Atypical miniature end plate currents in neuromuscular synapses of the rat under normal conditions, after acetylcholinesterase inhibition and cooling

In the end-plates of rat diaphragm among atypical miniature end-plate currents (MEPCs) 2.9% were giant and 5.1% were slowly rising. The frequency of the giant MEPCs was decreased when temperature was lowered and increased when acetylcholinesterase (AChE) was inhibited; the latter effect was reversed if d-tubocurarine was added. Frequency of the slowly rising MEPCs changed insignificantly by all conditions. It is suggested that a highly temperature-dependent presynaptic mechanism of giant MEPC generation does exist which is activated by acetylcholine (ACh). Data about changes in the time course of the slowly rising MEPCs by AChE inhibition and lowering of temperature make it possible to suggest that the slowly rising MEPCs may be accounted for either slow release of ACh quanta or release of quanta on large distances from synaptic cleft and postsynaptic cholinoreceptors. The latter is possible if ACh quanta are released from synaptic Schwann cell to periaxonial space.     

Effects of hyaluronidase on miniature endplate potentials and currents at the frog neuromuscular junction

The effects of 0.1% testicular hyaluronidase on miniature endplate potentials and currents (MEPP and MEPC) were investigated in frog pectorocutaneous muscle. The action of hyaluronidase on preparations with armine-induced blockade of acetylcholinesterase was associated with decreased amplitude and duration of MEPP and MEPC half-decay time and rising phase. The correlation between amplitude and half-decay time of MEPP and MEPC declined at the same time, while MEPC decay remained exponential. Treating preparations having intact acetylcholinesterase with hyaluronidase increased the length of MEPC halfdecay, with duration of the rising phase and amplitude remaining constant. It is suggested that enzymatic breakdown of a proportion of the glycocalix of cells forming the neuromuscular junction and a portion of the extracellular matrix at the synaptic cleft leads to attenuation of nonspectific acetylcholine binding, thus facilitating acetylcholine diffusion into the synaptic cleft.A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 113–119, January–February, 1988.     

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP_8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.     

Analysis of miniature potentials in reinnervated rat muscle]

Miniature endplate potentials (MEPPs) are regarded as the expression of release of a single quantum of acetylcholine by motor nerve endings in the muscle. Mepp frequency is dependent on the presynaptic mechanism, but MEPP amplitudes and time courses are the result of the characteristics of pre- and postsynaptic structures and of the interaction between them. After post-traumatic reinnervation of skeletal muscles, MEPP frequency increases, reaching slowly normal values. Two groups of male, Sprague Dawley rats were used: in the first group left sciatic nerve was crushed and nerve fibres were allowed to regenerate, whereas the others were regarded as controls. MEPPs were intracellularly recorded in end plates of normal and reinnervated left extensor digitorum longus muscle. MEPPs were sampled and recorded on a personal computer, and, subsequently, amplitude, rise time and half decay time were computed. At early stage after reinnervation, MEPPs showed rise times and decay times longer than normal. Afterwards, we did not find differences between mepp time courses by normal and reinnervated end plates. The possible relationships between the results and changes in acetylcholine receptor number and type, and in acetylcholinesterase activity occurring during denervation and reinnervation are discussed.     

Postsynaptic end-plate ionic currents in fast and slow chick muscle fibers. Effect of cholinesterase inhibition

Miniature end-plate potentials (MEPPs) were recorded in fast and slow chick muscle fibers extracellularly (focally) and under voltage clamp conditions. The duration of the MEPPs in synapses of slow fibers was on average 2.5 times longer than their duration in synapses of fast fibers. Inhibition of acetylcholinesterase (AChE) lengthened MEPPs by a varied degree: by 1.5 times in synapses of slow fibers and by 3.5 times in those of fast fibers. As a result the difference in the decay time of MEPPs in synapses of these types disappeared almost completely, and only a small difference remained in the rise time of the MEPPs. The MEPP decay time during hyperpolarization in the slow fiber synapse was rather less dependent on potential than in synapses of fast fibers; after inhibition of AChE it became even less dependent. Similar changes in potential dependence were found after lengthening of the MEPP by the action of ethanol. The functional significance of differences in AChE activity and in the activating effect of the mediator for the kinetics of MEPPs in synapses of fast and slow fibers is discussed.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 390–397, July–August, 1981.     

Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA.

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.     

Changes in acetylcholine concentration, miniature end-plate potentials and synaptic vesicles in frog neuromuscular preparations during lanthanum treatment

ACh content and synaptic ultrastructure were compared in neuromuscular preparations (sartorius muscle of Rana esculenta) incubated in control saline and in saline containing 1 mM LaCl3. ACh concentrations remained constant for 6 hr in control preparations. La3+ caused a 38% depletion of ACh within the first 30 min with subsequent recovery to 120% of control values within 3-4 hr. Recovery was prevented by hemicholinium-3. At 23 degrees C La3+ caused complete loss of synaptic vesicles: no depletion was seen at 4 degrees C. Initially MEPP frequency increased 300- to 700-fold (23 degrees C), then declined. Mean vesicle diameter did not change, but SD increased. As the frequency of MEPPs declined, the percentage of s-MEPPs greatly increased. La3+ had a postsynaptic effect which increased the amplitudes of both s-MEPPs and bell-MEPPs within a few seconds. The s-MEPP mean did not change during the course of La3+ treatment although the bell-MEPP mean usually decreased. How the decrease in synaptic vesicles, decrease in MEPP frequencies, and changes in ACh levels relate to changes in the percentage of different classes of quanta is discussed.     

Effects of black widow spider venom and Ca2+ on quantal secretion at the frog neuromuscular junction

A modification of the classical procedure of fluctuation analysis is used to measure the waveform, w(t), mean amplitude, (h), and mean rate of occurrence, (r), of miniature endplate potentials (MEPPs) at frog cutaneous pectoris neuromuscular junctions treated with black widow spider venom (BWSV). MEPP parameters are determined from the power spectrum of the fluctuating potential and the second (variance), third (skew), and fourth semi-invariants (cumulants) of high-pass-filtered records of the potential. The method gives valid results even when the mean potential undergoes slow changes unrelated to MEPPs and when the MEPP rate is not stationary; it detects changes in the distribution of MEPP amplitudes and corrects for the nonlinear summation of MEPPs. The effects of Ca2+ on BWSV-induced secretion are studied in detail. When Ca2+ is absent, the power spectrum of the fluctuations is shaped like the spectrum of w(t) and secretion is quasi-stationary; (r) rises smoothly to peak values of approximately 1,500/s and then quickly subsides to levels near 10/s. Many relatively small and some "giant" MEPPs occur at the ends of the experiments, and the distribution of MEPP amplitudes broadens. When the effects of this broadening are corrected for, we find that approximately 0.7 X 10(6) MEPPs occurred during the 30 min of intense secretion. Since BWSV depletes nerve terminals of their quanta of transmitter and their synaptic vesicles, this figure is an upper limit for the quantal store in a resting terminal. When Ca2+ is present, the noise spectrum deviates from the spectrum of w(t) and secretion is nonstationary; (r) rises to similar peak values but is sustained at levels near 400/s for up to an hour and at least 1.5 X 10(6) quanta are secreted within this period. Thus, the quantal store must have turned over at least twice under this condition. Data previously obtained at junctions treated with La3+ are corrected for nonlinear summation and for the distribution of MEPP amplitudes. The two corrections roughly compensate each other, and the corrected results confirm the previous conclusion that the number of quanta secreted from La3+-treated terminals during 1 h is not strongly dependent upon the extracellular concentration of Ca2+; approximately 2 X 10(6) quanta are released even when Ca2+ is absent.     

Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.     

KCl stimulation and ultrastructural responses of tannic acid-stained cholinergic synaptic terminals

The quantal-vesicular hypothesis equates miniature end-plate potentials (MEPPs) with fusions of synaptic vesicles. MEPP production thus predicts vesicle losses, increases in vesicle fusions and increases in terminal plasma membrane. MEPP production and these ultrastructural parameters have been evaluated in the cholinergic presynaptic terminals of skate electric organ following tannic acid saline incubation, known to promote capture and selective staining of dense-core granule fusions, and KCl stimulation, known to elevate MEPP production dramatically in these cholinergic terminals. After pretreatment in tannic acid-elasmobranch saline, KCl stimulation produced MEPPs at 40/s/microm(2)of terminal surface for several minutes with gradual reduction to spontaneous levels by 25-30 min. No loss of vesicles, no vesicle fusions, no expansions of plasma membrane and no tannic acid enhanced staining of vesicles or vacuoles accompanied the generation of 800 MEPPs/microm(3)of terminals having densities of 567 vesicles/microm(3). No ultrastructural footprints were found to support the notion that unnaturally high rates of vesicular exocytosis had occurred.     

Acetylcholinesterase mobility and stability at the neuromuscular junction of living mice

Acetylcholinesterase (AChE) is an enzyme that terminates acetylcholine neurotransmitter function at the synaptic cleft of cholinergic synapses. However, the mechanism by which AChE number and density are maintained at the synaptic cleft is poorly understood. In this work, we used fluorescence recovery after photobleaching, photo-unbinding, and quantitative fluorescence imaging to investigate the surface mobility and stability of AChE at the adult innervated neuromuscular junction of living mice. In wild-type synapses, we found that nonsynaptic (perisynaptic and extrasynaptic) AChEs are mobile and gradually recruited into synaptic sites and that most of the trapped AChEs come from the perijunctional pool. Selective labeling of a subset of synaptic AChEs within the synapse by using sequential unbinding and relabeling with different colors of streptavidin followed by time-lapse imaging showed that synaptic AChEs are nearly immobile. At neuromuscular junctions of mice deficient in alpha-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that alpha-dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses.     

An analysis of the relationship between the current and potential generated by a quantum of acetylcholine in muscle fibers without transverse tubules

Summary Miniature end plate potentials (MEPPs) were recorded in glyceroltreated muscle fibers with four microelectrodes which were used to determine the passive electrical characteristics of the same fibers. Voltage responses which were computed from miniature end plate currents (MEPCs) and the passive cable properties of a fiber, agreed very closely with experimentally recorded MEPPs confirming the hypothesis that MEPPs spread passively along a muscle fiber. The model was used to analyze the effect of variations in synaptic current and the properties of a muscle fiber on the postsynaptic response. The decrement of MEPPs was exponential for distances up to 1 to 2 mm from an origin but then deviated from the initial exponential. Variations in the growth time of the input current up to 1 msec had little effect on computed MEPPs whereas an increase in the decay time constant caused a significant increase in MEPP amplitude and effective space constant. An increase in the internal resistivity of a muscle fiber increased MEPP amplitude at the origin but decreased the effective space constant. The amplitude of MEPPs was inversely proportional to the 1.5 power of the diameter of a muscle fiber, and the MEPP space constant increased as the square root of the diameter. The amplitude of MEPPs is not necessarily determined by the input resistance of the muscle fiber. Changes in input resistance caused by changes in membrane resistance would have little effect on te amplitude or decrement of MEPPs.     

Multimodal Distribution of Frog Miniature Endplate Potentials in Adult, Denervated, and Tadpole Leg Muscle

Amplitude histograms of spontaneous miniature endplate potentials (MEPPs) from adult sartorius muscle cells show a definite bimodality with the mean amplitude of the larger mode five to seven times that of the smaller mode which accounted for 2–5 % of the total MEPPs. Histograms were plotted after high frequency MEPP generation induced by increasing temperature, increasing external calcium or nerve stimulation. These plots showed a reversible left-shift of the major mode as well as a reversible increase in the proportion of small mode MEPPs. Repeated challenges shifted almost all MEPPs into the small mode. An increase in the percentage of small mode MEPPs also occurred spontaneously during the course of denervation before the quiescent period and some of the histogram profiles showed multiple modes whose means were integer multiples of the small mode mean. In the early stages of hind leg development the greatest proportion of MEPPs were of the small mode size; as metamorphosis progressed, the histograms showed a definite multimodality with the mean of each mode being an integer multiple of the small mode mean and with the proportion of MEPPs in each mode about the same. During tail resorption the percentage of larger MEPPs increased until the adult histogram profile was reached. Thus, the changes in MEPP amplitude histograms over the course of metamorphosis are the reverse of those found with denervation.     

Role of Acetylcholinesterase on the Structure and Function of Cholinergic Synapses: Insights Gained from Studies on Knockout Mice

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE^+/+) or AChE KO (AChE^−/−) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE^−/− mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE^−/− mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE^−/− hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE^−/− mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE^+/+ mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE^−/− mice to function in the complete absence of AChE.     

Acetylcholinesterase inhibition in neuromuscular synapses in different background states: model studies

Using mathematical modeling of the process of generation of a miniature end-plate current (MEPC), we studied the effect of acetylcholinesterase (AChE) inhibition on the amplitude and frequency parameters of synaptic signals in the neuromuscular junction. The density of acetylcholine receptors on the postsynaptic membrane and the number of acetylcholine molecules in its quantum were varied. AChE inhibition against the background of a decreased receptor density was shown to result in a much higher increase in the amplitude of modeled MEPC than that in control and in the case of the changed transmitter amount released in the synaptic cleft. The simulation data can be used as a theoretical background for interpretation of the reason for different efficiencies of AChE inhibitors in certain pathological states of the neuromuscular apparatus.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 186–192, July–October, 1996.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.     

Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization

Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K(+) channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over butyrylcholinesterase--a plasma-based high activity esterase. Future in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication as well as other neuromuscular disorders.     

A small deletion in the olive fly acetylcholinesterase gene associated with high levels of organophosphate resistance

Organophosphate resistance in the olive fly was previously shown to associate with two point mutations in the ace gene. The frequency of these mutations was monitored in Bactrocera oleae individuals of increasing resistance. In spite of the difference in resistance among the individuals, there was no correlation between mutation frequencies and resistance level, indicating that other factors may contribute to this variation. The search for additional mutations in the ace gene of highly resistant insects revealed a small deletion at the carboxyl terminal of the protein (termed Delta3Q). Significant correlation was shown between the mutation frequency and resistance level in natural populations. In addition, remaining activity of acetylcholinesterase enzyme (AChE) after dimethoate inhibition was higher in genotypes carrying the mutation. These results strongly suggest a role of Delta3Q in high levels of organophosphate (OP) resistance. Interestingly, the carboxyl terminal of AChE is normally cleaved and substituted by a glycosylphosphatidylinositol (GPI) anchor. We hypothesize that Delta3Q may improve GPI anchoring, thus increasing the amount of AChE that reaches the synaptic cleft. In this way, despite the presence of insecticide, enough enzyme would remain in the cleft for its normal role of acetylcholine hydrolysis, allowing the insect to survive. This provides a previously un-described mechanism of resistance.     

Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca2+, Ba2+, and Sr2+ at the frog neuromuscular junction

Miniature endplate potentials (MEPPs) were recorded from frog sartorious neuromuscular junctions under conditions of reduced quantal contents to study the effect of repetitive nerve stimulation on asynchronous (tonic) quantal transmitter release. MEPP frequency increased during repetitive stimulation and then decayed back to the control level after the conditioning trains. The decay of the increased MEPP frequency after 100-to 200-impulse conditioning trains can be described by four components that decayed exponentially with time constants of about 50 ms, 500 ms, 7 s, and 80 s. These time constants are similar to those for the decay of stimulation-induced changes in synchronous (phasic) transmitter release, as measured by endplate potential (EPP) amplitudes, corresponding, respectively, to the first and second components of facilitation, augmentation, and potentiation. The addition of small amounts of Ca2+ or Ba2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with Sr2+, led to a greater increase in the stimulation-induced increases in MEPP frequency. The Sr-induced increase in MEPP frequency was associated with an increase in the second component of facilitation of MEPP frequency; the Ba-induced increase with an increase in augmentation. These effects of Sr2+ and Ba2+ on stimulation-induced changes in MEPP frequency are similar to the effects of these ions on stimulation- induced changes in EPP amplitude. These ionic similarities and the similar kinetics of decay suggest that stimulation induced changes in MEPP frequency and EPP amplitude have some similar underlying mechanisms. Calculations are presented which show that a fourth power residual calcium model for stimulation-induced changes in transmitter release cannot readily account for the observation that stimulation- induced changes in MEPP frequency and EPP amplitude have similar time- courses.     

Depolarization reverses age-related decrease of spontaneous transmitter release.

The effect of increasing extracellular Ca concentration on spontaneous transmitter release was studied at soleus nerve terminals of young (10 mo) and old (24 mo) C57BL/6J mice depolarized by high extracellular K concentration ([K]o). By using intracellular recording, miniature end-plate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solutions with four different Ca concentrations: Ca-free/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 0.5, 1.5, and 2.5 mM Ca. In both the normal [K]o Krebs and the Ca-free-high [K]o Krebs solutions, MEPP frequency was lower at old than at young nerve terminals. In the three high [K]o Krebs solutions with Ca, MEPP frequency was progressively higher at old than at young nerve terminals with higher Ca concentrations. Periodic oscillations were observed in MEPP frequency of depolarized nerve terminals. The period of oscillation was inversely proportional to spontaneous transmitter release. These results demonstrate that when the nerve terminal is depolarized, permeability of the terminal membrane to Ca increases because of opening of voltage-dependent Ca channels. In the present study resting MEPP frequency was lower at old than at young terminals. On depolarization, MEPP frequency became higher at old than at young terminals. The study demonstrates that voltage-dependent Ca entry increases during aging at the soleus nerve terminal.