QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : I. The Number of Acetylcholinesterase Molecules and of Other DFP-Reactive Sites at Motor Endplates, Measured by Radioautography

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-³²P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10⁷ sites per endplate, in the diaphragm, about 3 x 10⁷. Reaction with DFP-³²P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10⁷ and 1.1 x 10⁷per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.     

QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : II. Confirmation of Radioautographic Measurement by Liquid-Scintillation Counting

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-³H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10⁷molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHY AS A QUANTITATIVE TOOL IN ENZYME CYTOCHEMISTRY : II. The Distribution of DFP-Reactive Sites at Motor Endplates of A Vertebrate Twitch Muscle

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-³H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per µ³ of cleft tissue or 12,000 sites per µ² of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)—a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.     

The ultrastructural localization and quantitation of cholinergic receptors at the mouse motor endplate

Summary The snake venom toxin, -bungarotoxin, is known to bind specifically to the acetylcholine receptor at skeletal muscle endplates. In this study, tritiated -bungarotoxin has been used in conjunction with electron-microscope autoradiography to visualize and enumerate acetylcholine receptor sites at the neuromuscular junctions of the mouse diaphragm. From an analysis of the grain distribution, the receptor sites appear to be located specifically on the postjunctional membrane. The density there is about 8,500/² of membrane surface. For comparison purposes, cholinesterases and related active centers were labeled using [³H] diisopropylfluorophosphate; they were shown to be at this same concentration over the synaptic membranes (or along the cleft). The 11 relationship of the receptors to the cholinesterase type of site, found previously to hold in studies on whole endplates, is also true at the ultrastructural level in this case. In fact, this 11 relationship is believed to be a characteristic of the postsynaptic membranes of endplates in other muscles and other vertebrates.Based on the constant density value thus arrived at, the total surface areas of postsynaptic and of presynaptic membranes are at once obtained from the known total numbers of these sites per endplate, available from previous studies in this laboratory. Examples of such synaptic surface area values are given. These values are only reliable for a given muscle type if the approximate fiber size is defined.     

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.     

Development of rat soleus endplate membrane following denervation at birth

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.     

The voltage-dependent sodium channel is co-localized with the acetylcholine receptor at the vertebrate neuromuscular junction

Isolated motor endplates from mouse intercostal muscles can be obtained after subcellular fractionation. On these motor endplates, localization of the nicotinic receptor and of the voltage-dependent Na+ channel coincides as demonstrated by double labeling with rhodamine alpha-bungarotoxin and a specific anti-Na+ channel monoclonal antibody. High density of Na+ channel at the motor endplate is confirmed by the enrichment in TTX binding sites as compared to the crude homogenate. In contrast isolated motor endplates are almost completely devoid of Ca2+ channel antagonist binding sites.     

Pectoralis muscle morphology in the little brown bat,Myotis lucifugus: A non-convergence with birds

Recent studies of muscle architecture demonstrate that many mammalian muscles are composed of short, interdigitating fibers. In addition, the avian pectoralis, a muscle capable of producing high frequency oscillations has been shown to possess a serially arranged pattern of muscle endplate in all sizes of birds studied. The pectoralis muscle of the little brown bat, Myotis lucifugus (Chiroptera: Vespertilionidae), is composed of fairly uniform fibers that span the length of the muscle and is characterized by a zone of motor endplates within the middle third of the muscle. The homogeneous fiber architecture of the bat pectoralis muscle is in contrast to the serial arrangement of endplates (and presumably muscle muscle fibers) in the avian pectoralis in species equivalent in size to Myotis. The short fiber organization and motor endplate pattern observed in most birds is thus not a requisite design for flying vertebrates. © 1994 Wiley-Liss, Inc.     

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm² and nerve terminal synaptophysin 168±14 µm² (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm² area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.     

Anti-curare action of stannous ion in the frog neuromuscular junction

For the purpose of elucidating the mechanism of action of stannous ion (Sn2+), we investigated effects of stannous chloride (SnCl2) on the twitch and on the electrical phenomena in the muscle fiber. Sciatic nerve-sartorius muscle preparations from the bullfrog were used as the material. Effect of SnCl2 was examined on the twitch partially inhibited by pretreatment with d-tubocurarine. SnCl2 (1-100 microM) antagonized d-tubocurarine and enhanced the twitch dose-dependently. Tartaric acid, which is the solvent used for SnCl2 solution, had no augmentative effect on the twitch, even at a concentration as high as 250 microM. SnCl2 (1-50 microM) increased the amplitude of the endplate potential; that is, it exerted an anti-curare action. The resting potential and the membrane resistance of the muscle fiber were not altered by 30 microM SnCl2. These findings lead to the conclusion that Sn2+ enhances the twitch by increasing the endplate potential of the muscle fibers.     

Effect of phencyclidine on post-tetanic twitch tension of the mouse diaphragm preparation

The effects of phencyclidine(PCP) on the post-tetanic potentiation(PTP) of twitch tension were studied on the isolated mouse phrenic nerve diaphragm preparation. Phencyclidine increased directly elicited twitch tension while it decreased post-tetanic potentiation of the indirectly elicited twitch tension. The maximal depression effect of the PTP was found after higher frequencies and longer durations of stimulation. After repetitive stimulation, the amplitude of endplate potential was potentiated. Phencyclidine decreased the post-tetanic potentiation of the amplitude of endplate potential while the quantal content of the endplate potential was not affected. 4-Aminopyridine increased both directly and indirectly elicited twitch tension while it did not inhibit the post-tetanic potentiation of the twitch tension. It is concluded that phencyclidine suppressed the post-tetanic potentiation of the indirectly elicited twitch tension. The depressant effect may be mainly due to its effect on the acetylcholine receptor-ionic channel complex of the motor endplate.     

Sternomastoid, rib cage, and expiratory muscle activity during weaning failure.

We hypothesized that patients who fail weaning from mechanical ventilation recruit their inspiratory rib cage muscles sooner than they recruit their expiratory muscles, and that rib cage muscle recruitment is accompanied by recruitment of sternomastoid muscles. Accordingly, we measured sternomastoid electrical activity and changes in esophageal (DeltaPes) and gastric pressure (DeltaPga) in 11 weaning-failure and 8 weaning-success patients. At the start of trial, failure patients exhibited a higher DeltaPga-to-DeltaPes ratio than did success patients (P = 0.05), whereas expiratory rise in Pga was equivalent in the two groups. Between the start and end of the trial, failure patients developed additional increases in DeltaPga-to-DeltaPes ratio (P < 0.0014) and the expiratory rise in Pga also increased (P < 0.004). At the start of trial, sternomastoid activity was present in 8 of 11 failure patients contrasted with 1 of 8 success patients. Over the course of the trial, sternomastoid activity increased by 53.0 +/- 9.3% in the failure patients (P = 0.0005), whereas it did not change in the success patients. Failure patients recruited their respiratory muscles in a sequential manner. The sequence began with activity of diaphragm and greater-than-normal activity of inspiratory rib cage muscles; recruitment of sternomastoids and rib cage muscles approached near maximum within 4 min of trial commencement; expiratory muscles were recruited slowest of all. In conclusion, not only is activity of the inspiratory rib cage muscles increased during a failed weaning trial, but respiratory centers also recruit sternomastoid and expiratory muscles. Extradiaphragmatic muscle recruitment may be a mechanism for offsetting the effects of increased load on a weak diaphragm.     

Stimulation of calcineurin Aalpha activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin Aalpha transgene (CnAalpha) was overexpressed in skeletal muscles of mdx (mdx CnAalpha*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnAalpha* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnAalpha* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnAalpha* than mdx mice. In the diaphragm, despite a slower phenotype and a approximately 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnAalpha* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.     

Motor and sensory reinnervation of fast and slow mammalian muscles

Summary 1. The formation of endplates outside the original endplate region of a muscle fibre was studied in slow and fast rat muscles. It was found that such new endplates are readily formed on the soleus muscle, whereas hardly at all in the fast extensor digitorum longus. Most new endplates appear to be morphologically normal within 2 months after nerve implantation.2. The time course of recovery of slow and fast cat muscles was followed after crushing the sciatic nerve. It was found that the slow soleus muscle recovers more rapidly than the fast flexor hallucis longus muscle.3. The endplates of reinnervated cat muscles are more complicated than those of the control muscles, but have nevertheless fewer nerve terminals per endplate. Reinnervated muscles are more sensitive to curare and it is suggested that this is due to a decrease in transmitter release, for it was found that less acetylcholine is released from reinnervated rat hemidiaphragms than from control ones.4. Motor and sensory reinnervation of spindles and tendon organs was studied. At the time when motor reinnervation is almost completed the sensory endings from spindles and tendon organs are highly abnormal. Thus sensory reinnervation proceeds much more slowly than motor.     

Contractile characteristics and operating lengths of canine neck inspiratory muscles

The neck inspiratory muscles are recruited to support breathing under numerous conditions. To gain insight into their synergistic actions we examined the isometric contractile properties of bundles from canine scalene and sternomastoid muscles. In addition, we also related the length of the neck muscles, measured sonomicrometrically in vivo at different lung volumes and body positions, to their optimal force-producing length (Lo) determined in vitro. We found that the speed of the sternomastoid is somewhat faster than that of the scalene owing to a shorter relaxation rate; the sternomastoid generates higher forces at submaximal stimulation frequencies than the scalene; the maximal tetanic force corrected for cross-sectional area is the same for both neck muscles; the neck muscles are significantly faster than the canine costal diaphragm; at supine functional residual capacity (FRC), the scalene is operating at a length corresponding to 85% Lo, whereas the sternomastoid is significantly shorter at 75% Lo; increasing lung volume shortens both muscles slightly, the length at supine total lung capacity being approximately 5% shorter than at FRC; and in the upright posture, both neck muscles lengthen toward their Lo, with the sternomastoid lengthening more than the scalene. We conclude that the scalene is a more effective force generator than the sternomastoid with the animal lying supine; the neck muscles appear to maintain their force-generating potential regardless of the lung volume; and the force-generating potential of the neck muscles is greatly enhanced with the animal in the upright vs. the supine position. This may contribute to the augmented rib cage motion characteristic of breathing in the upright posture.     

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.     

Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure

A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 micron of the neuromuscular junction in innervated as well as 3- and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 micron, continuing less sharply to between 25 and 50 micron. Beyond 50 micron (to 250 micron) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction. The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.     

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.     

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.     

Contact behaviour of setal tips in the hairy attachment system of the fly Calliphora vicina (Diptera, Calliphoridae): a cryo-SEM approach

The fly Calliphora vicina (Diptera, Calliphoridae) bears attachment pads (pulvilli) covered with setae on their ventral sides. These structures enable attachment to smooth vertical surfaces and ceilings. The contact between the terminal setal tips (spatulae) and various substrates was visualised using various experimental techniques combined with conventional scanning electron microscopy (SEM) and cryo-SEM. The results show that the setal endplates are highly flexible structures that form contact with the surface by bending their tips in the distal direction. With conventional SEM, a comparison of partly attached endplates with unattached endplates demonstrated the presence of a distinct marginal bulge. As observed with cryo-SEM, the bulge continuously disappeared as a larger area of the endplate came into contact. Two explanations of this result are suggested. First, the volume between the bulge, the mid-part of the endplate and the substrate may be filled with a fluid secretion that is released into the contact area in the endplate region. Second, the flexible central part of the endplate may jump into contact with the substrate during contact formation.