Electrical Inexcitability of the Frog Neuromuscular Synapse

Frog muscle endplates were explored with an extracellular microelectrode. An intracellular microelectrode nearby simultaneously monitored invasion of the endplate by a spike directly evoked by a third microelectrode placed away from the endplate in the same fiber. External positivities were seen only at sites generating miniature endplate potentials. The external positivity reached a maximum prior to the internally recorded potential and was followed by a small late negativity. Small movements away from active synaptic sites resulted in positive-negative-positive potential sequences characteristic of activity and propagation. Since the external potential is a function of membrane current, the absence of negativity associated with the rising phase of the spike indicates the absence of inward current at synaptic sites. Thus, the synaptic membrane appears not to be excited by a depolarization of the magnitude of an action potential. In an Appendix it is shown that the late negativity and earlier maximum of the external potential can be accounted for by capacitative current through passive membrane.     

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.     

Phenobarbital Increases Spontaneous Transmitter Release at the Frog Neuromuscular Junction

Phenobarbital (1-2 × 10^-4M) markedly increases the frequency of miniature end-plate potentials at the neuromuscular synapse of the frog. This effect was seen in calcium free media containing EGTA. The drug probably acts presynaptically at an intracellular locus to increase the presynaptic free calcium concentration.     

免疫突触的形成及其介导的分子识别

T细胞和APC细胞相互作用形成免疫突触涉及到连续发生的一系列的分子识别事件,最初APC细胞在趋化因子的作用下向T细胞移动,相遇后在抗原非依赖性的弱的黏附力作用下发生最初的黏附,同时伴随着TCR在APC表面俘获特异性抗原;抗原识别之后,由多种机制使T细胞和APC紧密接触并维持一段时间,随后分开,最终引起T细胞的增殖和分化。对免疫突触形成过程中的分子识别机制目前尚无定论,拓扑模式和数学模式的解释,脂筏和细胞骨架蛋白的重排以及接头蛋白的连接为免疫突触形成中分子的识别提供了一定的依据。     

Perisynaptic Schwann Cells at the Neuromuscular Synapse: Adaptable,Multitasking Glial Cells

The neuromuscular junction (NMJ) is engineered to be a highly reliable synapse to carry the control of the motor commands of the nervous system over the muscles. Its development, organization, and synaptic properties are highly structured and regulated to support such reliability and efficacy. Yet, the NMJ is also highly plastic, able to react to injury and adapt to changes. This balance between structural stability and synaptic efficacy on one hand and structural plasticity and repair on another hand is made possible by the intricate regulation of perisynaptic Schwann cells, glial cells at this synapse. They regulate both the efficacy and structural plasticity of the NMJ in a dynamic, bidirectional manner owing to their ability to decode synaptic transmission and by their interactions via trophic-related factors.The vertebrate neuromuscular junction (NMJ), arguably the best characterized synapse in the peripheral nervous system (PNS), is composed of three closely associated cellular components: the presynaptic nerve terminal, the postsynaptic specialization, and nonmyelinating Schwann cells. These synapse-associated glial cells are called perisynaptic Schwann cells (PSCs), or terminal Schwann cells (see reviews by Todd and Robitaille 2006; Feng and Ko 2007; Griffin and Thompson 2008; Sugiura and Lin 2011). Multiple roles of PSCs have gained great appreciation since the 1990s and, along with the novel roles of astrocytes in central synapses, have led to the concept of the “tripartite” synapse (Araque et al. 1999, 2014; Volterra et al. 2002; Auld and Robitaille 2003; Kettenmann and Ransom 2013).Thus, to fully understand synaptic formation and function, it is critical to also consider the active and essential roles of synapse-associated glial cells. We will discuss evidence supporting the existence of a synapse–glia–synapse regulatory loop that helps maintain and restore synaptic efficacy at the NMJ. We will also explore the multiple functions that PSCs exert, functions that are adapted to a given situation at the NMJ (e.g., synapse formation, stability, and reinnervation). This will highlight the great adaptability and plasticity of the morphological and functional properties of PSCs.In this review, we will focus on the multiple roles PSCs play in synaptic formation, maintenance, remodeling, and regeneration, as well as synaptic function and plasticity. Based on the evidence presented, we propose a model in which PSCs, through specific receptor activation, play a prominent role in a continuum of synaptic efficacy, stability, and plasticity at the NMJ. These synaptic-regulated functions allow PSCs to orchestrate the stability and plasticity of the NMJ and, hence, are important for maintaining and adapting synaptic efficacy.     

Differences between Spontaneous and Evoked Monoquantum Signals in the Frog Neuromuscular Junction

In experiments on the frog cutaneous-pectoris muscle, the amplitude-temporal parameters of monoquantum end-plate currents (EPC) and miniature EPC (mEPC) were investigated using extracellular recording. A significant dependence of the risetime of the signals on their amplitude was found after analyzing mEPC; at the same time, such dependence was absent for EPC. Approaches leading to disorganization of the active zones (AZ) of the nerve ending (NE), prolonged action of a Ca-free solution, and denervation resulted in an increased dependence of the risetime of the monoquantum signals on their amplitude; moreover, these dependences were similar for both mEPC and monoquantum EPC. Mathematical simulation showed that the obtained data could be explained by the spatial heterogeneity of the sites of spontaneous and evoked transmitter release within the regions of the AZ. A new hypothesis interpreting spontaneous and evoked transmitter release is proposed.     

Localization of Butyrylcholinesterase at the Neuromuscular Junction of Normal and Acetylcholinesterase Knockout Mice

At the mouse neuromuscular junction (NMJ), there are two distinct cholinesterases (ChE): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Until now, it has been difficult to determine the precise localization of BChE at the NMJ. In this study, we use a modification of Koelle''s method to stain AChE and BChE activity. This method does not interfere with fluorescent co-staining, which allows precise co-localization of ChE and other synaptic molecules at the NMJ. We demonstrate that AChE and BChE exhibit different localization patterns at the mouse NMJ. AChE activity is present both in the primary cleft and in the secondary folds, whereas BChE activity appears to be almost absent in the primary cleft and to be concentrated in subsynaptic folds. The same localization for BChE is observed in the AChE-knockout (KO) mouse NMJ. Collagenase treatment removed AChE from the primary cleft, but not from secondary folds in the wild-type mouse, whereas in the AChE-KO mouse, BChE remains in the secondary folds. After peripheral nerve injury and regeneration, BChE localization is not modified in either normal or KO mice. In conclusion, specific localization of BChE in the secondary folds of the NMJ suggests that this enzyme is not a strict surrogate of AChE and that the two enzymes have two different roles. (J Histochem Cytochem 58:1075–1082, 2010)     

Recycling of Synaptic Vesicles at the Frog Neuromuscular Junction in the Presence of Strontium

Abstract: In these experiments, we followed the exocytosis and endocytosis of synaptic vesicles with the vital dye FM1-43 and asked whether calcium is important for membrane retrieval at the frog neuromuscular junction. We replaced calcium with equimolar amounts of strontium and monitored the staining of recycling vesicles by inducing exocytosis with electrical stimulation. Trains of 2,400 (2 or 20 Hz) or 4,200 (20 Hz) pulses failed to induce FM1-43 internalization in the presence of strontium, but they did in the presence of calcium. This effect of strontium was not due to a decrease in exocytosis, because FM1-43 release was similar in the presence of calcium or strontium. The impairment in endocytosis, observed as inhibition of FM1-43 internalization, could be overcome by longer periods of stimulation (6,000 pulses at 2 or 20 Hz) in the presence of strontium (1.8 m M ) or by increasing the extracellular concentration of strontium to 10 m M (2,400 action potentials at 20 Hz). It is suggested that endocytosis is dependent on calcium influx and that strontium is much less effective in replacing calcium for endocytosis than it is for exocytosis.     

Mechanisms of the Effect of Dimephosphone on Synaptic Transmission in the Frog Neuromuscular Junction

We studied the influence of dimephosphone, an organophosphorus drug with a broad spectrum of therapeutic effects on the peripheral and central nervous systems, on postsynaptic end-plate currents (EPC) in the frog neuromuscular junction. Dimephosphone was demonstrated to decrease in a voltage-independent manner the EPC amplitude and to prolong the EPC decay. These effects are not related to inhibition of acetylcholinesterase. We propose a theoretical interpretation of the observed phenomena based on the model of blockade of an open ion channel of the acetylcholine receptor and conclude that postsynaptic receptors are one of the most probable targets for the action of dimephosphone.     

“Disjunction” of Frog Neuromuscular Synapses by Treatment with Proteolytic Enzymes

VERTEBRATE skeletal muscle fibres are surrounded by the ectolemma or basement membrane, a thin sheath of filamentous material. At the neuromuscular junction, the ectolemma occupies the synaptic cleft and is continuous with a similar material which surrounds the axon terminal and its Schwann cell covering¹ (Fig. I A). Changes in the ectolemma of atrophic muscles have been observed^{2, 3}, but little is known about the structure and function of this material. The study described here demonstrates that certain proteolytic enzymes selectively digest the ectolemmal sheath and that concomitantly the motor nerve terminals and their associated Schwann cells dissociate from the muscle fibres.     

Statistical Estimation of Distribution of Quantal Release of Transmitter in Neuromuscular Synapse of the Lobster Homarus americanus

A new approach to estimation of quantal release distribution of transmitter under conditions of high synaptic activity is presented. Postsynaptic responses of neuromuscular excitatory synapse in muscle-opener of nipper of the lobster, which are obtained by focal extracellular recording, are used as original data set. Based on two data groups (value of evoked and spontaneous postsynaptic responses), the linear regression model is constructed. Parameters of this model describe completely the quantal release distribution. To evaluate the parameters, biased modifications of the least squares method—the penalized least squares method and the principal components method—were applied. As a result, it was possible to achieve estimations of the quantal release distribution with sufficiently low standard errors. Modeling studies have shown that the gain of accuracy of the estimation due to a decrease of the standard error exceeds considerably losses caused by its bias.     

Estimation of the Temporal Course of Evoked Secretion of Transmitter Quanta in the Frog Neuromuscular Junction

In experiments on the cutaneothoracic muscle of the frog, we recorded, using the technique of two-electrode voltage clamp at a normal Ca²⁺ concentration (1.8 mM), multiquantum end-plate currents (EPC) and miniature uniquantum EPC (mEPC). Multiquantum signals, when compared with uniquantum currents, were characterized by longer leading and trailing edges. The quantum composition of multiquantum signals estimated according to the ratios of EPC and mEPC amplitudes was, on average, 27% lower than that calculated according to the ratios of their integral values (areas). These data demonstrate that stimulus-evoked transmitter secretion from the motor nerve endings is noticeably asynchronous. Based on the parameters of the experimental EPC and mEPC, we estimated the temporal course of evoked secretion using various techniques: spectral analysis, a system of linear equations, and Van der Kloot's method. Using convolution with uniquantum signals, we found that spectral analysis is the best technique for such estimation. Calculated parameters of the temporal course of secretion were the following: risetime 0.20 msec and decay time constant 0.33 msec. The respective distribution significantly differed from that of the synaptic delays of extracellularly recorded uniquantum EPC by longer durations (150-200%) of the leading and trailing edges. We hypothesize that these differences are related to the geometry of the junction and the temporal sequence of switching on of the active zones in the nerve ending upon their activation by spreading action potentials. Factors influencing the temporal course of evoked secretion of the transmitter in the junction under study (its asynchronicity, in particular) are discussed.     

Effects of Glucocorticoids and Epinephrine on the Modulatory Action of Purines in the Frog Neuromuscular Junction

The effect of corticoids (hydrocortisone and dexamethasone) and epinephrine on the presynaptic action of purines was studied at the neuromuscular junction of the frog under two-electrode voltage-clamp conditions. Daily administration of hydrocortisone/dexamethasone (100 mg/kg into the lymphatic system) increased initially and later depressed the amplitude of multiquantum end-plate currents evoked by motor nerve stimulation. An initial facilitatory phase of the hormone action was accompanied by removal of the presynaptic action of ATP (for hydrocortisone only). Within the later phase (2 weeks of hydrocortisone administration), the inhibitory action of ATP was restored once again. The counteraction of ATP effect was reproduced under superfusion of the isolated muscle by a physiological solution containing hydrocortisone (not dexamethasone), indicating the nongenomic nature of the action of the hormone on presynaptic P2 receptors. This proved to be true in experiments on animals, which were stressed 30 min prior to the beginning of the experiment by electrical stimulation in a special cage. Independently of acute or chronic administration of hydrocortisone, the presynaptic action of another purine, adenosine, was preserved. Epinephrine only partially abolished the inhibitory effect of purines, which is indicative of the difference in the paths of incorporation of the biological effects of these agents. We suggest that prevention of the inhibitory action of ATP might be one of the components of a facilitatory acute stress reaction, while such an inhibitory feedback action is missing under chronic stress conditions.     

Localization of the Repetitive Firing Generation Site Induced by 4-Aminopyridine at the Frog Neuromuscular Junction

In the neuromuscular junction, blockade of potassium channels can produce multiple discharges after single nerve stimulation. Multielectrode recording from the nerve trunk and myelinated and non-myelinated parts of the nerve ending demonstrated that repetitive presynaptic spikes elicited by 4-aminopyridine arise earliest within the part of the axon proximal to the motor nerve ending.     

The Location of Photopigment Molecules in the Cross-Section of Frog Retinal Receptor Disk Membranes

The location of the photopigment molecules relative to the lipid hydrocarbon core of retinal receptor disk membranes was unknown. The photopigment molecules could occur entirely on the surface of the membrane, completely embedded in its hydrocarbon core, or at some intermediate location protruding into both the aqueous surface layer and the lipid core of the disk membrane. To resolve this uncertainty, we collected X-ray intensity data diffracted by the photopigment molecules in wet pellets of oriented frog retinal receptor disk membranes as a function of the electron density of the sedimentation medium. These data were fitted to a model which predicted the integrated intensity diffracted from the photopigment molecules as a function of the electron density of the sedimentation medium and the extent to which the molecule protruded into the aqueous surface layer and the lipid core of the disk membrane. This analysis showed that for the photopigment molecular diameter of ~42 A, about 28 A protrudes into the aqueous layer, and about 14 A into the lipid core for unbleached photopigment. Bleaching causes the photopigment to “sink” into the lipid core some 7 A. The partial embedding of the photopigment molecules in the lipid core introduces a correlation of the photopigment molecules with lipid hydrocarbon chains in the plane of the disk membranes.     

Temperature Effect on Carbachol-Induced Depression of Spontaneous Quantal Transmitter Release in Frog Neuromuscular Junction

The effects of carbachol (CCh) on the frequency (f) of the miniature endplate potentials were tested at temperatures between 5 and 30°C. Higher CCh concentrations, 1 × 10^–5 and 5 × 10^–6 M, reduced the f to 60% and the temperature dependence was negligible. However, an inverse temperature dependence was found when low concentrations 3 × 10^–7 and 6 × 10^–7 M were applied. The depression of f was 40–50% in 5–10°C but only 10–20% of the control in the 25 and 30°C. During application of CCh, the new steady of f was reached at temperatures between 5 and 30°C within 17–20 min (Q₁₀ = 1.07). Much greater temperature dependence of recovery was observed during washing out CCh (Q₁₀ = 1.6). The temperature-independence of the steady state effects of CCh, good agreement with Langmuir adsorption-desorption theory and non-steady kinetics indicate that physical rather than receptor-mediated events are responsible for the depression of f.