Injection Of Tetraethylammonium Research Articles

Effects of dietary or injected organic cations on larvalDrosophila melanogaster: Mortality and elimination of tetraethylammonium from the hemolymph

This study of larval Drosophila melanogaster examined the effects of injecting the prototypical organic cation tetraethylammonium (TEA) into the hemocoel or adding TEA and/or other organic cations to the diet. Mortality, hemolymph TEA levels, and Malpighian tubule TEA secretion rates were measured. The LD50 for dietary TEA was 158.4 mM and mortality increased if competitive inhibitors of organic cation transporters were also included in the diet. Mortality increased from 24% on TEA (100 mM) alone to 83 and 67% when the diet contained both TEA and quinidine (10 mM) or cimetidine (100 mM), respectively. TEA-selective microelectrode measurements indicated that hemolymph TEA concentration was approximately 3% of that in the diet for larvae maintained on TEA-enriched diet for 24 h. Malpighian tubules isolated from larvae exposed to dietary TEA excreted more TEA than did tubules from controls fed a TEA-free diet. However, the rate of decline of hemolymph TEA concentration following ingestion or injection of TEA into the hemocoel was greater than that explicable by rates of active transport by the gut and Malpighian tubules (MTs). We propose that TEA concentrations in the hemolymph are reduced not only by active transport across the MTs and gut, but also by diffusion into the gut. The latter pathway is particularly important when larvae previously maintained upon TEA-enriched diet are transferred to a TEA-free diet. The ingestion of TEA-free food not only clears the gut lumen, but also creates a TEA-free compartment into which TEA may passively diffuse from the hemolymph.

Effect of tetraethylammonium intracellular microiontophoretic injection on activity of the crayfish stretch receptor

Our investigation of the slow-adapting neurons of the crayfish abdominal receptor organ has revealed that intracellular microiontophoretic injection of tetraethylammonium induces no changes in the receptor potential of the neurons. The results obtained show that tetraethylammonium-sensitive potassium channels are not important for adaptation manifested by primary receptor responses of mechanosensitive neurons.

Guanosine 5'-triphosphate analogue activates potassium current modulated by neurotransmitters in Aplysia neurones.

1. Identified neurones in the abdominal ganglion of Aplysia californica were voltage clamped in order to investigate how guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), a GTP analogue that irreversibly activates guanine nucleotide-binding (G) proteins, modifies activation by the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) of a slow K+ current resembling the serotonin- and adenosine 3',5'-cyclic monophosphate (cyclic AMP)-sensitive 'S' current, and a similar response to acetylcholine. 2. Ionophoretic or pressure injection of GTP-gamma-S into the cell triggered the slow and irreversible development of a large K+ current, rendered the K+ current responses to FMRFamide and acetylcholine irreversible, and finally, once the GTP-gamma-S-induced current had fully developed, occluded the neurotransmitter responses altogether. 3. The K+ currents activated by GTP-gamma-S and acetylcholine had properties identical to those previously found for the FMRFamide-induced 'S'-like K+ current: they were Ca2+ and voltage independent, relatively insensitive to block by extracellular tetraethylammonium (TEA) and 4-aminopyridine (when high concentrations of acetylcholine were used to overcome an additional block by these agents of the receptor), and suppressed in Ba2+-containing solution, by injection of TEA+ or Cs+ into the cell, and by serotonin and elevation of the intracellular concentration of cyclic AMP. 4. The K+ current responses to FMRFamide and acetylcholine were not additive when the agonist concentrations used were high enough to activate most of the available current. 5. Desensitization of either response did not affect the other, and the effect of acetylcholine, but not that of FMRFamide, could be blocked by the known acetylcholine-receptor blockers phenyltrimethylammonium and TEA. 6. These results suggest that FMRFamide and acetylcholine, acting through different receptors, activate the same 'S'-like K+ current by a mechanism involving a G protein. 7. In addition to activating the slow K+ current, FMRFamide and acetylcholine each activate a faster current in these cells, carried by Na+ in the case of FMRFamide, and by Cl- in the case of acetylcholine. Neither fast response was affected by GTP-gamma-S.

Acetylcholine suppresses calcium current in neurons of Aplysia californica

1. The left upper quadrant neurons L2-L6 in the abdominal ganglion of Aplysia californica were voltage clamped in order to examine effects of acetylcholine on voltage-dependent Ca and Ca-dependent K currents. 2. "Puffed" application of 10-100 microM acetylcholine reduced both the early inward and late outward phases of the current elicited by depolarizing voltage steps. An identical effect of the peptide FMRFamide was previously found to result from a suppression of the Ca and Ca-dependent K currents. 3. This effect of acetylcholine was obscured by the simultaneous activation of a previously described K current resembling the "S" current. Extracellular tetraethylammonium (TEA) and 4-aminopyridine could not be used to eliminate this current, because both compounds also appeared to block the acetylcholine receptor mediating the putative suppression of Ca and Ca-dependent K currents. 4. The acetylcholine-induced "S"-like and other K currents could, however, be reduced or eliminated by injection of TEA+ or Cs+ into the cell, replacement of extracellular Ca2+ with Ba2+, and by shifting the K+ equilibrium potential so as to null K currents at the potential used to record Ca current, revealing in each case a partial (10-40%) suppression of the Ca (or Ba) current by acetylcholine. 5. The reduction of the outward phase of depolarization-activated current was confirmed to represent suppression of the Ca-dependent K current by acetylcholine. This effect was indirect, secondary to the suppression of Ca current, since acetylcholine had no effect on Ca-dependent K current elicited by direct injection of Ca2+ into the cell. 6. Activation of the "S"-like K current and suppression of the Ca current by FMRFamide are likely to be important in its proposed role as an agent of presynaptic inhibition in Aplysia. Since acetylcholine has identical effects, it too may have such a function.

Intracellular tetraethylammonium ions enhance group Ia excitatory post‐synaptic potentials evoked in cat motoneurones.

Single fibre group Ia excitatory post-synaptic potentials (e.p.s.p.s) were recorded in cat spinal motoneurones after the neurones were injected with tetraethylammonium (TEA) ions. TEA injection increased the peak amplitude of most e.p.s.p.s. The time course of e.p.s.p.s generated at the soma was unaffected, but the time course of e.p.s.p.s generated in the dendrites was prolonged. The membrane time constant did not change after TEA injection. Somatic e.p.s.p.s were voltage clamped after TEA was injected. The reversal potential for these e.p.s.p.s was more positive than for e.p.s.p.s unaffected by TEA. Composite e.p.s.p.s added linearly, or greater than linearly, whereas in motoneurones without TEA they added linearly or less than linearly. The enhanced amplitude and prolonged time course observed in dendritic e.p.s.p.s after TEA injection was reduced by small hyperpolarizing currents. Greater than linear summation of composite e.p.s.p.s was converted to linear summation by small hyperpolarizing currents. The increase in somatic e.p.s.p.s was attributed to a more positive reversal potential for the e.p.s.p.s. We suggest that TEA decreases the relative permeability of K+ in the subsynaptic channels. We propose that in the presence of TEA, dendritic depolarization activates an inward current which amplifies and prolongs synaptic potentials spreading towards the soma.

Two components of Ca-dependent potassium current in identified neurones ofAplysia californica

Outward tail currents measured in Aplysia neurones after termination of depolarizing voltage-clamp pulses consist of rapidly decaying voltage-dependent K currents and slow tail currents of much slower time course. The rapidly decaying voltage-dependent tail currents were blocked with aminopyridines, and measurements of the slow tail currents were made following decay of any residual rapid tail currents. The slow tail current exhibited two components of differing sensitivity to externally applied tetraethylammonium (TEA) ions. In some neurones of the abdominal ganglion (L-2, L-4), virtually all of the slow tail current was resistant to blockage by TEA, while in others (L-3, L-6) 80% or more of the slow tail current was blocked by low TEA concentrations (KD less than 1 mM), the remaining slow tail current being resistant to TEA. This TEA-resistant slow tail current was identified as a K current because it reversed near the K equilibrium potential (EK), the reversal potential was shifted by changes in the external K concentration, and it could be blocked by injection of Cs+. It was abolished by replacement of external Ca2+ by Co2+ or Ba2+, by addition of Cd2+, or by injection of EGTA, and thus determined to be a Ca-dependent current. Intracellular injection of TEA or external application of aminopyridine or apamine had little or no effect on the TEA-resistant slow tail current. Quinidine reduced the TEA-sensitive, but not the TEA-resistant current. Both the TEA-sensitive and the TEA-resistant components of the slow tail current exhibited similar time courses of decay.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of tetraethylammonium on potassium currents in a molluscan neurons.

The effects of tetraethylammonium (TEA) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free ASW containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External TEA blocks the delayed K+ current reversibly in a dose-dependent manner. The experimental results are well fitted with a Michaelis-Menten expression, assuming a one-to-one reaction between TEA and a receptor site, with an apparent dissociation constant of 6.0 mM. The block depends on membrane voltage and is reduced at positive membrane potentials. The Ca2+-activated K+ current was measured in Ca2+-free artificial seawater (ASW) containing TTX, using internal Ca2+ ion injection to directly activate the K+ conductance. External TEA and a number of other quaternary ammonium ions block the Ca2+-activated K+ current reversibly in a dose-dependent manner. TEA is the most effective blocker, with an apparent dissociation constant, for a one-to-one reaction with a receptor site, of 0.4 mM. The block decreases with depolarization. The Ca2+-activated K+ current was also measured after intracellular iontophoretic TEA injection. Internal TEA blocks the Ca2+-activated K+ current (but the block is only apparent at positive membrane potentials), is increased by depolarization, and is irreversible. The effects of external and internal TEA can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW.

Amplitude and rate of decay of post-tetanic potentiation are controlled by different mechanisms

Evidence is presented that post-tetanic potentiation (PTP) of the cholinergic, fast, C1 − dependent IPSP seen in cell L5 of the abdominal ganglion of Aplysia californica upon eliciting a spike in cell L10 is due to an increase in spike-evoked transmitter release. The magnitude of the post-tetanic change in spike-evoked release is inversely correlated with the amount of transmitter released by an isolated presynaptic spike. This was found whether the latter was increased by injection of tetraethyl ammonium (TEA) into the soma of L10 or decreased by hyperpolarization of the soma of L10. Neither of these manipulations affected the rate of decay of PTP. The magnitude of PTP was increased and the rate of decay reduced by increasing either the number of frequency of stimuli in the tetanus. Under all conditions PTP decayed with a single exponential time course, asymptotically approaching the unpotentiated magnitude. It is concluded that while both the amplitude and rate of decay of PTP are affected by the frequency and number of stimuli in the tetanus, the underlying mechanism controlling the amplitude of PTP is different from the mechanism controlling the rate of decay of PTP.

Synaptic connexions and related postsynaptic pharmacology studied in the cerebral ganglion of aplysia

Interneuronal connexions have been studied within the cerebral ganglion of Aplysia californica. Excitatory monosynaptic connexions between two groups of cells located on the dorsal surface of the ganglion near the cerebropleural connectives were analyzed in detail. The monosynapticity of these connexions was established not only by the strict one-to-one correlation between presynaptic action potential and excitatory postsynaptic response (EPSP) and the constant latency for any given cell pair, but also by the following criteria: (a) gradual change in the EPSP following tetraethylammonium injection into the presynaptic neurone, (b) sustained EPSP in the presence of a high external calcium ion concentration, (c) sensitivity of the EPSP amplitude to presynaptic polarization. Iontophoretic application of acetylcholine, 5-hydroxytryptamine and glutamate on the postsynaptic cells elicited excitatory responses in many cases. Inhibitory responses were obtained by local iontophoresis of dopamine, γ-aminobutyric acid and occasionally also by acetylcholine. The only agent found to block the EPSP was bufotenine, which also readily blocked the 5-hydroxytryptamine response. Bufotenine was completely ineffective on the acetylcholine or glutamate excitatory responses. Of the various cholinolytics tested, none had an effect on the EPSP. Our data all point to 5-hydroxytryptamine as a transmitter in the studied synaptic connexions. However, it must be emphasized that in the absence of biochemical and histological evidence the role of the 5-hydroxytryptamine cannot be regarded as conclusive.

Further studies of periodic miniature response in squid giant axons.

Electric responses of extremely small amplitudes (1-30 muV peak-to-peak) repeating with more-or-less definite periodicity could be induced by a variety of chemical stimulants applied to squid giant axons either extracellularly or intracellularly. The chemical stimulants studied include allethrin, aminopyridines, N-bromosuccinimide, dimethylamino-pyridine, glutaraldehyde, osmium tetroxide, parachloromercuribenzoate, rose bengal, scorpion venoms and veratridine. With several mild stimulants, it was possible to evoke periodic miniature responses unaccompanied by a fall in the resting membrane potential. The frequency of miniature responses could be lowered by intracellular injection of TEA (tetraethylammonium). These miniature responses could readily be suppressed by external application of TTX (tetrodotoxin). The miniature responses evoked by 4-aminopyrine were characterized by a large variation in the frequency of responses. Cross-linking of the membrane proteins with a dilute solution of glutaraldehyde produced miniature responses repeating at progressively falling frequencies. Analyses of the processes of production of miniature responses with these and other stimulants have clarified several aspects of the physicochemical properties of the excitable sites of the axon membrane.

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia.

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...

Monosynaptic connexions among Aplysia neurones examined by the intracellular application of tea.

1. Several assumptions underlying the use of intracellularly applied tetraethylammonium (TEA) for assessing monosynaptic connexions were evaluated in identified neurones of Aplysia. 2. In the R2 neurons, intrasomatic TEA application prolongs the duration of the intrasomatically recorded action potential. Subsequently, the action potential in the axon of R2, recorded extracellularly 4-7 mm from the soma, was also prolonged. 3. Intracellular application of TEA into the somata of the multi-action interneurone L10 enhances the duration of the L10 AP and results in larger and more prolonged post-synaptic potentials (p.s.p.s) recorded from neurones believed to be connected monosynaptically with L10. The action potential duration and wave form of p.s.p.s elicited by nerve stimulation in these same post-synaptic neurones were unaffected during the time L10-mediated p.s.p.s were potentiated. 4. Following TEA injection into L10 the p.s.p. recorded in neurone L7 changes wave form in a manner similar to that observed when L10 is tetanized. 5. It is concluded that TEA migrates from its intracellular site of application, does not leave the injected neurone in significant quantities, and alters the wave form of the p.s.p in only those neurones connected monosynaptically to the injected neurone.

Effects of trans-membrane polarization and TEA injection on monosynaptic actions from motor cortex, red nucleus and group Ia afferents on lumbar motoneurons in the monkey.

Abstract The monosynaptic EPSPs evoked from supraspinal structures and muscle afferents in alpha-motoneurons were investigated in the rhesus monkeys with the aid of intracellular recording. There was a statistically significant difference in the time course of cortico-motoneuronal, rubro-motoneuronal and group Ia monosynaptic EPSPs but a relatively small difference between these inputs in the sensitivity to the artificial polarization. No correlation was found between the sensitivity of monosynaptic actions to injected currents and the rise time of EPSP. Tetraethylammonium (TEA) injection into the neuroplasm induced a large increase in the ability of the applied currents to affect the magnitude of the monosynaptic EPSPs, and shifted the extrapolated equilibrium potential in the negative direction. TEA accentuated the trend of cortico-motoneuronal EPSPs toward a more negative equilibrium potential as compared with the group Ia and rubro-motoneuronal EPSPs recorded in the same cell. The early phase of all monosynaptic EPSPs could not be reversed by depolarizing currents in normal and TEA-treated motoneurons and when superimposed on the plateau of depolarization of TEA-prolonged action potential. The results are discussed in relation to the ionic mechanisms of excitatory subsynaptic membrane and to the location of the synaptic sites.

Electrical interactions between normal and TEA-treated zones of squid axon.

By stepwise injection of tetraethylammonium (TEA) chloride into a squid giant axon, it was shown that the degree of prolongation of the action potential by TEA depends on the length of the TEA-treated zone of the axon. Near the boundary between normal and TEA-treated zones of the axon, oscillatory potentials were often observed during the plateau of the prolonged action potential. It was shown that repetitive firing of impulses at the boundary originated in the TEA-treated zone, not in the normal zone. At the boundary between normal and TEA-treated zones, where the concentration of TEA changed continuously, there was a discontinuous change in duration of the action potential, indicating the existence of a sharp delineation between the zones developing long and short responses. Propagation of abolition of an action potential was demonstrated in uniformly TEA-treated axons.

The Action of Tetraethylammonium (TEA) on the Kitten Diaphragm

Summary.1. The action of tetraethylammonium (TEA) on the contractions of the isolated phrenic nerve diaphragm preparation from kittens has been investigated and compared with the action on other mammalian nerve‐muscle preparations.2. TEA in a concentration of 1 mM increased the height of isotonically recorded muscle twitches about 20 to 30% after stimulation of the muscle both indirectly and directly after full curarization. No change in the muscle action potentials was observed after addition of TEA to a concentration of 1 mM. The β‐hydroxy‐derivative of TEA had a similar but slightly less potent effect on the twitch height.3. Addition of TEA to the bath fluid of the kitten diaphragm increased the isometric peak twitch tension and the time from stimulus to twitch peak without significant increase in the rate of rise of tension. The maximal tetanic tension was unaltered or lowered after addition of TEA. These changes can be accounted for by a prolongation of the active state of the muscle by TEA.4. TEA in a concentration of 1 mM did not significantly change the twitch height of isolated diaphragm preparations from rats, young guinea pigs or young rabbits. Injection of TEA (5 mg/kg) in rabbits and cats did not increase the twitch height of the hind limb muscles. The respiratory excursions of rabbits were unchanged while those of the cat increased markedly after this dose of TEA. It is concluded that the diaphragm muscle of kittens and of cats is more sensitive to TEA than the other mammalian muscles investigated.

Electrical responses of frog spinal ganglion cells in sodium-free media.

SINCE Lorente de No1 found that a group of small axons (“Et fibres”) in the frog are able to conduct prolonged action potentials when the external sodium is replaced by certain quaternary ammonium ions, special attention has been directed to the effects of these compounds on other excitable tissues. Fatt and Katz2 observed that the electrical responses of crustacean muscle fibres are maintained and even intensified when choline, tetramethylammonium, tetraethylammonium or tetrabutylammonium ions are substituted for the sodium in the external medium. No other excitable tissue, however, has been reported to show active responses after complete replacement of sodium by these ions. Choline, in particular, has repeatedly been used as an ‘inert’ osmotic substitute in experiments on sodium depletion in the extracellular spaces. At low concentrations, for example, 10–20 per cent replacement of sodium, tetraefchylammonium lengthens the falling phase of the action potential of crustacean nerve3 and of frog twitch muscle4. The intracellular injection of tetraethylammonium into the giant axon of the squid prolongs the duration of the action potential5. In frog nerve fibres, the enhancement of the negative after-potential produced by addition of tetraethylammonium to the Ringer's solution is more prominent in the terminal endings than in the nodes6, suggesting that quaternary ammonium ions might act in different degrees on the various parts of the neurone.

The action of tetraethylammonium (TEA) on the rat diaphragm.

Summary. 1. The action of tetraethylammonium (TEA) on the contractions of the isolated phrenic nerve diaphragm preparation from kittens has been investigated and compared with the action on other mammalian nerve-muscle preparations. 2. TEA in a concentration of 1 mM increased the height of isotonically recorded muscle twitches about 20 to 30% after stimulation of the muscle both indirectly and directly after full curarization. No change in the muscle action potentials was observed after addition of TEA to a concentration of 1 mM. The β-hydroxy-derivative of TEA had a similar but slightly less potent effect on the twitch height. 3. Addition of TEA to the bath fluid of the kitten diaphragm increased the isometric peak twitch tension and the time from stimulus to twitch peak without significant increase in the rate of rise of tension. The maximal tetanic tension was unaltered or lowered after addition of TEA. These changes can be accounted for by a prolongation of the active state of the muscle by TEA. 4. TEA in a concentration of 1 mM did not significantly change the twitch height of isolated diaphragm preparations from rats, young guinea pigs or young rabbits. Injection of TEA (5 mg/kg) in rabbits and cats did not increase the twitch height of the hind limb muscles. The respiratory excursions of rabbits were unchanged while those of the cat increased markedly after this dose of TEA. It is concluded that the diaphragm muscle of kittens and of cats is more sensitive to TEA than the other mammalian muscles investigated.

Renal Hemodynamic Effects of Adrenaline and "Isuprel": Potentiation of Effects of Both Drugs by Tetraethylammonium

Comment and SummaryThe increased pressor responsiveness to adrenaline caused by injection of Et4N is shown to be associated with an increase in renal vasoconstriction. The depressor response to injection of “Isuprel,” the N-isopropyl homologue of adrenaline, is shown to be associated with renal vasodilation. The depressor, cardioaccelerator and renal vasodilator effects of injection of “Isuprel” are augmented by prior injection of tetraethylammonium. On the assumption that the effects of tetraethylammonium are due to blockinq of autonomic ganglia,4 we conclude that inhibition of these ganglia increases vascular responsiveness to typical vasoconstrictor and vasodilator sympatheticomimetics.