Quantal potential fields around individual active zones of amphibian motor-nerve terminals

The release of a quantum from a nerve terminal is accompanied by the flow of extracellular current, which creates a field around the site of transmitter action. We provide a solution for the extent of this field for the case of a quantum released from a site on an amphibian motor-nerve terminal branch onto the receptor patch of a muscle fiber and compare this with measurements of the field using three extracellular electrodes. Numerical solution of the equations for the quantal potential field in cylindrical coordinates show that the density of the field at the peak of the quantal current gives rise to a peak extracellular potential, which declines approximately as the inverse of the distance from the source at distances greater than about 4 microm from the source along the length of the fiber. The peak extracellular potential declines to 20% of its initial value in a distance of about 6 microm, both along the length of the fiber and in the circumferential direction around the fiber. Simultaneous recordings of quantal potential fields, made with three electrodes placed in a line at right angles to an FM1-43 visualized branch, gave determinations of the field strengths in accord with the numerical solutions. In addition, the three electrodes were placed so as to straddle the visualized release sites of a branch. The positions of these sites were correctly predicted on the basis of the theory and independently ascertained by FM1-43 staining of the sites. It is concluded that quantal potential fields at the neuromuscular junction that can be measured with available recording techniques are restricted to regions within about 10 microm of the release site.     

Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors.

The time course of most quantal currents recorded with a small diameter electrode placed over visualized varicosities of sympathetic nerve terminals that secrete ATP was determined: these had a time to reach 90% of peak of 1.3-1.8 ms and a time constant of decay of 12-18 ms; they were unaffected by blocking ectoenzymes or the uptake of adenosine. Monte Carlo methods were used to analyze the stochastic interaction between ATP, released in a packet from a varicosity, and the underlying patch of purinoceptors, to reconstitute the time course of the quantal current. This leads to certain restrictions on the possible number of ATP molecules in a quantum (about 1000) and the density of purinoceptors at the junctions (about 1000 microns-1), given the known geometry of the junction and the kinetics of ATP action. The observed quantal current has a relatively small variability (coefficient of variation < 0.1), and this stochastic property is reproduced for a given quantum of ATP. Potentiation effects (of about 12%) occur if two quanta are released from the same varicosity because the receptor patch is not saturated even by the release of two quanta. The simulations show that quantal currents have a characteristically distinct shape for varicosities with different junctional cleft widths (50-200 nm). Finally, incorporation of an ectoenzyme with the known kinetics of ATPase into the junctional cleft allows for a quantal current of the observed time course, provided the number of ATP molecules in a quantum is increased over the number in the absence of the ATPase.     

Synaptic transmission at visualized sympathetic boutons: stochastic interaction between acetylcholine and its receptors.

Excitatory postsynaptic currents (EPSCs) were recorded with loose patch electrodes placed over visualized boutons on the surface of rat pelvic ganglion cells. At 34 degrees C the time to peak of the EPSC was about 0.7 ms, and a single exponential described the declining phase with a time constant of about 4.0 ms; these times were not correlated with changes in the amplitude of the EPSC. The amplitude-frequency histogram of the EPSC at individual boutons was well described by a single Gaussian-distribution that possessed a variance similar to that of the electrical noise. Nonstationary fluctuation analysis of the EPSCs at a bouton indicated that about 120 ACh receptor channels were available beneath boutons for interaction with a quantum of ACh. The characteristics of these EPSCs were compared with the results of Monte Carlo simulations of the quantal release of 9000 acetylcholine (ACh) molecules onto receptor patches of density 1400 microns-2 and 0.41 micron diameter, using a kinetic scheme of interaction between ACh and the receptors similar to that observed at the neuromuscular junction. The simulated EPSC generated in this way had temporal characteristics similar to those of the experimental EPSC when either the diffusion of the ACh is slowed or allowance is made for a finite period of transmitter release from the bouton. The amplitude of the simulated EPSC then exhibited stochastic fluctuations similar to those of the experimental EPSC.     

The bioelectric field of the catfish Ictalurus nebulosus.

The variability of the bioelectric field of the electrosensitive catfish, Ictalurus nebulosus, was investigated by recording the potential variation occurring when the fish passed a stationary electrode, and by recording the field of a stationary fish by a 15-electrode array. A good first order approximation of the recorded field of a 20 cm long fish is a dipole dc source with the source and sink about 7 cm apart, carrying a current of about 1 microA in water with a specific resistivity of 3.3 kohm cm. At 5 cm distance from the dipole axis such a source generates an electric potential swing in the order of 50 microV in free space, head negative, tail positive. Superimposed on the basic component are respiration related fluctuations, and fluctuations related to the activity of the alimentary canal, gills, and skin. Novel stimuli, or stressors like investigators approaching the aquarium, evoke sudden increases in field strength. which last about 15 min. Demineralization of the aquarium water causes changes in field strength and reversal of field polarity. The administration of food causes field variations in the vicinity of the anal opening. The bioelectric field shows diurnal fluctuations of 100 microV. The peak is at about 04:00, the dip at 14:00. The fluctuations of the bioelectric field are sufficiently strong and specilic to serve as electrical stimuli to other electrosensitive catfish. It is suggested that the field changes allow a simple form of electrocommunication. i.e. inform conspecifics about some physiological properties of the field source. The cellular mechanisms underlying the fluctuations of the bioelectric field are homeostatic processes mediated by ion pumps and ion channels.     

Latency of transmitter release at crayfish motor nerve endings examined by intracellular depolarization

Latency of release of individual quanta of transmitter was studied at neuromuscular junctions of a crayfish (Procambarus clarkii). Postsynaptic quantal currents were recorded at individual motor nerve endings with a macropatch electrode while the subterminal axon branch was depolarized by current passed through an intracellular microelectrode. For depolarizing currents of moderate size, the latency of transmitter release did not change when the duration of the depolarizing current was altered. Previous studies in which a contrary result was obtained may have been compromised by artefacts or by the sampling methods employed. The present results do not support the hypothesis of a depolarization-induced "repressor" of quantal release.     

Membrane potential and its electrode-recorded counterpart in an electrical model of an olfactory sensillum

Insect receptor neurons are surrounded with auxiliary cells and encased in a hair. Their electrical activity is usually recorded with an electrode located at the tip of the hair. Analytical expressions giving the membrane potential along the sensory dendrite and the tip-recorded potential are derived for a neuron in steady-state conditions. They formally close the gap between theoretical models and experimental measurements, when transduction mechanisms and active membrane properties are not taken into account. It is shown that the tip-recorded potential reflects correctly the relative variations of the dendritic membrane potential as a function of stimulus intensity over a large range of parameters. The geometric and electrical characteristics of the sensillum that need be known to compute the dendritic membrane potential from the tip-recorded potential are given.     

An endplate potential due to potassium released by the motor nerve impulse

A small endplate potential can be recorded in frog muscle fibres, after all acetylcholine-mediated transmission has been eliminated by pre- or postsynaptic blocking agents (botulinum toxin, calcium lack, manganese, curare, alpha-bungarotoxin). It is usually necessary to hyperpolarize the muscle membrane to detect this 'non-cholinergic' endplate potential. Below--100 mV little or no response is seen; a maximum is reached at about--140 mV, when the amplitude can be as large as 100 microV (endplate current up to about 1 nA). Other characteristic features are: the response shows no quantal fluctuations; its amplitude is not facilitated by repetitive impulses; its size and time course are not noticeably affected by prostigmine, curare or alpha-bungarotoxin; the half-time of decline of the endplate current is approximately 1.7 ms at 20 degrees C, and is lengthened by lowering the temperature with a Q10 of about 1.3; the response is abolished by barium. When iontophoretic pulses of potassium are applied to the endplate, local depolarization is recorded whose amplitude varies with membrane potential similarly to that of the nerve-evoked response. These observations strongly indicate that this 'non-cholinergic', 'non-quantal' endplate potential arises from a rapid synaptic transfer of potassium ions, released by the active nerve terminal into the synaptic cleft and entering the muscle fibre through 'anomalous rectifier' channels in the endplate membrane.     

Cell-compatible array of three-dimensional tip electrodes for the detection of nitric oxide release

An array of electrodes on which cells could be grown directly was fabricated using silicon anisotropic etching and a thick-photoresist process and employed for the detection of nitric oxide (NO) released from a population of adherently growing human umbilical vein endothelial cells (HUVEC). The electrodes are tip-shaped and are 40 microm high of which only the top 15 microm are exposed Pt-tips. After electrochemical induced modification of the exposed Pt tips using Ni phthalocyanine the individual addressable electrode tips were sensitive and selective for the detection of NO at an applied constant potential of 750 mV. The silicon nitride insulation of the lower part of the tip electrodes prevented the death of the cells upon the application of the working potential at which NO was detected. It also helped to avoid the perturbation of the integrity of the sensing chemistry imparted on the electrode surface that could have resulted from the contact of the adherently growing cells with the active electrode surface. The release of nitric oxide from HUVEC was successfully monitored with different numbers of tip electrodes simultaneously connected as combined working electrode.     

The synaptic bouton acts like a slat shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

The synaptic bouton acts like a salt shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

Amplitude of miniature potentials of motor neurons in the frog Rana ridibunda

Miniature synaptic potentials have been recorded from motoneurones of the isolated spinal cord of the frog Rana ridibunda. In normal Ringer's solution, their frequency varied from 5 to 50/sec, whereas their amplitude reached 2-5 mV. Only 50-300 microV (rarely 0.5-1.5 mV) potentials persisted when TTX was added to Ringer's solution and/or Ca was replaced by Mn. However, in Ca-free solution, TTX in combination with Mn did not decrease the amplitude of miniature potentials, provided the initial values varied within 50-300 microV. Noise fluctuations did not exceed 40-50 microV, and the ratio of the number of miniature potentials of 50 microV to the number of 50 microV noise potentials was about 10:1. The observed miniature potentials with an amplitude of 50-100 microV coincide with the quantal units calculated by other authors from statistical analysis of the unitary EPSPs evoked by primary afferents or by ventrolateral tract fibers.     

Do mental events cause neural events analogously to the probability fields of quantum mechanics?

If non-material mental events, such as the intention to carry out an action, are to have an effective action on neural events in the brain, it has to be at the most subtle and plastic level of these events. In the first stage of our enquiry an introduction to conventional synaptic theory leads on to an account of the manner of operation of the ultimate synaptic units. These units are the synaptic boutons that, when excited by an all-or-nothing nerve impulse, deliver the total contents of a single synaptic vesicle, not regularly, but probabilistically. This quantal emission of the synaptic transmitter molecules (about 5000-10 000) is the elementary unit of the transmission process from one neuron to another. In the second stage this refined physiological analysis leads on to an account of the ultrastructure of the synapse, which gives clues as to the manner of its unitary probabilistic operation. The essential feature is that the effective structure of each bouton is a paracrystalline presynaptic vesicular grid with about 50 vesicles, which acts probabilistically in vesicular (quantal) release. In the third stage it is considered how a non-material mental event, such as an intention to move, could influence the subtle probabilistic operations of synaptic boutons. On the biological side, attention is focused on the paracrystalline presynaptic vesicular grids as the targets for non-material mental events. On the physical side, attention is focused on the probabilistic fields of quantum mechanics which carry neither mass nor energy, but which nevertheless can exert effective action at microsites. The new light on the mind-brain problem came from the hypothesis that the non-material mental events, the 'World 2' of Popper, relate to the neural events of the brain (the 'World 1' of matter and energy) by actions in conformity with quantum theory. This hypothesis that mental events act on probabilistic synaptic events in a manner analogous to the probability fields of quantum mechanics seems to open up an immense field of scientific investigation both in quantum physics and in neuroscience.     

l-3,4-Dihydroxyphenylalanine Increases the Quantal Size of Exocytotic Dopamine Release In Vitro

Abstract: The catecholamine precursor l -3,4-dihydroxyphenylalanine ( l -DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of l -DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 m M potassium-containing media, l -DOPA (100 µ M for 1 h) increased DA release by >10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, l -DOPA (50 µ M for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that l -DOPA (50 µ M for 40–70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that l -DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.     

Electrophysiological properties of Achlya hyphae: ionic currents studied by intracellular potential recording

The electrical properties of the water mold Achlya bisexualis were investigated using intracellular microelectrodes. Hyphae growing in a defined medium maintained a membrane potential (Vm) of -150 to -170 mV, interior negative. Under the conditions used here, this potential was insensitive to changes in the inorganic ion composition of the medium. Changes in external pH did affect Vm, but only outside the physiological pH range. By contrast, the addition of respiratory inhibitors caused a rapid depolarization without affecting the conductance of the plasma membrane. Taken together these findings strongly suggest that the membrane potential is governed by an electrogenic ion pump rather than by an ionic diffusion potential. Previous work from this laboratory showed that Achlya hyphae generate a transcellular proton current that enters the growing tip, flows along the hyphal length, and exits distally from the trunk. These initial experiments used an extracellular vibrating electrode, and I now report intracellular electrical recordings which support the hypothesis that protons enter the tip by symport with amino acids and are expelled distally by a proton-translocating ATPase. Most significantly, current flowing intracellularly along the hyphal length is associated with a cytoplasmic electric field of 0.2 V/cm or greater. Conditions that inhibit the current also abolish the internal field, suggesting that these two phenomena are closely linked.     

A study of the connection between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron of the snailHelix pomatia

The connection between an interneuron initiating pacemaker activity in the bursting RPa1 neuron and the bursting neuron itself (Pin and Gola, 1983) has been analyzed in the snail Helix pomatia. Prolonged depolarization of the interneuronal membrane produced in it a series of action potentials as well as a parallel initiation or enhancement of bursting activity in the RPa1 neuron. If the discharge in the interneuron was evoked by short current pulses of threshold amplitude, no bursting activity was seen in the RPa1 neuron. However, short stimuli delivered on the background of subthreshold depolarization of the interneuronal membrane produced bursting activity in the RPa1 neuron. Under voltage-clamp conditions a slow inward current could be recorded in the RPa1 neuronal membrane after stimulation of the interneuron with a latency of about 2 sec. Short shifts of the holding potential in the hyperpolarizing direction at the maximum of this current produced a transient outward current. Replacement of extracellular Ca2+ by Mg2+ ions, as well as addition of 1 mM CdCl2 to the external solution, prevented the response to the interneuronal stimulation in the RPa1 neuron. Electron microscopic investigation of the interneuron has shown the abundance of Golgi complexes in its cytoplasm with electron-dense granules in their vicinity. It is concluded that the connection between the interneuron and the bursting neuron is of chemical origin, based on secretion by the former of some substances which activate at least two types of ionic channels in the membrane of the RPa1 neuron.     

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.     

The pulse current pattern generated by developing fucoid eggs

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.     

Inter-operator agreement in decomposition of motor unit firings from high-density surface EMG.

High-density surface EMG can be used to obtain a spatially selective representation of several motor unit action potentials. Recently, a decomposition of the signal into the underlying motor neuron firing patterns has been described. The reliability of the algorithm has not yet been tested. Eleven healthy subjects participated. High-density surface EMG was recorded from the vastus lateralis muscle during an isometric knee extension. Two independent operators analyzed the signals. After operator-supervised cluster analysis of spikes, motor unit action potential templates were constructed and an automatic template matching was performed. The decomposition was adjusted by hand. Agreement between operators was calculated for the number of coincident firings. Bland-Altman plots of peak-to-peak amplitude were constructed and limits of agreement were calculated. For completely decomposed motor unit action potential trains the between-operator agreement of firing events was very high. The peak-to-peak amplitude of monopolar motor unit action potentials was 115microV (SD 74microV). The agreement was within 3microV and independent of amplitude. With partial decomposition agreement within 26microV was achieved. For bipolarly derived motor unit action potentials the peak-to-peak amplitude was 54microV (SD 49microV), the agreement was within 3microV. Only for recordings obtained from a force level below 5% of the maximum voluntary contraction full decomposition was possible. It was concluded that when full decomposition is achieved, two independent operators are likely to arrive at nearly identical firing patterns.     

Mechanism of trace hyperpolarization of the action potential of snail giant neurons]

Depolarization current decreases and hyperpolarization current increases the amplitude of tracing hyperpolarization of the neuron action potential. Calcium-defficient solution supresses the tracing depolarization, and turns the rhythmical activity of the neuron into the flashlike one. An increase of outer concentration of potassium ions decreases the tracing depolarization. The latter is suppressed completely when the membrane behaves as a potassium electrode. The suppressing effect of the increase of potassium outer concentration on tracing hyperpolarization decreases with a decrease of calcium ions content in the medium. When an active release of sodium ions from the cell is inhibited with DNP and substitution of sodium ions by lithium ions the tracing hyperpolarization of the action potential is suppressed. The tracing hyperpolarization is also suppressed during the shunting of the electrogenic effect of potassium pump with the outcoming current of chlorine ions. It is suggested that the tracing hyperpolarization of the single action potential is due to the calcium-dependent fraction of electrogenic release of sodium ions from the cell.