The development and evaluation of a new aerosol irritant assay with minimal animal stress.

Current aerosol irritant assays trap animals in noxious atmospheres and put a lot of stress on them. For this reason, the Minimal Animal Stress Irritant Assay Chamber (MASIAC) was developed based on the principle of avoidance, and evaluated. The MASIAC reproducibly detected citric acid with more sensitivity than conventionally used assays. With a group of mice tested simultaneously, the responses were not significantly affected by the presence of other mice. In addition, following multiple exposures to citric acid, the mice either sensitized to the irritant, or learned to avoid it. This suggests a number of areas where the MASIAC could be applied, including behavioral and asthma research. If this new method turns out to be as good as currently used assays, it could provide investigators with an alternative, more humane method of evaluating pulmonary irritants.

The binomial model in fluctuation analysis of quantal neurotransmitter release.

The mathematics of the binomial model for quantal neurotransmitter release is considered in general terms, to explore what information might be extractable from statistical aspects of data. For an array of N statistically independent release sites, each with a release probability p, the compound binomial always pertains, with = N

, p' identical to 1 - var(m)/ =

(1 + cvp2) and n' identical to /p' = N/(1 + cvp2), where m is the output/stimulus and cvp2 is var(p)/

2. Unless n' is invariant with ambient conditions or stimulation paradigms, the simple binomial (cvp = 0) is untenable and n' is neither N nor the number of "active" sites or sites with a quantum available. At each site p = popA, whereas po is the output probability if a site is "eligible" or "filled" despite previous quantal discharge, and pA (eligibility probability) depends at least on the replenishment rate, po, and interstimulus time. Assuming stochastic replenishment, a simple algorithm allows calculation of the full statistical composition of outputs for any hypothetical combinations of po's and refill rates, for any stimulation paradigm and spontaneous release. A rise in n' (reduced cvp) tends to occur whenever po varies widely between sites, with a raised stimulation frequency or factors (tending to increase po's. Unlike and var(m) at equilibrium, output changes early in trains of stimuli, and covariances, potentially provide information about whether changes in reflect change in or in . Formulae are derived for variance and third moments of postsynaptic responses, which depend on the quantal mix in the signals. A new, easily computed function, the area product, gives noise-unbiased variance of a series of synaptic signals and its peristimulus time distribution, which is modified by the unit channel composition of quantal responses and if the signals reflect mixed responses from synapses with different quantal time course.

The relation between transmitter release and Ca2+ entry at the mouse motor nerve terminal: role of stochastic factors causing heterogeneity.

The relation between quantal transmitter release and presynaptic Ca2+/Ba2+ entry at the mouse neuromuscular junction was studied, making use of the finding that in the presence of Ba2+ trains of nerve stimuli or brief nerve terminal depolarizations elicit "tails" of raised miniature end-plate potential frequency (fm) that reflect entry of Ba2+ per pulse, and hence effectiveness of pulses in opening Ca2+/Ba2+ channels; at the same time these pulses elicit end-plate potentials. With nerve stimulation in the presence of Ba2+ and Ca2+ and modulation of release by raised Mg2+ or bekanamycin, slopes of log quantal content (m) vs log apparent Ba2+ entry per pulse were close to 4, which is the same as the Hill coefficient for Ba2+ cooperativity derived from other data. With depolarizing pulses of varied intensity, however, similar plots gave slopes close to 2, with Ba2+ alone or in a mixture of Ca2+ and Ba2+. Thus, the relation between transmitter release and Ca2+ (or Ba2+) entry apparently depends upon how entry is varied; varying the numbers of channels opened is not the same as varying ion entry per channel. A mathematical model was developed to examine the consequences of heterogeneity of local Ca2+ (or Ba2+) between release sites, arising because of stochastic variation of number and time course of Ca2+ channels opened per site; the experimental results were consistent with this model. It was therefore concluded that release is normally governed by intracellular Ca2+ close to points of Ca2+ entry through channels; stochastic factors give rise to more release than if Ca2+ were homogeneously distributed. If Ca2+ channels are uniformly close to release sites the average number of channels opened per site per action potential may be as low as 4.

Multiplicative and additive Ca(2+)-dependent components of facilitation at mouse endplates.

1. Facilitation of endplate potentials (EPPs) and frequency of miniature endplate potentials (MEPPs) were studied, in the presence of low Ca2+/raised Mg2+, in isolated mouse hemidiaphragm, using pseudo-random sequences of nerve stimulation and automated (computer) counting of MEPPs and quantal components of EPPs. 2. The facilitation in quantal content of EPPs (m) produced by one or more antecedent stimuli was accompanied by facilitation of MEPP frequency (fm) that was similar in magnitude and substantially less than expected if facilitation reflects persistent (residual) intraterminal Ca2+. The time course of 'phasic' quantal release, associated with the EPP, was little if at all altered with facilitation. 3. The magnitude and time course of facilitation was consistent with two distinct presynaptic processes, each manifest both in m and fm, (i) an effect to multiply transmitter release, and (ii) residual Ca2+ which adds to Ca2+ brought in by nerve impulses. These have distinct time courses. 4. After loading nerve terminals with bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), facilitation of m and fm became very small. 5. At sufficiently low Ca2+/raised Mg2+ facilitation of m and fm became very small although latency histograms showed clear EPPs. However, the multiplicative component of facilitation became maximal at Ca2+/Mg2+ concentrations giving an average m value less than 0.1, corresponding to about 5% of normal Ca2+ entry per pulse. At lower Ca2+, facilitation was restored when EPPs were made larger using 4-aminopyridine. 6. With EPPs elicited by brief 'direct' nerve terminal depolarizations, facilitation was graded with pulse intensity (and m) and could be much less than with EPPs with similar m evoked by nerve stimuli at lower Ca2+ and/or higher Mg2+. 7. It was concluded that fast facilitation is primarily multiplicative and reflects activity within the nerve terminal of a Ca(2+)-sensitive process distinct from that generating Ca(2+)-dependent release.

Quantal transmitter release mediated by strontium at the mouse motor nerve terminal.

1. In isolated mouse diaphragm, nerve stimulation in the presence of Sr2+ evokes phasic quantal transmitter release (endplate potentials, EPPs) with the same time course as in the presence of Ca2+. 2. Brief tetanic trains of nerve stimuli in the presence of Sr2+ cause an increase in quantal content of EPPs accompanied by an increase in the frequency of miniature EPPs (MEPPs); the latter persists as a 'tail' that subsides within about a second. Pseudo-random stimulation sequences were used to characterize these changes. 3. The fourth root of MEPP frequency during or after stimulation rose and fell in accordance with first order kinetics with the same time constants for rising and falling phases, in agreement with a 'residual ion' model in which (a) each nerve impulse causes the same entry of Sr2+ into the nerve terminal, (b) transmitter release (MEPP frequency) is proportional to the fourth power of [Sr2+] at release sites, and (c) Sr2+ removal is a first order process with a time constant of about 250 ms. 4. After exposure to bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, acetoxymethyl ester form (BAPTA AM), 'Sr2+ tails' of MEPP frequency were reduced in magnitude and prolonged. 5. During stimulation trains, growth of phasic transmitter release rates (and quantal content of EPPs) were related to growth of MEPP frequency in almost exact agreement with a residual ion model, in which 'phasic' release (EPPs) and MEPP frequency are governed by the same equation, with the same parameters, and without any effect of depolarization per se to affect phasic release. 6. Prolonged (1 s) nerve terminal depolarizations in the presence of Sr2+ produce increased MEPP frequency with a time course corresponding to a model in which depolarization per se has little or no effect to increase transmitter release. 7. It was concluded that in the presence of Sr2+ the intense 'phasic' acceleration of quantal release induced by nerve impulse manifest in an EPP can be attributed to a transient rise of intracellular [Sr2+] in the vicinity of release sites, while the modulation of 'phasic' release by antecedent nerve impulses can be attributed to residual Sr2+ which is also manifest in a rise in MEPP frequency.

Actions of lead on transmitter release at mouse motor nerve terminals.

The actions of lead (Pb2+) on transmitter release were studied at neuromuscular junctions in mouse diaphragm in vitro. The quantal content of end-plate potentials (EPPs) was reduced by Pb2+ in a dose-related manner consistent with inhibition of Ca2+ entry into nerve terminals, with a half-maximal effect at 1.4 microM (in 0.5 mM Ca2+ and 2 mM Mg2+). Pb2+ also inhibited the increased frequency of MEPPs (fMEPP where MEPPs denotes miniature EPPs) produced by Ba2+ in the presence of raised K+, blocking the calculated Ba2+ entry half-maximally at 170 microM. However, at concentrations of 50-200 nM, Pb2+ often increased fMEPP in 20 mM K+ in the presence of Ca2+ and acted to promote the irreversible effect of lanthanum (La3+) to raise fMEPP. In nominally Ca(2+)-free solution with 20 mM K+, brief (1 min) application of Pb2+ (20-320 microM) caused rapid dose-dependent reversible rises in fMEPP. With prolonged exposure to Pb2+, fMEPP rose and then slowly declined; after removal of Pb2+, once fMEPP had fallen to low levels, fMEPP responded nearly normally to Ca2+ or ethanol, but not to Pb2+ itself. In 5 mM K+, 0 mM Ca2+ and varied [Pb2+] (where [] denotes concentration), nerve stimulation caused no EPPs, but prolonged tetanic stimulation produced increases in fMEPP graded with [Pb2+] that persisted as a "tail"; results were consistent with growth of fMEPP with the 4th power of intracellular Pb2+ and removal of intracellular Pb2+ with a time constant of about 30 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple actions of zinc on transmitter release at mouse end-plates.

In mouse diaphragm, the increase in frequency of mini end-plate potentials (fmepp), by Ca2+ or Ba2+ in 20 mM K+, was reversibly inhibited by Zn2+ in a manner consistent with competition between Zn2+ and Ca2+ at a site which interacts with only one atom of Zn with an apparent dissociation constant (Ki) of about 0.015 mM. Between 0.5 mM and 2 mM, Zn2+ caused a rapid and reversible dose-dependent increase in fmepp in 20 mM K+/0 Ca2+. Prolonged or repeated exposure to Zn2+ produced a slow increase in fmepp followed by a decline, which once started, was not modified by of Zn2+. The time course was prolonged in raised Mg2+, bekanamycin, or in 5 mM K+ solution, and graded with Zn2+ concentration, but total numbers of MEPPs induced by 0.1 mM, 1 mM or 4 mM Zn2+ were not significantly different. When fmeppp fell it became insensitive to Ca2+, Ba2+, La3+ (in 20 mM K+), ethanol and raised osmotic pressure. Before complete block of responses to Ca2+, the Ca2+/fmepp dose/response curve in 20 mM K+ was shifted to the right. These results indicate that Zn2+ enters the terminal via voltage-gated Ca2+ channels that interact in a complex way with these ions and then acts (a) as a partial agonist at sites where Ca2+ normally governs transmitter release, and (b) to produce irreversible changes in the nerve terminal, associated with a rise and subsequent fall of fmepp and loss of sensitivity of the release mechanism to Ca2+ and other agents.

Transmitter release at mouse motor nerve terminals mediated by temporary accumulation of intracellular barium.

1. In isolated mouse diaphragm, tetanic nerve stimulation in the presence of Ba2+ causes an increase in frequency of MEPPs which continues as an after-discharge or 'tail' of raised MEPP frequency that subsides over a period of seconds, in addition to EPPs of low quantal content. 'Ba2+ tails' are also seen with focal depolarization of nerve terminals in the presence of tetrodotoxin. 2. The development of 'Ba2+ tails' could be inhibited or blocked by neomycin, raised Mg2+, or Cd2+ present at the time of stimulation; the presence of the blocking substance during the tail itself had no effect. 3. The time course of MEPP frequency changes during and after stimulation could be expressed as a simple exponential process, with the same time constant for both the rise and the fall, by taking as the time-dependent variable the nth root of MEPP frequency, n being 4 or 5. The time constant (tau) derived from the rate of fall of the 1/4 power of MEPP frequency during the tail was at most junctions between 3 and 6 s, and apparently unaffected by concentration of Ba2+, or of Ca2+, or by tonic depolarization of the nerve terminal. 4. The intensity of 'Ca2+ tails' was graded steeply with the number of stimuli applied, but was nearly independent of stimulus frequency, when train duration was kept brief compared to tau, i.e. about a second or less. The nth root of the number of MEPPs at a given time period in the tail was linearly related to the number of stimuli, when n was chosen to be 4 or 5. 5. The above data are consistent with a model in which (a) with each nerve impulse in a train there occurs the same entry of Ba2+ into the terminal, (b) transmitter release (MEPP frequency) is proportional to the fourth or fifth power of [Ba2+] at critical sites within the nerve terminal, (c) the Ba2+ leaves these sites as a first-order process with a time constant of a few seconds. Compared to Ca2+, Ba2+ persists longer but has lower potency. 6. With variation of external [Ba2+] over the range 50 microM to 6.4 mM, apparent Ba2+ entry per nerve impulse grew linearly with concentration. 7. Evidence is presented indicating that intraterminal Ba2+ can 'co-operate' with Ca2+ or La3+ in promoting transmitter release.

Receptor kinetics pertaining to blockade of nerve-released transmitter at synapses.

The question is raised as to whether competitive inhibitors should block responses of tissue to nerve-released neurotransmitter to the same extent as they block equivalent responses to exogenous agonist. From a simple dynamic model of synaptic events, which takes into account non-constancy of transmitter concentration in space and time, it is deduced that equal blockade of responses to nerve-released and exogenous transmitter substance will occur if: (i) there are locally many more receptor molecules than transmitter molecules; (ii) the active agonist-receptor complex, AnR, has n = 1; and (iii) tissue response is insensitive to spatial or temporal inhomogeneity of AR. In such a case there will also be equal sensitivity of responses to other modes of inhibition: irreversible competitive, uncompetitive, and non-competitive. Equal blockade of responses to equi-effective endogenous and exogenous agonist will also occur if nerve stimulation gives rise to a steady uniform concentration of agonist, so that equilibrium kinetics are applicable. When n greater than 1 and/or when tissue responses reflect local peak AnR, response to nerve-released transmitter will be relatively insensitive to receptor blockade by a competitive inhibitor. The same is true for irreversible competitive blockade or for modulation of receptor density. However, an uncompetitive inhibitor (e.g. a 'channel blocker') may be more effective against nerve-released agonist than against exogenous agonist.

A voltage clamp study of the glutamate responsive neuromuscular junction in Drosophila melanogaster.

The point and single electrode voltage clamp methods have been used to study the characteristics of junctional currents in Drosophila melanogaster larvae muscle fibers and the modulation of these currents by excitatory amino acids, short and long chain n-alkanols, and pentobarbital. The decay phase of junctional currents in Drosophila was found to be dominated by cooperativity in transmitter binding associated with reverberation, that is, repeated binding of transmitter with receptors as the transmitter molecules diffuse away from the active region. The current decay does not directly reflect the closure of ion channels and is qualitatively similar to the decay of miniature end-plate currents at the mouse neuromuscular junction after poisoning of acetylcholinesterase by paraoxon. In Drosophila an increase in membrane hyperpolarization both slows the time course of current decay and increases the degree of reverberation. The application of excitatory amino acids including glutamate, N-methyl-D-aspartate, quisqualate, and kainate causes a significant decrease in the amplitude of the junctional currents, a prolongation of the decay time course, and a reduction in reverberation of transmitter. The height of junctional currents is also diminished by the n-alkanols ethanol, pentanol, and octanol and by the barbiturate pentobarbital; ethanol also hastened the time course of decay of the currents.

Single Ca2+ entry and transmitter release systems at the neuromuscular synapse.

The possibility that there exists more than one voltage-gated Ca2+ channel system subserving quantal release of neurotransmitter at nerve endings was examined by measuring the sensitivity of quantal release to agents that block Ca2+ or Ba2+ entry, namely Cd2+, Mg2+, neomycin, and bekanamycin. The results show equal effectiveness vs. release evoked by presynaptic action potentials, brief intense presynaptic depolarizations, or prolonged relatively mild depolarizations, from which it is concluded that the same channel system is involved in each case. In the presence of Ba2+ and no Ca2+, there occur essentially normal (but small) endplate potentials (epps), while in the presence of Ba2+ and Ca2+ epp amplitude and frequency of miniature epps co-modulate with stimulation frequency in a manner which corresponds to a residual Ba2+ model for augmentation. It is therefore also concluded that a single transmitter release system is responsible for normal phasic release and the asynchronous release that is mediated by Ba2+.

Multiple potassium conductances at the mammalian motor nerve terminal.

The possibility that multiple K+ conductances are present in mammalian motor nerve terminals was investigated by measuring the differential effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on transmitter release and on nerve terminal excitability in mouse phrenic nerve-diaphram preparations. Neither 4-AP nor TEA alter the spontaneous frequency of miniature end-plate potentials (fmepp) and therefore evidently do not affect calcium channels directly. Both 4-AP and TEA increased the quantal content of end-plate potentials (epps) evoked by nerve stimulation but the effects were not mutually exclusive; TEA continued to act in the presence of a maximally effective concentration of 4-AP. The increase in transmitter release evoked by focal polarisation of the terminal was not affected by 4-AP, whereas TEA exerted an effect consistent with a reduction in membrane conductance. Similarly, threshold for nerve terminal action potential generation was not affected by 4-AP, but TEA caused a reduction in threshold and alteration of the 'accommodation curve' indicative of a reduction of membrane conductance. Under conditions where one would predict no contribution of calcium K+ conductance, i.e., when release was evoked by Ba2+ in the absence of Ca2+, the different and non-competing effects of TEA and 4-AP were still apparent. It is concluded, therefore, that the motor nerve terminal possesses at least two K+ conductances, not including calcium activated gK, which may be distinguished pharmacologically.

Multiple actions of cadmium on transmitter release at the mouse neuromuscular junction.

The action of cadmium ions on transmitter release was studied at the neuromuscular junction in mouse diaphragm. In the presence of raised K+, Cd2+ caused a parallel shift to the right of the graph of transmitter release rate (frequency of miniature end-plate potentials, fmepp) versus log [Ca2+], with no change in maximum or slope, indicating a competitive mode of action of Cd2+. The apparent dissociation constant for Cd2+ was 3 microM. In calcium-free solutions containing 15 mM K+, Cd2+ caused a rise in the fmepp, which subsequently slowly declined despite the continued presence of Cd2+. The rise in fmepp caused by Cd2+ could be interrupted, but not reversed, by washing out the Cd2+ with EDTA. Exposure of the preparation to 100 microM Cd2+ for 15 min or more resulted in a raised fmepp that persisted despite the removal of Cd2+ and exposure to 200 microM EDTA. Following such treatment, the graph of fmepp versus log [Ca2+] continued to be shifted to the right. The interaction of Ca2+ with the residual effect of Cd2+ indicates that Cd2+, in addition to its action to block Ca2+ entry into the terminal, may act as a competitor and perhaps as a partial agonist at intracellular sites that normally bind Ca2+ and govern transmitter release. If this is the case, then it must be supposed that, in raised K+, quantal release of transmitter represents intermittent intense activation of release sites with local high levels of Ca2+ rather than continuous low level activation.

Anion permeability of motor nerve terminals.

Motor nerve terminals in mouse and frog display behavior consistent with an appreciable permeability of the nerve terminal membrane to chloride. In mouse diaphragm, in the presence of 15 mM K+ and 2 mM or 8 mM Ca2+, replacement of Cl- by NO3-, Br- or acetate causes a transient increase in the quantal release of acetylcholine, measured as the frequency of spontaneously occurring miniature end plate potentials (FMEPP); a rapid rise in FMEPP is followed by a slow decline, with a half-time of about 4 min, to an equilibration level close to the control level. After equilibration in a solution in which the Cl- is replaced by another anion, return to Cl- -containing solution causes a transient decrease in FMEPP with a subsequent slow recovery. The data are consistent with transient nerve terminal depolarization or hyperpolarization, reflecting a nerve terminal permeability to anions in the sequence Cl- greater than Br- greater than NO3- greater than acetate. In 5 mM K+, changes in nerve terminal excitability, determined using focal stimulation, are also consistent with alteration of nerve terminal membrane potential as a consequence of anion substitution. The time course of relaxation of FMEPP after a change from Cl- to an anion of lower permeability, or vice versa, is considerably slower than that expected if Cl- permeability of nerve terminals is similar to that of skeletal muscle fibres, and if the nerve terminal behaves as a single compartment. In frog cutaneous pectoris, transient changes in FMEPP produced by substitution of anions in the bathing solution were similar to those produced in mouse diaphragm, but more rapid in time course.

The actions of dimethyl sulfoxide on neuromuscular transmission.

The effects of dimethyl sulfoxide (DMSO) on subsynaptic response and quantal release of transmitter have been studied at the mammalian neuromuscular junction. Subsynaptically, at low concentrations (up to 1% by volume), DMSO prolongs the time course of decay of miniature endplate currents, (MEPCs), with no significant effect on the amplitude of the currents, which is consistent with an action of DMSO to inhibit acetylcholinesterase. At higher concentrations of DMSO (in excess of 1% by volume) the amplitude of MEPCs and the steady state response to carbamoylcholine (carbachol) are significantly reduced, which suggests an additional action of DMSO other than pure anticholinesterase activity. After pretreatment of the preparation with a low concentration of paraoxon, higher concentrations of DMSO decrease MEPC height and cause highly variable changes in the decay time course of the MEPC. The results suggest that DMSO concentrations in excess of 1% by volume have two distinct and opposite actions on the subsynaptic response; a pure anticholinesterase activity to enhance the response and a depressant effect which is similar to that caused by d-tubocurarine. Presynaptically, DMSO increased both the spontaneous release (measured as the frequency of miniature endplate potentials, fMEPP) and the evoked release (measured as the quantal content of endplate potentials). Both types of release were increased as an exponential function, with the same slope, of the DMSO concentration, suggesting a common mode of action on these two types of release. This action appeared not to be due to an effect on the disposition or effectiveness of calcium ions inside the terminal but, rather, was due to a fusogenic or global effect. In addition, the increase in fMEPP with DMSO was the same when external calcium was replaced by barium. At the concentrations studied, up to 8% by volume, DMSO did not cause any substantial depolarization of the nerve terminal or any appreciable change in the nerve terminal action potential. In a few experiments facilitation was studied at the frog neuromuscular junction and was unchanged by DMSO at concentrations which considerably enhanced transmitter release.

Modification of motor nerve terminal excitability by alkanols and volatile anaesthetics.

A method of local polarization-excitation was used to study changes in motor nerve terminal excitability produced by n-alkanols and volatile anaesthetics in mouse diaphragm preparations. Ethanol and propanol caused an exaggeration of 'accommodation', i.e., the increase in excitation threshold produced by a conditioning depolarization. Butanol, hexanol and octanol had mixed effects, producing a rise in the minimum threshold (threshold after removal of resting accommodation) in addition to an increase in accommodation. Volatile anaesthetics produced effects on excitability at concentrations comparable to minimum alveolar concentration. The action of enflurane was essentially only to increase accommodation while methoxyflurane produced an increase in threshold insensitive to conditioning polarization. Halothane and isoflurane produced intermediate effects. Accommodation curves were little affected by Ba2+ or 4-aminopyridine and were consistent with accommodation being a reflection of inactivation of the Na+ current system. We conclude that volatile anaesthetics, at concentrations comparable to those producing anaesthesia, may substantially modify Na+ channel gating and inactivation.

Lanthanum as a surrogate for calcium in transmitter release at mouse motor nerve terminals.

The mechanism by which lanthanum (La3+) causes an increased frequency of miniature end-plate potentials (m.e.p.p.s) was studied at the mouse neuromuscular junction. At concentrations as low as 0.25 microM, La3+ caused a progressive rise in m.e.p.p. frequency, to a maximum of several hundred per second. 'Washing' with solution containing EDTA arrested the rise, but did not substantially reduce the raised m.e.p.p. frequency. At partially 'lanthanized' junctions high frequencies of m.e.p.p.s were maintained indefinitely, even in 0 Ca2+/EDTA solutions. The rate of development of high m.e.p.p. frequency was increased by repetitive nerve stimulation or by depolarization of the nerve terminal (high K+ or focally applied current), and appeared to be proportional to the concentration of La3+ over the range of 0.25-5 microM. At low concentrations of La3+ the rise of m.e.p.p. frequency depended upon the co-presence of a small amount of Ca2+ (greater than 10 microM) and was slowed and partially blocked by Cd2+, or by Ca2+ at about 10 microM. The quantal content of end-plate potentials was usually reduced in the presence of La3+, but was increased over control values after removal of La3+ by 'washing' with solution containing EDTA, once a raised m.e.p.p. frequency had developed. At partially lanthanized junctions the absolute increases in m.e.p.p. frequency produced by Ca2+ (in raised K+), ethanol, or by nerve stimulation in the presence of Ba2+, were greater than at control junctions, but in each case the increases in the logarithm of m.e.p.p. frequency were less than at control junctions. It is concluded that La3+ causes transmitter release only after entry into the nerve terminal via voltage-sensitive channels, probably those that normally admit Ca2+, that La3+ and Ca2+ may co-operate at internal sites to induce transmitter release, and that these ions both co-operate and compete at external sites that regulate their entry into the nerve terminal.

Effect of a volatile anesthetic upon presynaptic excitability in mammalian hippocampus.

Changes in presynaptic terminal axon excitability produced by enflurane in the rat hippocampal slice preparation were investigated by stimulation of Schaeffer collateral terminal axons and by recording single unit antidromic action potentials. Stimulating pulses were preceded by conditioning hyperpolarizing or depolarizing current pulses. A plot of net threshold for action potential generation against the conditioning pulse yields an "accommodation curve;" changes in this curve can be used to assess the mechanism by which changes in excitability are produced. Enflurane, at a concentration equivalent to approximately equal to 1.3 times the minimum alveolar concentration, reduced excitability of terminal axons and increased accommodation in a manner consistent with a possible change in the inactivation of gNa.