Nerve Terminal Action Potential Research Articles

Effects of odontobuthus doriae scorpion venom on mouse sciatic nerve.

Temporary paralysis is a rare manifestation of envenoming following the yellow Iranian scorpion, Odontobuthus doriae (O. doriae). Thus, to elucidate the underlying mechanism, we investigated the neurotoxic effect of venom in the sciatic nerve, the possible mechanism in a mice model. The neurotoxicity and temperature effects in the venom-induced neurotoxicity were examined using the mouse sciatic nerve and mouse phrenic nerve-hemidiaphragm (MHD) preparations. O .doriae venom (1 μg/mL) caused changes in the perineural waveform associated with nerve terminal action potentials. Venom affected on both negative and positive components of the waveform which is known as a compound action potential. The timeresponse relationship of venom-induced depression of resting membrane potential (RMP) was significant (p < 0.05). No significant difference in augmentation was seen in room temperature in comparison with 37°C. In conclusion, although there was no evidence that the venom had any specific curarizing action at the neuromuscular junction, the results suggest that the venom exerts its neuromuscular transmission on the sciatic nerve through potassium and sodium ionic-currents. Furthermore, the influence of temperature on neurotoxicity was ineffective on blockade of the neuromuscular transmission in-vitro.

Presynaptic Resurgent Na+Currents Sculpt the Action Potential Waveform and Increase Firing Reliability at a CNS Nerve Terminal

Axonal and nerve terminal action potentials often display a depolarizing after potential (DAP). However, the underlying mechanism that generates the DAP, and its impact on firing patterns, are poorly understood at axon terminals. Here, we find that at calyx of Held nerve terminals in the rat auditory brainstem the DAP is blocked by low doses of externally applied TTX or by the internal dialysis of low doses of lidocaine analog QX-314. The DAP is thus generated by a voltage-dependent Na(+) conductance present after the action potential spike. Voltage-clamp recordings from the calyx terminal revealed the expression of a resurgent Na(+) current (I(NaR)), the amplitude of which increased during early postnatal development. The calyx of Held also expresses a persistent Na(+) current (I(NaP)), but measurements of calyx I(NaP) together with computer modeling indicate that the fast deactivation time constant of I(NaP) minimizes its contribution to the DAP. I(NaP) is thus neither sufficient nor necessary to generate the calyx DAP, whereas I(NaR) by itself can generate a prominent DAP. Dialysis of a small peptide fragment of the auxiliary β4 Na(+) channel subunit into immature calyces (postnatal day 5-6) induced an increase in I(NaR) and a larger DAP amplitude, and enhanced the spike-firing precision and reliability of the calyx terminal. Our results thus suggest that an increase of I(NaR) during postnatal synaptic maturation is a critical feature that promotes precise and resilient high-frequency firing.

Prejunctional and postjunctional actions of heptanol and 18β-glycyrretinic acid in the rodent vas deferens

Heptanol and 18β-glycyrrhetinic acid (18βGA) block gap junctions, but have other actions on transmitter release that have not been characterised. This study investigates the prejunctional and postjunctional effects of these compounds in guinea pig and mouse vas deferens using intracellular electrophysiological recording and confocal Ca2+ imaging of sympathetic nerve terminals. In mice, heptanol (2 mM) reversibly decreased the amplitude of purinergic excitatory junction potentials (EJPs; 52±5%, P<0.05) while having little effect on spontaneous excitatory junction potentials (sEJPs). Heptanol (2 mM) reversibly abolished the nerve terminal Ca2+ transient in 52% of terminals. 18βGA (10 μM) decreased the mean EJP amplitude, and increased input resistance in both mouse (137±17%, P<0.05) and guinea pig (354±50%, P<0.001) vas deferens indicating gap junction blockade. Further, 18βGA increased the sEJP frequency significantly in guinea pigs (by 71±25%, P<0.05) and in 5 out of 6 tissues in mice (19±3%, P<0.05). Moreover, 18βGA depolarised cells from both mice (11±1%, P<0.01) and guinea pigs (8±1%, P<0.005). Therefore, we conclude that heptanol (2 mM) decreases neurotransmitter release (given the decrease in EJP amplitude) by abolishing the nerve terminal action potential in a proportion of nerve terminals. 18βGA (10 μM) effectively blocks the gap junctions, but the increase in sEJP frequency suggests an additional prejunctional effect, which might involve the induction of spontaneous nerve terminal action potentials.

The effect of the venom of the yellow Iranian scorpion Odontobuthus doriae on skeletal muscle preparations in vitro

The yellow Iranian scorpion Odontobuthus doriae can cause fatal envenoming, but its mechanism of action is unclear. One of the reported manifestations of envenoming is moderate to severe involuntary tremor of skeletal muscle. In order to understand better the mechanism of action of this venom on skeletal muscle function, we examined the effects of the venom in vitro on chick biventer cervicis (CBC) and mouse hemidiaphragm (MHD) nerve muscle preparations. O. doriae venom (0.3–10 μg/ml) initially increased and then decreased twitch height. The venom also caused contracture in both preparations. In mouse triangularis sterni preparations, used for all intracellular recording techniques, the venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials and endplate potentials in response to single-shock stimulation. With extracellular recording techniques, scorpion venom (1 μg/ml) was found to cause changes to the perineural waveform associated with nerve terminal action potentials consistent with effects on Na + and K + currents. The main facilitatory effects of O. doriae venom are likely to be due to toxins that affect Na + channels in nerve–muscle preparations similar to most Old World scorpion venoms, but blocking effects on K + channels are also possible. Such effects could lead to initial enhancement of transmitter release that could underlie the muscle tremors seen in victims. Toxins acting on Na + and K+ currents have been isolated from the venom [Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E., Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat, J., 2005. OD1, the first toxin isolated from the venom of the scorpion Odontobuthus doriae active on voltage-gated Na + channels. FEBS Lett. 579, 4181–4186; Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian scorpion Odontobuthus doriae. FEBS Lett. 580, 6254–6258]; however, the muscle paralysis seen at higher concentrations of venom may be due to additional, as yet uncharacterised, components of the venom.

Proportion of N-Type Calcium Current Activated by Action Potential Stimuli

N-type calcium currents are important in many neuronal functions, including cellular signaling, regulation of gene expression, and triggering of neurotransmitter release. Often the control of these diverse cellular functions is governed by the spatial and temporal patterns of calcium entry in subcellular compartments. Underlying this issue is the effectiveness of action potentials at triggering calcium channel opening. Chick ciliary ganglion neurons were used as model cells to study the activation of N-type calcium current during action potential depolarization. Several different action potential shapes were recorded, used as voltage command templates, and altered such that control action potential-evoked currents could be compared with those elicited by broadened action potential commands. Depending on the action potential shape used to activate calcium currents in chick ciliary ganglion neurons, and the temperature at which recordings were performed, varying proportions (I/I(max)) of N-type calcium current could be activated. The largest proportion measured occurred using a broad ciliary ganglion cell soma action potential to activate calcium current at 37 degrees C (100%). The smallest proportion measured occurred using a fast, high-temperature-adjusted frog motoneuron nerve terminal action potential to activate calcium current at room temperature (10%). These data are discussed with respect to the impact on cellular signaling and the regulation of transmitter release.

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, Cyclorana alboguttata

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.

Depression by isoflurane of the action potential and underlying voltage-gated ion currents in isolated rat neurohypophysial nerve terminals.

We characterized the effects of the volatile anesthetic isoflurane on the ion currents that contribute to the action potential (AP) in isolated rat neurohypophysial (NHP) nerve terminals using patch-clamp electrophysiology. Mean resting membrane potential and AP amplitude were -62.3 +/- 4.1 and 69.2 +/- 2.9 mV, respectively, in NHP terminals. Two components of outward K(+) current (I(K)) were identified in voltage-clamp recordings: a transient I(K) and a sustained I(K) with minimal inactivation. Some terminals displayed a slowly activating I(K), probably the big Ca(2+)-activated K(+) current (BK). Isoflurane reversibly inhibited AP amplitude and increased AP half-width in normal extracellular Ca(2+) (2.2 mM). In high extracellular Ca(2+) (10 mM), isoflurane also reduced the afterhypolarization peak amplitude. A transient tetrodotoxin-sensitive Na(+) current (I(Na)) was the principal current mediating the depolarizing phase of the AP. A slowly inactivating Cd(2+)-sensitive current (probably a voltagegated Ca(2+) current; I(Ca)) followed the initial I(Na). Isoflurane reversibly inhibited both I(Na) and I(Ca) elicited by a voltage-stimulus based on an averaged AP waveform. The isoflurane IC(50) for AP waveform-evoked I(Na) was 0.36 mM. Isoflurane (0.84 +/- 0.04 mM) inhibited AP waveform-evoked I(Ca) by 37.5 +/- 0.16% (p < 0.05). The isoflurane IC(50) for peak I(K) was 0.83 mM and for sustained I(K) was 0.73 mM, with no effect on the voltage dependence of activation. The results indicate that multiple voltage-gated ion channels (Na(+) > K(+) > Ca(2+)) in NHP terminals, although not typical central nervous system terminals, are inhibited by the volatile general anesthetic isoflurane. The net inhibitory effects of volatile anesthetics on nerve terminal action potentials and excitability result from integrated actions on multiple voltage-gated currents.

An electrophysiological study on the effects of Pa-1G (a phospholipase A 2) from the venom of king brown snake, Pseudechis australis, on neuromuscular function

The effects of Pa-1G, a phospholipase A 2 (PLA 2) from the venom of the Australian king brown snake ( Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58±6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to α-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.

Synchronization of evoked secretion of quanta of mediator as a mechanism facilitating the action of sympathomimetics.

Experiments on frog neuromuscular junction preparations with extracellular recording of nerve terminal action potentials and single-quantum end-plate currents (EPC) were used to assess the time course of evoked quantum secretion of mediator by analyzing histograms of the distribution of true synaptic delays. These studies showed that noradrenaline, isoproterenol, and dobutamine change the kinetics of secretion of quanta, leading to synchronization of the process of mediator release; substances blocking beta-adrenoceptors (atenolol, propranolol) blocked this effect. Clonidine and phenylephrine, which activate alpha-receptors, had no effect on the kinetics of secretion, while the alpha-blocker phentolamine had no effect on the synchronizing action of noradrenaline. Reconstruction of multiquantum EPC from changes in the level of synchronization in the release of individual quanta, showed that EPC amplitude increased in response to noradrenaline by 17%, and that this was due only to alterations in the time course of secretion. These data led to the conclusion that there is a special presynaptic mechanism which facilitates the action of sympathomimetics, acting via beta-adrenoceptors.

Characterisation of the effects of depolarising toxins on nerve terminal action potentials: apparent block of presynaptic potassium currents

Previous studies showed that toxic phospholipases A 2 (Pa-8 and Pa-10F) from the venom of Pseudechis australis, the Australian king brown snake, reduced acetylcholine release at mouse neuromuscular junctions and depressed motor nerve terminal action potentials [ Fatehi et al. (1994a), Toxicon 32, 1559–1572], and it was postulated that these toxins induced their effect on the action potential waveforms through nerve terminal depolarisation. To test this hypothesis, the effects of Pa-11 (another phospholipase A 2 from the venom of Pseudechis australis), and the known depolarising agents, myotoxin a, from the venom of the rattlesnake, Crotalus viridis viridis, and ouabain on these waveforms were compared with the changes induced in the nerve terminal action potentials by Pa-8 and Pa-10F. The experiments were performed on the isolated mouse triangularis sterni preparation, using extracellular recordings. Pa-11 (0.1 μM) decreased the component of nerve terminal action potential related to Na + and K + currents to about 80% and 40% of control, respectively, after 60 min. Myotoxin a (5 μM) and ouabain (50 μM) produced similar, time-dependent changes in the nerve terminal action potential. These effects are similar to those produced by Pa-8 and Pa-10F, and are consistent with a slow but partial loss of membrane potential at the nerve terminal. In addition, whole-cell patch-clamp recording was employed to investigate possible direct actions of Pa-8, Pa-10F and Pa-11 on Na + and K + currents in NG108 and PC12 cells in culture. None of these toxins (0.8 μM) reduced the Na + and K + currents in these cells. There was also no displacement of [ 125I] α-dendrotoxin bound to voltage-sensitive potassium channels on rat synaptosomal membranes induced by Pa-8, Pa-10F and Pa-11 (up to 100 μM). These results support the hypothesis that the alteration of nerve terminal waveforms by these toxic phospholipases A 2 is mediated by nerve terminal depolarisation.

Characteristics of Transmitter Secretion from Individual Sympathetic Varicosities

Electrophysiological recording techniques have been developed to study action potential propagation and neurotransmitter release, at the level of the varicosity, in postganglionic sympathetic nerve terminals. Several techniques have been employed, including differentiation of the rising phases of intracellularly recorded excitatory junction potentials (discrete events) and focal extracellular recording of the nerve terminal action potential and associated excitatory junction currents (NTIs, EJCs). This chapter focuses on the majority of this chapter on results obtained using this preparation. Researchers have shown that transmitter release from individual varicosities occurs intermittently following low-frequency nerve stimulation. The probability of transmitter release increases with stimulation frequency, that is, frequency-dependent facilitation. The mechanisms underlying frequency-dependent facilitation remain fairly poorly understood, but it is clear that calcium plays a pivotal role. One surprising finding is that residual release is inhibited by ryanodine in a use- and time-dependent manner, suggesting that this pharmacologically distinct voltage-dependent calcium channel is located close to, or is in some way coupled to, ryanodine receptors (RyRs). Ryanodine receptors are involved in excitation-contraction coupling in cardiac and skeletal muscle but are not thought to play any role in action potential-evoked transmitter release. The critical determinant of neurotransmitter release is an increase in the intraneuronal calcium concentration following invasion of the nerve terminal by the action potential. This transient increase in the intraneuronal calcium level is thought to arise solely by calcium entering through clusters of voltage-dependent calcium channels located close to the sites of neurotransmitter release, that is, active zones.

Modification of ionic currents underlying action potentials in mouse nerve terminals by the thioloxidizing agent diamide

The effect of diamide, a thiol-oxidizing agent, was tested using electrophysiological techniques to determine whether its ability to alter neuromuscular transmission in vitro could be attributed to alterations of ion channels controlling neuronal excitability and/or acetylcholine release. In mouse triangularis sterni preparations, diamide transiently increased the evoked release of acetylcholine and then blocked release. Extracellular recording of perineural waveforms associated with neuronal action potentials at motor nerve terminals showed that diamide reduced the waveforms associated with the delayed rectifier K + current, a Ca 2+ current and a Ca 2+-activated K + current ( i k, ca ). Inhibition of quantal transmitter release was not associated with failure of action potentials to invade nerve terminals. Thus, diamide modifies the ionic currents underlying the nerve terminal action potential, some of these changes probably account for the complex effects of diamide on quantal transmission.

Exposure of the population to chlorinated phenols

The yellow Iranian scorpion Odontobuthus doriae can cause fatal envenoming, but its mechanism of action is unclear. One of the reported manifestations of envenoming is moderate to severe involuntary tremor of skeletal muscle. In order to understand better the mechanism of action of this venom on skeletal muscle function, we examined the effects of the venom in vitro on chick biventer cervicis (CBC) and mouse hemidiaphragm (MHD) nerve muscle preparations. O. doriae venom (0.3–10 μg/ml) initially increased and then decreased twitch height. The venom also caused contracture in both preparations. In mouse triangularis sterni preparations, used for all intracellular recording techniques, the venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials and endplate potentials in response to single-shock stimulation. With extracellular recording techniques, scorpion venom (1 μg/ml) was found to cause changes to the perineural waveform associated with nerve terminal action potentials consistent with effects on Na+ and K+ currents. The main facilitatory effects of O. doriae venom are likely to be due to toxins that affect Na+ channels in nerve–muscle preparations similar to most Old World scorpion venoms, but blocking effects on K+ channels are also possible. Such effects could lead to initial enhancement of transmitter release that could underlie the muscle tremors seen in victims. Toxins acting on Na+ and K+ currents have been isolated from the venom [Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E., Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat, J., 2005. OD1, the first toxin isolated from the venom of the scorpion Odontobuthus doriae active on voltage-gated Na+ channels. FEBS Lett. 579, 4181–4186; Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian scorpion Odontobuthus doriae. FEBS Lett. 580, 6254–6258]; however, the muscle paralysis seen at higher concentrations of venom may be due to additional, as yet uncharacterised, components of the venom.

Effects of waglerin-I on neuromuscular transmission of mouse nerve-muscle preparations

The effects of waglerin-I, a toxin from Trimeresurus wagleri, on neuromuscular (NM) transmission were studied on the phrenic nervediaphragm preparation and triangularis sterni nerve-muscle preparation of mice. The toxin (1.2–4.0 μM) reversibly inhibited the indirectly elicited twitch tension of the diaphragm and decreased the ACh-elicited muscle contracture of chronically denervated diaphragm, while the directly elicited twitch tension was not affected. The toxin reversibly decreased the amplitude of miniature endplate potential (MEPP) at 0.52 μM and endplate potential (EPP) at 1.2–4 nM. The toxin (120nM–0.4 μM) also decreased the quantal content of EPP. The perineural waveforms were recorded with an extracellular electrode placed into the perineural sheaths of motor nerves of M. triangularis sterni. The toxin (4 μM) did not alter the amplitudes of waveforms related to sodium and potassium currents of the nerve terminal action potential, while the waveform related to calcium current was decreased. It is concluded that the toxin acts on both presynaptic and postsynaptic sites of the mouse motor endplate, and that the presynaptic effect is apparently more potent than the postsynaptic effect.

Dual contractile effects of ATP released by field stimulation revealed by effects of alpha,beta-methylene ATP and suramin in rat tail artery.

1. The field stimulation-induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2-purinceptor antagonist, suramin, on these parameters were compared with those of alpha,beta-methylene ATP, a P2X-purinoceptor desensitizing agent. 2. Neither alpha,beta-methylene ATP (10 microM) nor suramin (100-500 microM) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP-induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3. Combined blockade of both postjunctional alpha 1- and alpha 2-adrenoceptors by prazosin and yohimbine (both 0.1 microM) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by alpha,beta-methylene ATP (10 microM) and inhibited by suramin in a concentration-dependent manner (10-500 microM; IC50 75 microM) and was hence probably caused by ATP or a related nucleotide. 4. When added first, alpha,beta-methylene ATP (10 microM) or suramin (100-500 microM) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 microM). 5. The K+ channel blocker, tetraethylammonium (10 mM), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic. Addition of alpha,beta-methylene ATP (10 microM)or suramin (100-500 microM) abolished the large initial twitch component of this contraction and depressed the tonic phase.6. Like alpha,beta-methylene ATP, suramin (500 microM) had no effect on the contraction caused by exogenous NA (1O nM-l10 microM) or KCI (60 mM); both agents almost abolished the contraction caused by ATP(100 microM).7. In conclusion, (i) the contractile response of rat tail artery to electrical field stimulation is mediated by both ATP and NA, and is thus an expression of ATP-NA co-transmission, (ii) the released ATP exerts two opposite effects via 'P2x-like' purinoceptors, triggering the initial rapid phase of the neurogenic contraction and restricting the NA-mediated component of the contraction; and (iii) the source and possible physiological role of the ATP which causes the inhibitory effect are unknown at present.

K + and Ca 2+ channel blockers may enhance or depress sympathetic transmitter release via a Ca 2 +-dependent mechanism “upstream” of the release site

Extracellular recording of the pre- and postjunctional electrical activity in guinea-pig or mouse vas deferens or rat tail artery was employed to study the mechanisms by which the K + channel blockers, tetraethylammonium and 4-aminopyridine and the Ca 2 + channel blockers, Cd 2 + , Mn 2 + or nifedipine influence the nerve stimulation-induced release of adenosine 5′-triphosphate as a sympathetic co-transmitter. The K + and Ca 2+ channel blocking agents examined had no effect on the spontaneous quantal release of adenosine 5′-triphosphate. However, addition of tetraethylammonium and 4-aminopyridine inside the recording electrode broadened the nerve terminal action potential and caused it to become more resistant to local application of tetrodotoxin, and dramatically increased the magnitude and tetrodotoxin resistance of adenosine 5′-triphosphate release within the patch. Surprisingly, tetraethylammonium and 4-aminopyridine were equally effective when added outside the recording electrode; now they did not increase the duration of the nerve terminal action potential inside the patch but increased its resistance to locally applied tetrodotoxin and dramatically increased the magnitude as well as the tetrodoxin resistance of adenosine 5'-triphosphate release from sites inside the patch. Both tetraethylammonium and 4-aminopyridine contributed to these effects, with a strong potentiating interaction. Nifedipine was without effect, but application of 1–100 μM Cd 2+ or 1–5 mM Mn 2+ either inside or outside the recording electrode blocked adenosine 5′-triphosphate release inside the patch. The results indicate: (i) that the nerve terminal action potential is generated by activation of voltage- gated, regenerative Na + channels but also has a small component carried by influx of Ca 2+ and that it is “normally” terminated by activation of voltage- as well as Ca 2 +-dependent K + channels; (ii) that the release probability is tonically depressed by the resting K + efflux, and promoted by the resting Ca 2+ influx, “upstream” of the release sites; (iii) that the upstream control of the release probability may involve both changes in properties of ionic channels in the nerve terminal membrane, and effects on the cytoskeleton leading to changes in the availability of releasable quanta in varicosities within the patch.

Effects of butanedione monoxime on neuromuscular transmission

1. The amplitude of endplate potentials was increased by concentrations of butanedione monoxime (BDM, 5-20 mM) that typically caused muscle paralysis. 2. Although BDM slowed the decay of spontaneous miniature endplate currents, the effect was insufficient to explain most of the large increase in amplitude of endplate potentials. 3. The quantal content of endplate potentials was increased by BDM in a reversible, concentration-dependent manner. 4. The frequency of miniature endplate potentials was not changed by 10 mM BDM in the presence of normal or raised potassium concentrations, indicating that BDM does not change quantal content by a direct effect on calcium channels or on steady-state intracellular calcium concentration. 5. A change in the time course of the extracellularly recorded nerve terminal action potential caused by BDM was similar to the change produced by 4-aminopyridine (4-AP). 6. The increase in quantal content produced by BDM was only slightly reduced in the presence of 1 mM tetraethylammonium (TEA) but was significantly reduced in the presence of 0.5 to 1 mM 4-AP. 7. It was concluded that BDM blocks a 4-AP-sensitive potassium conductance in motor nerve terminals and, by increasing the duration of the action potential in this way, increases evoked transmitter release.

On the blockade of acetylcholine release at mouse motor nerve terminals by β‐bungarotoxin and crotoxin

1. beta-Bungarotoxin and crotoxin are phospholipose A2 neurotoxins, which block irreversibly the evoked release of acetylcholine from motor nerve terminals of mouse triangularis sterni preparations. 2. Extracellular recording of nerve terminal action potentials reveal that inhibition of transmitter release is not associated with failure of the action potential to invade nerve terminals. 3. When evoked transmitter release (measured as intracellularly recorded endplate potentials) was blocked by beta-bungarotoxin, spontaneous acetylcholine release was stimulated as in control experiments by K(+)-induced depolarization and by the Ca2(+)-ionophore A23187. 4. The site of action of the toxins remains to be elucidated but would appear to be associated with the coupling of action potential induced-depolarization to the release mechanism, rather than with the release mechanism itself.

Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn 2+, verapamil (40 μM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg 2+, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca 2+ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca 2+ (nominally Ca 2+-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn 2+ and Mg 2+, as well as Ca 2+, enter the nerve terminal through Ca 2+ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca 2+ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na + channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.

Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to omega-conotoxin and relatively insensitive to dihydropyridines. Optical studies with and without voltage-sensitive dyes.

Extrinsic absorption changes exhibited by potentiometric dyes have established the ionic basis of the action potential in synchronously activated populations of nerve terminals in the intact neurohypophyses of amphibia and mammals (Salzberg et al., 1983; Obaid et al., 1983, 1985b). Also, large and rapid changes in light scattering, measured as transparency, have been shown to follow membrane depolarization and to be intimately associated with the release of neuropeptides from the nerve terminals of the mouse neurohypophysis (Salzberg et al., 1985; Gainer et al., 1986). We report some experiments that help to define the pharmacological profile of the calcium channels present in intact neurosecretory terminals of vertebrates. For these, we used the peptide toxin omega-conotoxin GVIA (1-5 microM) and the dihydropyridine compounds Bay-K 8644 and nifedipine (2-5 microM), together with the after-hyperpolarization of the nerve terminal action potential. This undershoot depends upon the activation of a calcium-mediated potassium channel, as suggested by its sensitivity to [Ca++]o and charybdotoxin. omega-conotoxin GVIA substantially reduced the after-hyperpolarization in neurosecretory terminals of Xenopus, while neither of the dihydropyridine compounds had any effect under conditions that mimic natural stimulation. The effects of these calcium channel modifiers on the action potential recorded optically from the terminals of the Xenopus neurohypophysis were faithfully reflected in the behavior of the light-scattering changes observed in the neurohypophysis of the CD-1 mouse. omega-conotoxin GVIA (5 microM) reduced the size of the intrinsic optical signal associated with secretion by 50%, while the dihydropyridines had little effect. These observations suggest that the type of calcium channel that dominates the secretory behavior of intact vertebrate nerve terminals is at least partially blocked by omega-conotoxin GVIA and is insensitive, under normal conditions, to dihydropyridines.