照相比,空间常数减小29.4％(p<0.01),膜时间常数减小30.1％(p<0.01),细胞内纵向电阻增大100％(p<0.01),动作电位传导速度减小28％(p<0.01)。低Ca~(2 )(0.6mmol/L)加缺氧时,上述变化更加明显,与正常对照相比,空间常数减小39.4％(p<0.01),膜时间常数减小25.8％(p<0.05),细胞内纵向电阻增大140.9％(p<0.01),动作电位传导速度减小37.7％(p<0.01)。     

Electrotonic measurements by electric field-induced polarization in neurons: theory and experimental estimation

We present a theory for estimation of the dendritic electrotonic length constant and the membrane time constant from the transmembrane potential (TMP) induced by an applied electric field. The theory is adapted to morphologically defined neurons with homogeneous passive electric properties. Frequency characteristics and transients at the onset and offset of the DC field are considered. Two relations are useful for estimating the electrotonic parameters: 1) steady-state polarization versus the dendritic electrotonic length constant; 2) membrane time constant versus length constant. These relations are monotonic and may provide a unique estimate of the electrotonic parameters for 3D-reconstructed neurons. Equivalent tip-to-tip electrotonic length of the dendritic tree was estimated by measuring the equalization time of the field-induced TMP. For 11 turtle spinal motoneurons, the electrotonic length from tip to tip of the dendrites was in the range of 1-2.5 lambda, whereas classical estimation using injection of current pulses gave an average dendrite length of 0.9-1.1 lambda. For seven ventral horn interneurons, the estimates were 0.7-2.6 lambda and 0.6-0.9 lambda, respectively. The measurements of the field-induced polarization promise to be a useful addition to the conventional methods using microelectrode stimulation.     

Differences in Membrane Properties in Simulated Cases of Demyelinating Neuropathies: Internodal Focal Demyelinations without Conduction Block

The membrane properties (intracellular, extracellular, electrotonic potentials, strength-duration time constants, rheobasic currents and recovery cycles), which can now be measured in healthy subjects and patients with demyelinating neuropathies, are investigated in simulated cases of focal reduction (70%) of the myelin sheath in one, two and three successive internodal segments along the length of human motor fibres. The internodally focally demyelinated cases (termed as IFD1, IFD2 and IFD3, respectively) are simulated using our previous double cable model of the fibres. The results show that the intracellular potentials are with reduced amplitude and slowed conduction velocity in the vicinity of demyelinated segments, however the segmental conduction block is not achieved. The radial decline of the extracellular potential amplitudes slightly increases with the increase of the radial distance and demyelination. In contrast, the electrotonic potentials, strength-duration time constants and rheobases are normal. In the recovery cycles, the refractoriness, supernormality and less late subnormality are close to the normal, showing that the pathology is relatively minor. The obtained abnormalities in the potentials and excitability properties provide new information about the pathophysiology of the demyelinated human motor axons and can be observed in vivo in patients with acquired demyelinating neuropathies.     

Cyclic nucleotides and calcium: their role in the control of cell communication in the heart

The effects of theophylline and di-butyryl cyclic adenosine 3',5'-monophosphate (db-cAMP) on the electrical coupling of heart cells were investigated in rat trabeculae. Theophylline (4 X 10(-4) M) and db-cAMP (5 X 10(-5) M) increased both the space constant and conduction velocity. The time constant of the membrane was not changed by either drug. Measurements of the time constant of the foot of the action potential and conduction velocity were used to calculate the intracellular longitudinal resistance. Both theophylline and db-cAMP were found to enhance cell-to-cell communication in the heart by decreasing the intracellular longitudinal resistance.     

Membrane potential clamping in horizontal cells of the fish retina

The membrane potential of horizontal cells of the retina was clamped by uniform polarization of the layer of these cells by a current passed through extracellular electrodes. The volt-ampere characteristic curve of the synaptic membrane of the horizontal cells in some cases had segments with negative slope. With a sharp change in the level of voltage clamping the time taken for the resistance of the membrane to change was under 20 msec. Comparison of responses to photic stimulation recorded with and without voltage clamping showed that participation of the nonsynaptic membrane in the generation of responses to photic stimulation can affect their shape substantially.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 402–407, July–August, 1977.     

Gap junction uncoupling and discontinuous propagation in the heart. A comparison of experimental data with computer simulations.

The effects of octanol on longitudinal propagation in guinea pig papillary muscles were measured by intracellular microelectrodes. These data were compared with alterations in conduction induced by stepwise removal of gap junction channels in computer simulations of propagation based on a discontinuous cable model. Octanol reduced the velocity (theta) of propagating action potentials (APs) from 53.2 +/- 3.5 to less than 6.6 +/- 2.1 cm/s before block occurred. The maximal rate of rise (Vmax) changed in a biphasic manner, increasing from 133.1 +/- 5.4 in controls to 201.7 +/- 11.0 V/s when theta was 20.5 +/- 2.8 cm/s, and then declining to less than 58.6 +/- 15.2 V/s just before block. The input resistance and time constant of the AP foot increased, and the ascending limb of phase-plane loops became increasingly nonlinear and notched during octanol treatment. All effects of octanol reversed upon washout. A strand of cardiac tissue was modeled as a discontinuous cable composed of 40 cells, each with 10 isopotential membrane segments described by Beeler-Reuter kinetics, and coupled by a variable number of gap junction channels (156 pS). Decreasing the number of channels from 40,000 to 400 to 60 slowed conduction from 62.6 to 16.4 to 3.1 cm/s. As noted in the experimental data, Vmax increased from 103 to 130 and then fell to less than 96 V/s. The AP foot increased and became nonexponential. Distinct notches developed during phase 1 of the APs at slower propagation velocities in the experiments and simulations. The close similarities between the experimental and theoretical data obtained in this study supports the applicability of a discontinuous cable model for describing longitudinal propagation in the heart.     

On the electrotonic spread in cardiac muscle of the mouse

As an appropriate model which can simulate the cardiac working muscle with respect to the passive electrical spread, a lattice whose sides have linear cable properties is presented, and the passive potential spread on the model is mathematically analyzed in the fiber direction. Distribution of electrotonic potential in the fiber direction was measured with a pair of intracellular microelectrodes in the cardiac muscle fiber of mouse. By employing “pencil type” microelectrodes potential distribution in the transverse direction within a fiber was also measured. This transverse effect was differentiated from the longitudinal potential distribution. A tonically applied potential at any point of a cell interior spreads continuously in a manner described by a Bessel function. Using appropriate electrical and morphological parameters the experimental results proved to fit the curve obtained from numerical calculation on the model. The apparent length constant obtained for smaller distances (less than 100 μ) from the current source was 70 μ, and it increases as the distance becomes larger. At a point inside the fiber the resistance to the extracellular fluid ranged from 200 to 600 KΩ. The influence of coupling resistance between current and recording electrodes on the measurement of electrotonic potential was examined for small interelectrode distance.     

Electrophysiological Studies of the Antrum Muscle Fibers of the Guinea Pig Stomach

The membrane potentials of single smooth muscle fibers of various regions of the stomach were measured, and do not differ from those measured in intestinal muscle. Spontaneous slow waves with superimposed spikes could be recorded from the longitudinal and circular muscle of the antrum. The development of tension was preceded by spikes but often tension appeared only when the slow waves were generated. Contracture in high K solution developed at a critical membrane potential of -42 mv. MnCl₂ blocked the spike generation, then lowered the amplitude of the slow wave. On the other hand, withdrawal of Na⁺, or addition of atropine and tetrodotoxin inhibited the generation of most of the slow waves but a spike could still be elicited by electrical stimulation. Prostigmine enhanced and prolonged the slow wave; acetylcholine depolarized the membrane without change in the frequency of the slow waves. Chronaxie for the spike generation in the longitudinal muscle of the antrum was 30 msec and conduction velocity was 1.2 cm/sec. The time constant of the foot of the propagated spike was 28 msec. The space constants measured from the longitudinal and circular muscles of the antrum were 1.1 mm and 1.4 mm, respectively.     

Time Constants and Electrotonic Length of Membrane Cylinders and Neurons

A theoretical basis is provided for the estimation of the electrotonic length of a membrane cylinder, or the effective electrotonic length of a whole neuron, from electrophysiological experiments. It depends upon the several time constants present in passive decay of membrane potential from an initially nonuniform distribution over the length. In addition to the well known passive membrane time constant, τ_m = R_mC_m, observed in the decay of a uniform membrane potential, there exist many smaller time constants that govern rapid equalization of membrane potential over the length. These time constants are present also in the transient response to a current step applied across the membrane at one location, such as the neuron soma. Similar time constants are derived when a lumped soma is coupled to one or more cylinders representing one or more dendritic trees. Different time constants are derived when a voltage clamp is applied at one location; the effects of both leaky and short-circuited termination are also derived. All of these time constants are demonstrated as consequences of mathematical boundary value problems. These results not only provide a basis for estimating electrotonic length, L = [unk]/λ, but also provide a new basis for estimating the steady-state ratio, ρ, of cylinder input conductance to soma membrane conductance.     

Some Electrical Measurements of Motoneuron Parameters

Electrical properties of the membrane of cat spinal motoneurons have been studied using pulses of current and sinusoidally varying currents applied with intracellular microelectrodes. Hyperpolarization of the motoneuron membrane produces time and voltage dependent changes in membrane resistance and E. M. F. The voltage transients produced by steps of current have been analyzed in order to determine the effective electrotonic length of the dendrites. In a sample of 16 motoneurons, the average total length of the dendrites was 1.5 times the electrotonic length constant of the dendrites. The phase relationship between applied sinusoidal currents and the resultant transmembrane voltage was studied to determine the dendritic to somatic conductance ratio, ρ. In a sample of seven cells the best estimate for ρ was in the range between 5 and 10.     

The study of photoconduction of artificial lipid membranes incorporating rhodopsin. The simultaneous changes of membrane conduction and rhodopsin fluorescence

The protein fluorescence changes of rod outer segment fragments during bleaching were studied. Flash caused a fluorescence intensity drop by about 6%. The time constant of this process was30 msec and coincided with the time constant of increasing the permeability of an artificial lipid membrane containing rhodopsin and of Metarhodopsin I decay. In the presence of hydroxylamine the fluorescence intensity increases after the initial drop. The second process time constant was about 300 msec and coincided with the conduction drop time constant of the artificial membrane containing rhodopsin. A new intermediate — Metarhodopsin II₁ is proposed. It has the Metarhodopsin II absorption spectrum, lives for about 300 msec at room temperature, does not react with hydroxylamine, and increases the permeability of a disk membrane.     

Electrophysiological properties of human oviduct smooth muscle cells in dissociated cell culture.

Intracellular recordings were made from human oviduct smooth muscle maintained in cell culture. Solitary cells isolated from one another and cells in contact with one another retained electrical properties of smooth muscle in vivo. Membrane potential of solitary cells and connected cells was -35 mV. Connected cells formed electrotonic junctions which transmitted current from one cell to another. This current spread was responsible for differences in input resistance and time constant in solitary cells, 66 Momega and 96 msec, compared to connected cells, 26 Momega and 56 msec. All cells expressed delayed rectification to depolarizing current pulses. Some cells generated action potentials spontaneously or in response to intracellular current pulses. Action potentials were abolished by cobalt or by EGTA. Slow wave potentials, 5 . 20 mV in amplitude, occurred continuously once every 15 to 45 seconds in connected cells.     

Effects of phlorizin and phloretin on passive and dynamic electrical properties in muscle membrane

The effects of phlorizin and phloretin on the cable properties were investigated in frog sartorius muscle by conventional cable analysis. Actions of phloretin on voltage-dependent ionic conductances were also studied by analysis of the phase plane trajectories. Both drugs evoked a significant decrease in specific membrane resistance (Rm) in chloride-containing Ringer's solution. The linear membrane capacitance increased by about 30%. On the contrary, in the presence of the non-penetrating anion, glutamate, a slight increase in Rm was induced by phlorizin. It is suggested that these drugs may increase the chloride conductance in the muscle membrane. Under the effect of phloretin the resting membrane potential remained unchanged but the amplitude of the action potential was lowered and the rate of repolarization was significantly reduced. The rate of depolarization during the "foot" of the action potential and the conduction velocity calculated from the rate constant of depolarization decreased. The maximum Na conductance was not altered by phloretin but K conductance was reduced. The time constant (tau K) reflecting the kinetic properties of K conductance was increased about seven-fold. It is suggested that great importance may be attributed to the dipole properties of these drugs in the actions presented above.     

Changes in membrane characteristics of heart muscle during inhibition

Membrane characteristics were studied in isolated muscle strands from auricles of frogs using the "square pulse" technique. Changes in the time course and spatial spread of subthreshold electrotonic potentials were measured. If acetylcholine is applied in concentrations which cause slowing or stoppage of the heart beat, the following changes are produced: (a) the length constant (λ) of the membrane is reduced, (b) the time constant is shortened. The effects are reversible and increase with acetylcholine concentration. The membrane changes caused by acetylcholine dimmish with time. It is concluded that during acetylcholine inhibition, as well as during vagal inhibition, the conductance of the muscle membrane is increased. Appreciable changes in the resting membrane potential need not accompany inhibition.     

Electrical Properties of Hypothalamic Neuroendocrine Cells

Goldfish hypothalamic neuroendocrine cells have been investigated with intracellular recordings. The cells showed resting potentials of 50 mv and action potentials up to 117 mv followed by a long lasting and prominent diphasic hyperpolarizing afterpotential. The action potential occurred in two steps indicating sequential invasion. "Total" neuron (input) resistance was measured to be 3.3 x 10⁷ Ω and total neuron time constant was 42 msec. Orthodromic volleys, produced by olfactory tract stimulation, generated graded excitatory postsynaptic potentials. These neuroendocrine cells seem, therefore, to have electrical membrane properties that are similar to those of other central neurons. Antidromic volleys (pituitary stimulation) produced inhibitory post-synaptic potentials whose latency was only slightly longer than that of the antidromic spike indicating the presence of recurrent collaterals. This finding suggests that the concept of the neuroendocrine cell as a neuron whose axon forms contacts only on blood vessels and not on other neurons or effector cells is too restrictive. Perfusion of the gills with dilute (0.3 per cent) sea water produced an inhibition of spontaneous activity. This inhibition is discussed in relation to recent work which demonstrates that goldfish hypothalamic hormones facilitate Na⁺ influx across the gill membrane.     

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.     

Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine

The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries (n = 23; diameter, 58 +/- 4 microm; segment length, 2-8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37 degrees C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 microm to 3.5 mm). Membrane potential (E(m)) (rest, approximately -30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (-0.8 nA, 1.5 s) evoked hyperpolarization (-10 +/- 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant (lambda) = 1.2 +/- 0.1 mm; spontaneous E(m) oscillations (approximately 1 Hz) decayed with lambda = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 microm tip) evoked hyperpolarization (-14 +/- 2 mV) that conducted with lambda = 1.9 +/- 0.1 mm, 60% further (P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.