Effects of ascorbic acid on lead induced alterations of synaptic transmission and contractile features in murine dorsiflexor muscle

Lead is a common environmental toxin that affects neuromuscular junction and potentially might cause muscle weakness. Antioxidants like ascorbic acid may protect against lead induced myopathy. The present study measured isometric twitch tensions (evoked either directly by muscle stimulation or indirectly by nerve stimulation) to study effects of ascorbic acid on lead induced alterations at murine dorsiflexor skeletal muscle. Resting membrane potentials (RMPs), endplate potentials (EPPs) and miniature endplate potentials (MEPPs) were also recorded. Forty animals were divided into four groups of n = 10 each. (10 control, 10 lead alone, 10 ascorbic acid alone, 10 lead treated plus ascorbic acid). Lead (1 mg/kg) i.p, was administered daily for 2 weeks before the recording day and ascorbic acid (200 mg/kg, i.p) was given daily for 3 weeks prior to the experiment day. Lead treatment reduced twitch tension significantly (from 4.3 +/- 0.5 g to 2.7 +/- 0.2 g) and delayed half time of decay compared to the control. Similarly MEPPs frequencies were reduced following lead treatment. Application of ascorbic acid prevented twitch tension reduction in lead treated mice (3.3 +/- 0.3 g) and reversed lead induced delay in half time of decay. The negative actions of lead treatment on MEPPs frequencies were also modified with ascorbic acid. It appears that ascorbic acid exerts a protective role against lead induced peripheral nerve and muscle dysfunction. This effect of ascorbic acid on lead induced neuromyopathy is probably mediated via a free radical scavenging mechanism or modification of Ca(2+) homeostasis.     

Dehydration affects synaptic transmission at flexor muscle in acute lead-treated mice.

The effect of 24 hrs. water deprivation on spontaneous and evoked transmitter release was studied at flexor nerve terminals of control and lead-treated male C57BL mice. Miniature endplate potentials (MEPPs) and endplate potentials (EPPs) were recorded intracellularly from urethane-anesthetized (2 mg/g, i.p.) control and lead exposed mice in both hydrated and dehydrated conditions. Exposure to lead was made by i.p. injection of lead acetate (1.0 mg/kg) dissolved in a 5% glucose solution 24 hrs. prior to the experiment. Unimodal and bimodal MEPP frequencies decreased with dehydration, while small mode MEPPs remained unchanged and large mode MEPPs increased in frequency. EPP amplitude and quantal content were unchanged by dehydration. Lead treatment by itself reduced the frequency of unimodal and bimodal MEPPs but had no effect on the amplitude of EPPs or of quantal content. However a combination of dehydration and acute lead treatment reduced the frequency of unimodal, bimodal and large mode MEPPs and significantly reduced both EPP amplitude and quantal content. Dehydration apparently reveals an underlying neurotoxic action of lead at the neuromuscular junction. This raises a health concern that people subjected to both lead pollution and dehydration are at greater risk to lead poisoning of the neuromuscular junction.     

Adenosine A2A Receptors Activation Facilitates Neuromuscular Transmission in the Pre-Symptomatic Phase of the SOD1(G93A) ALS Mice,but Not in the Symptomatic Phase

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to motor neuron dysfunction resulting in impairment of neuromuscular transmission. A_2A adenosine receptors have already been considered as a potential therapeutical target for ALS but their neuromodulatory role at the neuromuscular junction in ALS remains to be clarified. In the present work, we evaluated the effects of A_2A receptors on neuromuscular transmission of an animal model of ALS: SOD1(G93A) mice either in the pre-symptomatic (4–6 weeks old) or in the symptomatic (12–14 weeks old) stage. Electrophysiological experiments were performed obtaining intracellular recordings in Mg²⁺ paralyzed phrenic nerve-hemidiaphragm preparations. Endplate potentials (EPPs), quantal content (q. c.) of EPPs, miniature endplate potentials (MEPPs) and giant miniature endplate potential (GMEPPs) were recorded. In the pre-symptomatic phase of the disease (4–6 weeks old mice), the selective A_2A receptor agonist, CGS 21680, significantly enhanced (p<0.05 Unpaired t-test) the mean amplitude and q.c. of EPPs, and the frequency of MEPPs and GMEPPs at SOD1(G93A) neuromuscular junctions, the effect being of higher magnitude (p<0.05, Unpaired t-test) than age-matched control littermates. On the contrary, in symptomatic mice (12–14 weeks old), CGS 21680 was devoid of effect on both the amplitude and q.c. of EPPs and the frequency of MEPPs and GMEPPs (p<0.05 Paired t-test). The results herein reported clearly document that at the neuromuscular junction of SOD1(G93A) mice there is an exacerbation of A_2A receptor-mediated excitatory effects at the pre-symptomatic phase, whereas in the symptomatic phase A_2A receptor activation is absent. The results thus suggest that A_2A receptors function changes with ALS progression.     

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.     

Obidoxime antagonizes the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate via different mechanisms

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC₅₀: 300 µM) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC₅₀: 0.6 µM) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC₅₀ of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC₅₀ against DFP, at a concentration (30 µM) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation.     

alpha-Tocopherol modifies lead induced functional changes at murine neuromuscular junction

Lead impacts neuromuscular junction and might induce skeletal muscle weakness. Antioxidants may prevent toxic actions of lead on muscle. In this study, resting membrane potentials, endplate potentials, miniature endplate potentials (MEPPs) and isometric twitch tensions were recorded to investigate effects of alpha-tocopherol (Vitamin E) on lead induced changes at murine dorsiflexor muscle. Moreover, levels of endplate nicotinic receptors were measured by receptor autoradiography. Forty rats were divided into four groups (lead alone, alpha-tocopherol, lead plus alpha-tocopherol and saline). Lead (1 mg/kg, i.p.), was administered daily for 2 weeks and alpha-tocopherol (100 mg/kg, i.p.) was given daily for 3 weeks. Lead treatment significantly reduced twitch tension (from 4.4+/-0.4 to 2.2+/-0.3 g) and delayed half time of decay. MEPP frequencies and quantal content were also significantly reduced after lead treatment. Pretreatment with alpha-tocopherol reversed twitch tension reduction (4.1+/-0.3 g) and modified lead induced delay in half time of decay. Similarly, alpha-tocopherol modified the negative actions of lead exposure on MEPP frequencies and quantal content. Receptor autoradiographic studies revealed significant increase of nicotinic receptor levels at the endplate region of flexor muscle in lead treated mice. However, animals treated with lead plus alpha-tocopherol showed significantly decreased levels of nicotinic receptors. alpha-Tocopherol appears to protect against lead induced neuromuscular dysfunction. These effects of alpha-tocopherol are possibly mediated via a free radical mechanism or modification of calcium homeostasis.     

α-Tocopherol Modifies Lead Induced Functional Changes at Murine Neuromuscular Junction

Lead impacts neuromuscular junction and might induce skeletal muscle weakness. Antioxidants may prevent toxic actions of lead on muscle. In this study, resting membrane potentials, endplate potentials, miniature endplate potentials (MEPPs) and isometric twitch tensions were recorded to investigate effects of α-tocopherol (Vitamin E) on lead induced changes at murine dorsiflexor muscle. Moreover, levels of endplate nicotinic receptors were measured by receptor autoradiography. Forty rats were divided into four groups (lead alone, α-tocopherol, lead plus α-tocopherol and saline). Lead (1?mg/kg, i.p.), was administered daily for 2 weeks and α-tocopherol (100?mg/kg, i.p.) was given daily for 3 weeks. Lead treatment significantly reduced twitch tension (from 4.4±0.4 to 2.2±0.3?g) and delayed half time of decay. MEPP frequencies and quantal content were also significantly reduced after lead treatment. Pretreatment with α-tocopherol reversed twitch tension reduction (4.1±0.3?g) and modified lead induced delay in half time of decay. Similarly, α-tocopherol modified the negative actions of lead exposure on MEPP frequencies and quantal content. Receptor autoradiographic studies revealed significant increase of nicotinic receptor levels at the endplate region of flexor muscle in lead treated mice. However, animals treated with lead plus α-tocopherol showed significantly decreased levels of nicotinic receptors. α-Tocopherol appears to protect against lead induced neuromuscular dysfunction. These effects of α-tocopherol are possibly mediated via a free radical mechanism or modification of calcium homeostasis.     

In vitro reinnervation of adult rat muscle fibres by foreign neurons and transformed chromaffin PC12 cells

Adult rat muscle fibres were dissociated by using collagenase and maintained in culture. One to nine days later, neurons obtained from stages 22-30 Xenopus laevis embryos, or neonatal spinal cord, or pheochromocytoma (PC12) cells treated with nerve growth factor were added. Subsequently, the co-cultures were maintained for up to eight days. Functional synapses were formed with variable efficiency: 12% in rat-Xenopus nerve-muscle co-cultures, 23% in rat-rat and 33% in PC12 co-cultures. Miniature endplate potentials (MEPPs) and currents (MEPCs) were recorded, at frequencies ranging from 0.01 to 0.9 Hz. Their mean amplitude was smaller than in normal mammalian muscles. The rise time and time-constant of decay of MEPCs was about seven to ten times longer than that found in the original muscle, resembling immature synapses. (+)-Tubocurarine abolished the MEPPs in the rat-PC12 neuromuscular junctions. It is concluded that dissociated adult rat muscle fibres retain their ability of being reinnervated, and can form functional synapses with foreign neurons and transformed chromaffin cells.     

Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A_2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A₁ receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4–6 weeks old) and symptomatic (12–14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg²⁺ paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N⁶-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.     

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.     

The role of pannexin 1 in the purinergic regulation of synaptic transmission in mouse motor synapses

The role of pannexin 1 in the release to the extracellular space of ATP/adenosine modulating the acetylcholine (ACh) secretion was studied in mouse diaphragm motor synapses. Using neuromuscular preparations obtained from wild-type and pannexin-1 knockout mice, the miniature endplate potential (MEPPs) and evoked endplate potentials (EPPs) were recorded in combination with pharmacological modulation of P2-type ATP receptors and A₁-type adenosine receptors. Selective inhibition of A₁ receptors with DPCPX or P2 receptors with PPADS increased quantal content of EPPs in wild-type mice. MRS 2211, selective antagonist of P2Y13 receptors, produced the same effect. Activation of receptors A₁ or P2Y₁₃ by their agonists (2-CADO and IDP, respectively) decreased the EPP quantal content. It means that the activity of endogenous ATP and adenosine is synergistic and directed to depression of the ACh release. ARL67156, an inhibitor of synaptic ecto-ATPases, which blocks the hydrolysis of ATP to adenosine and increases the level of ATP in the synaptic cleft, prolonged EPPs without changing their quantal content. In pannexin-1 knockout mice there were no changes in the EPP quantal content and in other parameters of synaptic transmission as compared to wildtype mice. However, downregulation of purinergic effects with antagonists of A₁ or P2 receptors (DPCPX, PPADS, MRS 2211) did not change EPP quantal content and any other parameters of spontaneous or evoked ACh release in all cases. ARL67156 did not alter the temporal parameters of EPPs, either. Nevertheless, 2-CADO, the A₁-type receptor agonist, decreased the EPP quantal content, while the agonist of P2Y₁₃ receptors decreased the MEPP amplitude. Thus, in mice lacking pannexin 1, procedures revealing the presence and regulatory activity of synaptic ATP/adenosine did not change the parameters of synaptic transmission. The obtained data substantiate a mandatory role of pannexin 1 in the purinergic regulation of motor synapse activity by endogenous ATP/adenosine.     

LY 294002 inhibits adenosine receptor activation by a mechanism independent of effects on PI-3 kinase or casein kinase II

Adenosine reduces both evoked and spontaneous calcium-dependent acetylcholine (ACh) release through a mechanism downstream of calcium entry at amphibian motor nerve endings (Silinsky EM. J Physiol 1984; 346: 243–56). LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), an inhibitor of both phosphoinositide-3 kinase (PI-3 kinase) and casein kinase II, has been reported to increase spontaneous ACh release reflected in miniature endplate potential (MEPP) frequencies independently of intraterminal calcium at the frog neuromuscular junction (Rizzoli SO, Betz WJ. J Neurosci 2002; 22: 10680–9). It has been suggested that the increase in MEPP frequency caused by LY 294002, is mediated through an action on synaptotagmins, vesicle associated calcium sensors believed to trigger synaptic vesicle exocytosis. We thus examined the effects of adenosine on MEPP frequencies and evoked ACh release reflected as endplate potentials (EPPs) in order to determine if the presumed calcium-independent ACh release is affected by adenosine. We also wanted to determine if PI-3 kinase or casein kinase II is involved in mediating or modulating the inhibitory effects of adenosine. To these ends, we examined the effects of adenosine in the presence of LY 294002, wortmannin (a highly selective the PI-3 kinase inhibitor), or DRB (5,6-dichlorobenzimidazole riboside, an inhibitor of casein kinase II). LY 294002 reduced the sensitivity of both MEPP frequencies and the nerve-evoked calcium dependent EPPs to adenosine. The occlusive effects of LY 294002 on the actions of adenosine on MEPPs and EPPs were overcome by increasing adenosine concentration. Neither wortmannin nor DRB had any effect on the sensitivity of the EPPs to adenosine indicating that neither PI-3 kinase nor casein kinase II inhibition mediates the reduction in motor-nerve terminal sensitivity to adenosine produced by LY 294002. The results indicate a competitive relationship between LY 294002 and adenosine at A₁ receptors at the frog neuromuscular junction. This effect is independent of the previously described effects of LY 294002 on the exocytotic process, and is also independent of PI-3 kinase or casein kinase II.     

Mechanism of P2X7 receptor-dependent enhancement of neuromuscular transmission in pannexin 1 knockout mice

P2X7 receptors are present in presynaptic membranes of motor synapses, but their regulatory role in modulation of neurotransmitter release remains poorly understood. P2X7 receptors may interact with pannexin 1 channels to form a purinergic signaling unit. The potential mechanism of P2X7 receptor-dependent modulation of acetylcholine (ACh) release was investigated by recording miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in neuromuscular junctions of wild-type (WT) and pannexin 1 knockout (Panx1^?/?) mice. Modulation of P2X7 receptors with the selective inhibitor A740003 or the selective agonist BzATP did not alter the parameters of either spontaneous or evoked ACh release in WT mice. In Panx1^?/? mice, BzATP-induced activation of P2X7 receptors resulted in a uniformly increased quantal content of EPPs during a short stimulation train. This effect was accompanied by an increase in the size of the readily releasable pool, while the release probability did not change. Inhibition of calmodulin by W-7 or of calcium/calmodulin-dependent kinase II (CaMKII) by KN-93 completely prevented the potentiating effect of BzATP on the EPP quantal content. The blockade of L-type calcium channels also prevented BzATP action on evoked synaptic activity. Thus, the activation of presynaptic P2X7 receptors in mice lacking pannexin 1 resulted in enhanced evoked ACh release. Such enhanced release was provoked by triggering the calmodulin- and CaMKII-dependent signaling pathway, followed by activation of presynaptic L-type calcium channels. We suggest that in WT mice, this pathway is downregulated due to pannexin 1-dependent tonic activation of inhibitory presynaptic purinergic receptors, which overcomes P2X7-mediated effects.     

Changes in the Parameters of Quantal Acetylcholine Release after Activation of PAR1-Type Thrombin Receptors at the Mouse Neuromuscular Junctions

In mature and newly formed neuromuscular synapses of mouse skeletal muscles, miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by a single stimulation of the nerve were recorded using intracellular microelectrode technique. The mechanisms underlying the changes in spontaneous and evoked acetylcholine (ACh) release caused by the activation of PAR1-type muscle receptors induced by their peptide agonist TRAP6-NH₂ were studied. It has been shown for the first time that, in either mature or newly formed motor synapses, the activation of PAR1 that lack presynaptic localization causes a sustained increase in the MEPP amplitude due to the increase in the ACh quantal size at the presynaptic level. It was found that phospholipase C (PLC) participates in the signaling mechanism triggered by the PAR1 activation. Exogenously applied brain-derived neurotrophic factor (BDNF) mimics the effect of activation of PAR1 by TRAP6-NH₂. Moreover, an increase in the MEPP amplitude caused by the peptide PAR1 agonist was fully prevented by blocking the BDNF receptors–tropomyosin receptor kinases B (TrkB). Thus, it has been shown for the first time that the increase in ACh quantal size due to the activation of PAR1 in motor synapses is mediated by a complex signaling cascade that starts at the postsynaptic level of the motor synapse and ends at the presynaptic level. It is expected that the activation of PAR1 at the muscle fiber membrane followed by the PLC upregulation results in the release of neurotrophin BDNF as a retrograde signal. Its effect on the presynaptic TrkB receptors triggers the cascade leading to an increase in the quantal size of ACh.     

A computer system for real-time data acquisition and analysis of biopotentials and quantal content at the neuromuscular junction

A computerized data acquisition system for on-line analysis of the parameters of neuromuscular transmission is described. Both hardware usage and software methodologies are discussed with regard to sampling in real-time and analyzing miniature end-plate potentials (MEPPs), end-plate potentials (EPPs) and quantal content of the evoked transmitter release. Significant features of the program include: (1) automatic threshold determination for MEPP detection; (2) the use of a circular buffer to give pre-trigger information; (3) real-time noise spike rejection; (4) an automatic procedure for EPP failure detection; (5) rapid quantal content determinations by several methods as well as complete MEPP and EPP waveform analysis. The system has proven both accurate and reliable during more than two years of use. Advantages of the system over conventional methods include: (1) increased accuracy and efficiency in data analysis; (2) immediate availability of results; (3) conventional data storage; (4) flexibility to meet changing requirements.     

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.     

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.     

秋水仙素对大鼠神经肌肉接头传递的作用

在不均匀牵拉法固定的离体大鼠膈神经膈肌标本上,用秋水仙素探讨了突触后膜的微管在神经肌肉接头乙酰胆碱受体兴奋中的作用。秋水仙素使小终板电位的幅度下降;使串刺激(10Hz,50Hz)诱发的平均终板电位幅度和平均量子含量减少;并使有神经支配的膈肌终板区乙酰胆碱电位幅度降低;但不影响膜电位、小终板电位的频率及终板电位和乙酰胆碱电位的时程。结果表明该药对神经肌肉接头乙酰胆碱受体的兴奋有抑制作用。而其同分异构体光化秋水仙素则无此作用。据此本文提出突触后膜下的微管可能参与神经肌肉接头乙酰胆碱受体的反应过程。     

Habitual exercise enhances neuromuscular transmission efficacy of rat soleus muscle in situ.

Rat motor nerve terminals and the endplates they interact with exhibit changes to varying patterns of use, as when exposed to increased activation in the form of endurance exercise training. The extent to which these changes affect neuromuscular transmission efficacy is uncertain. In this study, the effects of habitual exercise on the electrophysiological properties of neuromuscular transmission in rat soleus muscle were investigated using a novel in situ approach. Consistent with previous reports, miniature endplate potential frequency was enhanced by habitual exercise. Other passive properties, such as resting membrane potential, miniature endplate potential amplitude, and "giant" miniature endplate potential characteristics were unaltered by the training program. Full-size endplate potentials were obtained by blocking soleus muscle action potentials with mu-conotoxin GIIIb. Quantal content values were 91.5 and 119.9 for control and active groups, respectively (P < 0.01). We also measured the rate and extent of endplate potential amplitude rundown during 3-s trains of continuous stimulation at 25, 50, and 75 Hz; at 50 and 75 Hz, we found both the rate and extent of rundown to be significantly attenuated (10--20%) in a specific population of cells from active rats (P < 0.05). The results establish the degree of activity-dependent plasticity as it pertains to neuromuscular transmission in a mammalian slow-twitch muscle.     

Development of the myotomal neuromuscular junction in Xenopus laevis: An electrophysiological and fine-structural study

The normal development of the myotomal neuromuscular junction in Xenopus embryos and tadpoles was investigated electrophysiologically as well as electron microscopically. Spontaneous potentials, considered to be miniature end-plate potentials (MEPPs), were detected by intracellular recording as early as stage 21 and by stage 24 they were observed in every embryo tested. Like MEPPS at later stages they were blocked by curare but not by tetrodotoxin. End-plate potentials (EPPs), subject to block by tetrodotoxin, were evoked by electrical stimulation of the spinal cord in embryos as young as stage 24 and occurred spontaneously as early as stage 22. The durations of MEPPs and EPPs were initially relatively long. Focal external recordings revealed an eightfold decrease in duration during the course of development. Nerve processes emerged from the spinal cord and contacted developing muscle cells as early as stage 21, but junctional specializations were not apparent and vesicles were rare even in stage 24 embryos. During the next 24 hr, between stages 25 and 36, vesicles increased in number and became localized toward the junctional surface of the nerve ending. Basement lamina developed in the cleft and postjunctional ridges and densities were observed. Individual muscle cells also became contacted by several nerve processes. By stages 48–52 there were fewer contacts on individual muscle cells and Schwann cell processes partially covered the nerve endings. Gap junctions were observed between the muscle cells throughout development but occurred less frequently at the later stages. It is concluded that by the time they reach the muscle cells, or very shortly thereafter, at least some of the growing nerve processes can release transmitter, and some of the muscle cells are sufficiently sensitive to acetylcholine in the region of contact to respond with millivolt depolarizations. These earliest functional contacts, however, are morphologically undifferentiated.