Slow-waves in rat small intestine

Rat small intestine generates rhythmic slow-wave activity. The slow-waves are not eliminated by ouabain application or incubation in potassium free solution. Exposure to low sodium or calcium free solution decreases slow-wave activity. Incubation in sodium and calcium free solution eliminates activity. It is concluded that rat small intestinal slow-waves may not result from the same mechanism as in the cat.     

Primitive nervous systems: Electrical activity in ventral nerve cords of the flatworm,Notoplana acticola

Electrical activity evoked in the major cords of the ventral submuscular nerve plexus were measured. Recordings and stimulation utilized suction electrodes attached directly to exposed nerve cords. Four categories have been recorded: (a) short latency spikes which have relatively high thresholds and appear to be single units; (b) short duration fast compound spikes that are made up of a large units; (c) long duration compound potentials that are made up from a large number of smaller units, and (d) small amplitude potentials with long latencies and a characteristic shape. These can be conducted diffesely through the nerve plexus. The first two categories of spikes are called “fast” potentials because of their characteristic rise and fall times and the last categories are known as “diffuse” potentials. The spikelike fast potentials were only recorded from the main trunks (nerves VI), while diffuse potentials could also be recorded from side branches of these nerves. The diffuse potential appears to be concluded throughout the plexus but preferential conducting pathways occur lesions. Both diffuse and fast potentials show facilitation of response to repeated stimulation. Facilitation can be demonstrated in the presence of high Mg²⁺ concentrations. Conductance of the diffuse potential also occurs in the presence of high ambient Mg²⁺. In Ca²⁺ free medium containing 1-mM EGTA one can also observe facilitatory events. The possibility of Mg²⁺-insensitive synapses is discussed.     

Primitive nervous systems: electrical activity in ventral nerve cords of the flatworm, Notoplana acticola.

Electrical activity evoked in the major cords of the ventral submuscular nerve plexus were measured. Recording and stimulation utilized suction electrodes attached directly to exposed nerve cords. Four categories of potentials have been recorded: (a) short latency spikes which have relatively high thresholds and appear to be single units; (b) short duration fast compound spikes that are made up of a small number of large units; (c) long duration compound potentials that are made up from a large number of smaller units, and (d) small amplitude potentials with long latencies and a characteristic shape. These can be conducted diffusely through the nerve plexus. The first two categories of spikes are called "fast" potentials because of their characteristic rise and fall times and the last categories are known as "diffuse" potentials. The spikelike fast potentials were only recorded from the main trunks (nerves VI), while diffuse potentials could also be recorded from side branches of these nerves. The diffuse potential appears to be conducted throughout the plexus but preferential conducting pathways occur around lesions. Both diffuse and fast potentials show facilitation of response to repeated stimulation. Facilitation can be demonstrated in the presence of high Mg2+ concentrations. Conductance of the diffuse potential also occurs in the presence of high ambient Mg2+. In Ca2+-free medium containing 1-mM EGTA one can also observe facilitatory events. The possibility of Mg2+-insensitive synapses is discussed.     

Influence of medium ionic composition on the electrophysiological properties of the intracardiac-neuron membrane

Replacement of the sodium and chloride ions in the solution bathing a frog heart with an osmotically equivalent sucrose solution leads to hyperpolarization of the neuronal membranes of the intracardiac ganglia and to an increase in their total resistance. The intracardiac neurons continue to respond to stimulation in this solution for some time, producing action-potential spikes, but the amplitude of the potentials is greatly reduced. The neurons also continued to generate action potentials in solutions where the sodium ions were replaced by an osmotically equivalent amount of calcium ions. The amplitude of the potentials increased at first, but action-potential generation was completely inhibited after 14–17 min. The presence of sodium ions in the solution bathing the heart is therefore responsible for development of action potentials in the neurons of the frog intracardiac ganglia.     

Ionic mechanisms of electrical activity in somatic muscle of the nematodeAscaris lumbricoides

Summary The ionic dependence of the myogenic spike potentials and slow waves recorded fromAscaris lumbricoides somatic muscles has been investigated. Spikes appear to be mediated exclusively by calcium ions; the spike active potential varies with calcium concentration as expected for a calcium electrode and spikes persist in sodium-free media (Fig. 2). Slow waves can be mediated either by sodium or calcium; they persist when calcium or sodium are removed separately, but not when both are removed together (Figs. 3, 4, 6).In rhythmically active preparations, a burst of slow waves and spikes accompanies each contraction. Two phenomena may be related to the mechanism of this modulation:1) TEA, although it does not prolong slow waves or spikes, induces rhythmic bursts of activity similar to spontaneous modulation (Fig. 5). This TEA-induced modulation appears to be myogenic. 2) Under conditions where calcium influx is reduced (either by addition of EGTA to the bath or by replacement of calcium with barium or strontium), very long-duration square waves are observed (Figs. 4. 7. 8). The square waves resemble slow waves in their ionic dependence, but differ in their sensitivity to TEA and to variation in the external potassium concentration. It is suggested that modulation and square waves involve the same channels. The significance of these results in understanding the role of myogenic activity in nematode locomotion is discussed.We thank Mr. Mac McGlaughlin for help in obtainingAscaris. This work was supported by a Sloan Foundation grant in Neuroscience and a U.S. Public Health Service grant (NS 09654) to R.L.R., by an NIH Traineeship on grant BCH Tol GM 01262-12 to D.A.W., and by an NIH Postdoctoral Fellowship (1 FO2 GM55347) to L.B.     

The ionic dependence of voltage-activated inward currents in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of Caenorhabditis elegans consists of a syncytium of radially orientated muscle cells that contract synchronously and rhythmically to ingest and crush bacteria and pump them into the intestine of the animal. The action potentials that support this activity are superficially similar to vertebrate cardiac action potentials in appearance with a long, calcium-dependent plateau phase. Although the pharyngeal muscle can generate action potentials in the absence of external calcium ions, action potentials are absent when sodium is removed from the extracellullar solution (Franks et al. 2002). Here we have used whole cell patch clamp recordings from the pharynx and show low voltage-activated inward currents that are present in zero external calcium and reduced in zero external sodium ions. Whilst the lack of effect of zero calcium when sodium ions are present is not surprising in view of the known permeability of voltage-gated calcium channels to sodium ions, the reduction in current in zero sodium when calcium ions are present is harder to explain in terms of a conventional voltage-gated calcium channel. Inward currents were also recorded from egl-19 (n582) which has a loss of function mutation in the pharyngeal L-type calcium channel and these were also markedly reduced in zero external sodium. Despite this apparent dependence on external sodium ions, the current was partially blocked by the divalent cations, cadmium, barium and nickel. Using single-channel recordings we identified a cation channel for which the open-time duration was increased by depolarisation. In inside-out patches, the single-channel conductance was highest in symmetrical sodium solution. Further studies are required to determine the contribution of these channels to the pharyngeal action potential.     

A Quick Preparative Method for Electron Microscopy Observations of Delicate Objects Using Alginate Embedding Medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

A quick preparative method for electron microscopy observations of delicate objects using alginate embedding medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

Calcium-activated conductance in skate electroreceptors: current clamp experiments

When current clamped, skate electroreceptor epithelium produces large action potentials in response to stimuli that depolarize the lumenal faces of the receptor cells. With increasing stimulus strength these action potentials become prolonged. When the peak voltage exceeds about 140 mV the repolarizing phase is blocked until the end of the stimulus. Perfusion experiments show that the rising phase of the action potential results from an increase in calcium permeability in the lumenal membranes. Perfusion of the lumen with cobalt or with a zero calcium solution containing EGTA blocks the action potential. Perfusion of the lumen with a solution containing 10 mM Ca and 20 mM EGTA initially slows the repolarizing process at all voltages and lowers the potential at which it is blocked. With prolonged perfusion, repolarization is blocked at all voltages. When excitability is abolished by perfusion with cobalt, or with a zero calcium solution containing EGTA, no delayed rectification occurs. We suggest that repolarization during the action potential depends on an influx of calcium into the cytoplasm, and that the rate of repolarization depends on the magnitude of the inward calcium current. Increasingly large stimuli reduce the rate of repolarization by reducing the driving force for calcium, and then block repolarization by causing the lumenal membrane potential to exceed ECa. Changes in extracellular calcium affect repolarization in a manner consistent with the resulting change in ECa.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.

Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of bursting in a thalamic neuron model

We extend a quantitative model for low-voltage, slow-wave excitability based on the T-type calcium current (Wang et al. 1991) by juxtaposing it with a Hodgkin-Huxley-like model for fast sodium spiking in the high voltage regime to account for the distinct firing modes of thalamic neurons. We employ bifurcation analysis to illustrate the stimulus-response behavior of the full model under both voltage regimes. The model neuron shows continuous sodium spiking when depolarized sufficiently from rest. Depending on the parameters of calcium current inactivation, there are two types of low-voltage responses to a hyperpolarizing current step: a single rebound low threshold spike (LTS) upon release of the step and periodic LTSs. Bursting is seen as sodium spikes ride the LTS crest. In both cases, we analyze the LTS burst response by projecting its trajectory into a fast/slow phase plane. We also use phase plane methods to show that a potassium A-current shifts the threshold for sodium spikes, reducing the number of fast sodium spikes in an LTS burst. It can also annihilate periodic bursting. We extend the previous work of Rose and Hindmarsh (1989a–c) for a thalamic neuron and propose a simpler model for thalamic activity. We consider burst modulation by using a neuromodulator-dependent potassium leakage conductance as a control parameter. These results correspond with experiments showing that the application of certain neurotransmitters can switch firing modes. Received: 18 July 1993/Accepted in revised form: 22 January 1994     

Induction of a sodium-dependent depolarization by external calcium removal in human sperm

Removal of external calcium with EGTA (from 2.5 mm to nanomolar levels) caused a remarkable depolarization in human sperm. This depolarization was initially fast. It was followed by a slow phase that brought the Vm to values of over 0 mV in 1-2 min. The slow and sustained phase correlated with a sustained decrease in intracellular calcium. However, calcium removal still induced depolarization in sperm with enhanced intracellular calcium (induced by progesterone), indicating that the sustained depolarization was not caused by a sustained intracellular calcium decrease. The depolarization was reduced as the external sodium content was substituted with choline, indicating that it was due to a sodium current, and was observed in lithium but not in tetramethylammonium-containing medium. In low sodium medium, the addition of sodium after calcium removal induced depolarization to the extent of which slightly increased in 2 min. The depolarization was completely inhibited by external magnesium (Ki = 1.16 mm). The addition of calcium or magnesium to calcium removal-induced depolarized sperm induced hyperpolarization that was inhibited by ouabain and was also prevented in medium without potassium, suggesting that the activity of the electrogenic Na+,K+-ATPase was involved. The conductance activated by calcium removal might unveil the presence of a calcium channel that in the absence of external calcium allows sodium permeation and that in normal conditions might contribute to the resting intracellular calcium concentration.     

Calcium spikes in cultured human reticular cells from peritoneal exudates

Intracellular recordings of cultured human peritoneal exudate cells reveal that cells within the culture exhibit an active depolarizing response to injected currents which can reach positive potentials and resemble slow spikes. The cells exhibiting spikes are similar to the reticular cells described by Stuart and Davidson (1971a,b) in that they are esterase(+), acid phosphatase(+), and internalize colloidal carbon but not opsonized red blood cells. The active depolarizing response is unaffected by either decreasing the external sodium concentration or by adding tetrodotoxin (3 X 10(-5) M), whereas increasing the external calcium concentration increases both the spike amplitude and rate of rise, and the addition of cobalt (3 mM) blocks the response. Addition of barium increases the duration and amplitude of the spikes but reduces the afterhyperpolarization. The data indicate that cultured human reticular cells from the peritoneal cavity exhibit a calcium spike.     

Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking

Gelsolin is a calcium binding protein that shortens actin filaments. This effect occurs in the presence but not in the absence of micromolar calcium ion concentrations and is partially reversed following removal of calcium ions. Once two actin molecules have bound to gelsolin in solutions containing Ca2+, one of the actins remains bound following chelation of calcium, so that the reversal of gelsolin's effect cannot be accounted for simply by its dissociation from the ends of the shortened filaments to allow for elongation. In this paper, the interactions with actin of the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stable 1:1 gelsolin-actin complexes are compared with those of free gelsolin. The abilities of free or complexed gelsolin to sever actin filaments, nucleate filament assembly, bind to the fast growing (+) filament ends, and lower the filament size distribution in the presence of either Ca2+ or EGTA were examined. The results show that both free gelsolin and gelsolin-actin complexes are highly dependent on Ca2+ concentration when present in a molar ratio to actin less than 1:50. The gelsolin-actin complexes, however, differ from free gelsolin in that they have a higher affinity for (+) filament ends in EGTA and they cannot sever filaments in calcium. The limited reversal of actin-gelsolin binding following removal of calcium and the calcium sensitivity of nucleation by complexes suggest an alternative to reannealing of shortened filaments that involves redistribution of actin monomers and may account for the calcium-sensitive functional reversibility of the solation of actin by gelsolin.     

Heterogeneity of brush-border-membrane vesicles from rat small intestine prepared by a precipitation method using Mg/EGTA

Brush border membrane vesicles from rat small intestine were isolated by a Mg/EGTA precipitation method. Further fractionation either by free flow electrophoresis or by sucrose density gradient centrifugation leads to subfractions which differ with respect to enzyme enrichment factors, transport properties for D-glucose and protein pattern analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A relative enrichment of (Na+ + K+)-ATPase is found in one fraction, whereas in another fraction maltase, aminopeptidase M and alkaline phosphatase are relatively enriched. The fractions show different properties of D-glucose transport under tracer exchange conditions and a different inhibition of D-glucose transport by phlorizin and phloretin. These results indicate that the vesicles obtained from rat small intestine by this cation precipitation method are not homogeneous. The inhomogeneity cannot be due to a crosscontamination by membranes other than from the cell envelopment, as none of the fractions show a significant enrichment of succinate--cytochrome c oxidoreductase, KCN-resistant NADH oxidoreductase or glucosaminidase. The inhomogeneity might be due either to a crosscontamination by basal-lateral membranes or to membranes derived from epithelial cells not yet fully differentiated.     

The influence of calcium-free EGTA solution upon membrane permeability in the crystalline lens of the frog

Potential difference, resistance, cation content, and 86Rb efflux were measured in frog lenses maintained in normal or calcium-free EGTA Ringer's solution. Exposure of the lens to calcium-free solution resulted in a rapid fall in potential and resistance, together with a twofold increase in 86Rb efflux rate. These rapid changes were not due to an alteration in cation distribution between the lens and its environment. However, the alteration in 86Rb efflux rate could be explained on the basis of the fall in potential. These findings suggested that removal of calcium from the bathing medium caused a rapid increase in sodium permeability alone. This suggestion was substantiated by the results of experiments where the response of the lens to low calcium solution was determined in a medium in which 90% of the sodium had been replaced by sucrose.     

Expanding the range of free calcium regulation in biological solutions

Many biological systems use ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca(2+)](free)) in the presence of physiological levels of free Mg(2+) ([Mg(2+)](free)). Frequently, it is necessary to work at [Ca(2+)](free) beyond EGTA's buffering capabilities. Therefore, we have developed methods to extend the buffering range by adding nitrilotriacetic acid (NTA) to solutions containing EGTA. This extension results from NTA having a lower K'(dCa) than EGTA. Such equilibria are solved by pCa Calculator, a computer program designed to aid in the study of Ca(2+)-dependent physiological processes while accounting for the effects of pH, temperature, and ionic strength. With multiple chelators and pH buffers from which to choose, pCa Calculator calculates the total concentration of each species required to achieve specified free concentrations of Ca(2+), ATP, and Mg(2+). The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP(2-)] and ionic strength. Moreover, it can account for increases in experimental volume from calcium addition. A comparative analysis is reported for testing solutions in the presence and absence of NTA by measuring the calcium binding affinity of fluorescent cardiac troponin C. These findings demonstrate that EGTA, when used in conjunction with NTA, improves and expands the regulation of free calcium in solution.     

Phenobarbital Increases Spontaneous Transmitter Release at the Frog Neuromuscular Junction

Phenobarbital (1-2 × 10^-4M) markedly increases the frequency of miniature end-plate potentials at the neuromuscular synapse of the frog. This effect was seen in calcium free media containing EGTA. The drug probably acts presynaptically at an intracellular locus to increase the presynaptic free calcium concentration.     

On the effects of divalent cations and ethylene glycol-bis-(beta- aminoethyl ether) N,N,N'',N''-tetraacetate on action potential duration in frog heart

Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or without ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) prolongs the action potential (AP). The change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 min. This prolongation can be reversed by other divalent cations in EGTA (Mg2+, Sr2+) or Ca-free (Mn2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. The cell membrane does not become excessively "leaky" in EGTA solutions.