Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – I. Naturally occurring variability in active zone structure

The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction (‘active zones’) are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 μm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.     

Synaptic competition and the persistence of polyneuronal innervation at frog neuromuscular junctions

Mechanisms governing the elimination of polyneuronal innervation were examined by correlating the morphology and physiology of competing nerve terminals at identified dually innervated neuromuscular junctions in sartorius muscles of adult frogs (Rana pipiens). Synaptic efficacy (endplate potential amplitude per unit nerve terminal length) was presumed to reflect the ability of a terminal to compete for synaptic space. The synaptic efficacies of two terminals at the same synaptic site were found to be surprisingly equal, with a median difference of 33%. Much more variation would be expected if dually innervated junctions were randomly innervated by pairs of terminals having the same range of synaptic efficacy as that found at singly innervated junctions in the same muscle. This finding supports the hypothesis that the weaker input is eliminated from dually innervated junctions when there is a large discrepancy in competitive efficacy, and that both inputs may persist if competitive efficacies are relatively equal. We also tested but failed to find support for the hypothesis that spatial proximity between competing terminals intensifies competition for synaptic space during synapse elimination.     

Pattern of arborization of the motor nerve terminals in the fast and slow mammalian muscles.

A silver impregnation method and a morphometric approach were used to define differences existing in the motor nerve terminal branching pattern between a fast-twitch muscle (extensor digitorum longus) and a slow-twitch one (soleus) of the normal adult rat. Because no single measure can describe precisely all geometrical properties (ie both topology and metrics) of the nerve terminals, we evaluated morphologic parameters defining length and angular characteristics in the different terminal segments classified according to their centrifugal order. The main results indicate that the distal free-end segments in the extensor digitorum longus muscle are shorter and less divergent than in the soleus nerve terminals. The endings in the two muscles have different fractal dimensions. Findings are discussed in the context of the hypothetical mechanisms governing motor nerve terminal size and complexity.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – II. Proximal–distal measurements

Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal–distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal–distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 μm in length and with sufficient information to determine their proximal–distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5–10 μm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure/μm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.     

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.     

Effects of high potassium solutions and caffeine on the processes of exo-endocytosis of synaptic vesicles at the frog motor nerve ending

In experiments on the frog motor nerve endings of cutaneous pectoris muscle using fluorescent microscopy it has been shown that initiation of massive transmitter release of synaptic vesicles by high potassium solutions in using endocytotic marker FM 1-43 at the nerve terminals light spots occurred only at some of the nerve terminals or at the some parts of nerve terminal. It has been revealed that application of caffeine increased the number of light terminals. Using extracellular microelectrode recording, we showed that both high potassium solutions and caffeine increased frequency of miniature end-plate potentials in a dose-dependent manner. However, high potassium solutions always increased the frequency of spontaneous transmitter release while caffeine increased it only in some experiments. It was concluded that processes of exo- and endocytosis can be caused both by entry of Ca ions at the nerve ending during depolarization (high potassium solutions) and by Ca release from endoplasmic reticulum (caffeine). Possible spatial localization of endoplasmic reticulum at the motor nerve ending is discussed. The hypothesis of its role at the remodeling of synapse was proposed.     

Release of packets of acetylcholine and synaptic vesicles elicited by brown widow spider venom in frog motor nerve endings poisoned by botulinum toxin

Homogenates of venom glands of brown widow spiders caused the release of packets of acetylcholine, observed as miniature end-plate potentials (mepps), from nerve terminals in sartorius and cutaneous pectoris muscles of the frog, even though the appearance of mepps which is normally induced by depolarization of the presynaptic membrane had been completely blocked by prior treatment with type A botulinum toxin (BotTX). The distribution of the amplitudes of the recorded mepps resembled that of the mepps in normal muscle and was quite different from the heavily skewed (to the left) distribution of the mepps recorded from BotTX-treated muscles. This suggests that the mepps elicited by brown venom glands homogenates in muscles blocked by BotTX were due to the release of a population of vesicles which is unaffected by BotTX and which is also released on hyperpolarization of nerve terminals.     

Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified alpha-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca2+-free solution with 1 mM EGTA and 4 mM Mg2+, the rate of quantal secretion rose to high levels but quickly subsided; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up. When BWSV was applied to other muscles bathed in a solution with 1.8 mM Ca2+ and 4 mM Mg2+, the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughot the hour of exposure. Neuromuscular transmission was blocked in fewer than 50% of these fibers. The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles. If HRP had been present, most of the synaptic vesicles were labeled with reaction product. These observations suggest that Ca2+ plays an important role in endocytosis at the frog neuromuscular junction.     

Changes in the topography of transmitter release in a neuromuscular synapse reflect plastic alterations occurring in active zones

The topography of transmitter release along the motor nerve terminals (NT) was studied on the frogcutaneous pectoris muscle under normal conditions and following denervation. Coordinates of release sites (RS) of transmitter quanta were determined by extracellular recording of postsynaptic signals using three microelectrodes. It was shown that RS form groupings that reflect transmitter release in individual active zones (AZ). The topography of transmitter release in the distal parts of the NT under normal conditions was shown to differ from that observed in the proximal parts. The difference consists in a lower probability of transmitter release in AZ and a higher probability of this process between AZ, as well as in a change of release profile in individual AZ. Similar differences were found following denervation. It is suggested that these properties may reflect plastic reorganization occurring in AZ in the course of remodelling of neuromuscular synapse and its degeneration.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 253–260, July–August, 1995.     

Structural changes in locust leg muscle fibres in response to tenotomy and joint immobilization

Tenotomy and immobilization at minimum physiological length resulted in degenerative changes in the ultrastructure of the locust metathoracic flexor tibiae muscle.Degeneration was observed in some muscle fibres 9 days after tenolomy and was progressive, affecting an increasing proportion of fibres at longer times after operation. Changes included disruption and loss of myofilaments, Z-disk streaming, degeneration of sarcoplasmic reticulum, formation of large membranous complexes and changes in number and appearance of mitochondria. Different muscle fibres followed different degenerative pathways. By 73 days very few intact muscle fibres remained.Joint immobilization resulted in similar but much less severe progressive degeneration. Degenerate fibres were largely localized in the distal and medial parts of the muscle. Both immobilized and tenotomized muscles were invaded by large numbers of fat body cells in the later stages of degeneration.Nerve terminals on degenerate tenotomized and immobilized muscle fibres were ultrastructurally normal. Tendinous cells attaching degenerate muscle fibres to the cuticle themselves degenerated.     

Scanning and freeze-fracture study of larval nerves and neuromuscular junctions in Manduca sexta

The nerves and nerve terminals to tonic larval muscle fibers in third and fifth instar caterpillars were studied to compare them with those formed by the same motor neurons on phasic flight muscles in adult moths. Scanning micrographs showed a primary nerve branch running the length of each fiber, with secondary nerve branches extending from it at intervals. There was a great deal of variability in both the length of the branches and the distance from the nerve at which the neuromuscular junctions were formed. The rapid increase in muscle fiber size during larval development may be responsible for this variability. The nerves and junctions were often found to be obscure by overlying fibroblasts and tracheoblasts or entering the deep muscle clefts. Those examined were similar in appearance to the adult junctions formed by the same neurons, although some may have formed single branches instead of y-shapes. The membrane specializations of the synapse seen in freeze-fractured specimens were similar to those of the adult junction. However, the overall shape of the nerve terminal within the junction differed. The larval nerve terminals appeared varicose instead of having a uniform diameter. The spacing of the nerve plaques varied, in contrast with the relatively straight alignment and even spacing of plaques found in adult junctions. Such differences could result from an interaction between the motor neuron and the two different types of muscle fiber that it innervates, an intrinsic change in the motor neurons themselves that occurs with metamorphosis, or a plastic functional response that occurs as a result of the different types of motor patterns that are used in the two stages.     

Complex nerve terminal formation in the phasic muscles of frogs

Synapses with complex nerve terminals consisting of several terminal arbors of a single axon divided by myelin segments were investigated using histological and electrophysiological techniques during experiments on the cutaneous-pectotoralis muscles of different aged frogs. Numbers of synapses with complex nerve terminals were shown to increase during the postnatal developmental process. The relationship between the complexity of nerve terminals, summated length of terminals, and size of muscle fiber is described. Some terminal arborizations at complex nerve terminals originate from nodes of Ranvier; these are marked by low quantal secretion and a distinctive pattern of sodium current decay along the path of the terminals. The causes and mechanisms governing increased complexity of nerve endings in phasic muscles are discussed, together with transmitter release patterns at these endings. It is postulated that growth and myelination processes occur in parallel at the nerve terminal.A. A. Ukhtomskii Physiological Institute, Leningrad State University; S. A. Kurashov Medical Institute, RSFSR Ministry of Health, Kazan'. V. I. Ul'yanov State University, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 99–107, January–February, 1990.     

Ultrastructural characteristics of rat neuromuscular junctions after physical load

It is interesting to ascertain the adaptive reaction of rat neuromuscular junctions (NMJ) of muscle fibers of different types to a chronic physical load. We examined ultrastructural changes in NMJ following both static load (pre- and postnatal ontogenesis of Wistar rats till a 2 month age took place under a constant rotation on the centrifuge at hypergravity conditions 2G), and after three kinds of dynamic loads (1/run on treadmill with a speed 35 m/min for 6 wks, 10-60 min/day; 2/swimmings, each 10 hrs/day for 10 days; 3/strength exercises on a vertical treadmill with load for 6 wks). Differences in NMJ reaction of muscle fibers of the same type to various loads were established. A low secretory activity of axonal terminals of type I muscle fibers of m. soleus was shown after the static load. The dynamic load (run) is accompanied with a high secretory activity of axonal terminals in m. soleus type I muscle fibers and of some axonal terminals of m. quadriceps femoris IIB type muscle fibers after strength exercises; the secretory activity of axonal terminals of m. quadriceps femoris IIA and IIB types muscle fibers is expressed in a lesser degree after swimming. The NMJ ultrastructure remodelling (terminal renewal) of type I muscle fibers of the 2 month old control rats increases after static and dynamic (run) loads. Some correlations between different kinds of physical load, muscle fiber type and the degree of NMJ ultrastructure transformation have been shown.     

Sexual differentiation of identified motor terminals in Drosophila larvae

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males. This is due to greater release of transmitter from female than male synaptic terminals because the amplitude of spontaneous miniature EPSPs was similar in male and female muscle fibers. This sexual difference exists throughout the third-instar: it is seen in both early (foraging) and late (wandering) third-instar larvae. The sexual differentiation appears to be neuron specific and not muscle specific because the same axon produces Is terminals on muscle fibers 2 and 4, and both terminals produce larger EPSCs in females than males. Whereas, the Ib terminals innervating muscle fibers 2 and 4 are not sexually differentiated. The differences in transmitter release are not due to differences in the size of the motor terminals. For the terminal on muscle fiber 5 and the Is terminal on muscle fiber 4, there were no differences in terminal length, the number of branches, or the number of synaptic boutons in males compared to females. These sexual differences in neuromuscular synaptic physiology may be related to male-female differences in locomotion.     

Stiffness of hip adductor myofibrils is decreased in children with spastic cerebral palsy

Cerebral palsy (CP) is the result of a static brain lesion which causes spasticity and muscle contracture. The source of the increased passive stiffness in patients is not understood and while whole muscle down to single muscle fibres have been investigated, the smallest functional unit of muscle (the sarcomere) has not been. Muscle biopsies (adductor longus and gracilis) from pediatric patients were obtained (CP n = 9 and control n = 2) and analyzed for mechanical stiffness, in-vivo sarcomere length and titin isoforms. Adductor longus muscle was the focus of this study and the results for sarcomere length showed a significant increase in length for CP (3.6 µm) compared to controls (2.6 µm). Passive stress at the same sarcomere length for CP compared to control was significantly lower in CP and the elastic modulus for the physiological range of muscle was lower in CP compared to control (98.2 kPa and 166.1 kPa, respectively). Our results show that CP muscle at its most reduced level (the myofibril) is more compliant compared to normal, which is completely opposite to what is observed at higher structural levels (single fibres, muscle fibre bundles and whole muscle). It is noteworthy that at the in vivo sarcomere length in CP, the passive forces are greater than normal, purely as a functional of these more compliant sarcomeres operating at long lengths. Titin isoforms were not different between CP and non-CP adductor longus but titin:nebulin was reduced in CP muscle, which may be due to titin loss or an over-expression of nebulin in CP muscles.     

Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

Ultrastructure of the corneal nerves in the Rat

Summary The ultrastructure of the corneal nerves of the rat was studied in tissue fixed by immersion in and by perfusion with glutaraldehyde-containing fixatives. Of the four types of axonal terminal identified in the nerves, those with the features of adrenergic and cholinergic terminals were confined to the nerves at the limbus and were concentrated in the perivascular plexuses. The remaining two types of terminal were found on axons located in all parts of the cornea and on both intraepithelial axons and axons in the stromal nerves. Of these, one contained the numerous mitochondria which occur in the terminals of axons associated with known mechanoreceptors and the second contained variable and often small numbers of both clear and large dense-cored vesicles. While most of the mitochondria-containing terminals were seen in nerves located near the periphery, vesicle-containing terminals were numerous in all of the nerves, and especially in those in the avascular cornea. In material fixed by immersion in glutaraldehyde-paraformaldehyde, the vesicle-containing terminals appeared to be dilated, but in material fixed by perfusion there was little evidence of any increase in the diameter of the axons in the terminal regions. The structure of the terminals was compared with that of the terminals of axons identified in the nerves of the skin and the urinary tract and the differences in the vesicle content of the terminals to those reported in other studies of the corneal nerves was related to the use of different fixation procedures. The possibility that axons possessing such terminals are identical with the beaded axons and both the cholinesterase-positive and fluorescent axons demonstrated in light microscopical studies of the corneal nerves is discussed, and the widespread distribution of the axons in the cornea is equated with the hypothesis that they are afferent in nature and represent the peripheral receptors for pain impulses.     

Catecholaminergic innervation of muscles in the hindgut of crustaceans

Summary The crustacean species Pacifastacus leniusculus and Gammarus pulex were investigated by electron microscopy in a search for possible neuromuscular junctions in the hindgut, which has a rich supply of catecholaminergic fibres. True neuromuscular synapses were found in both species between nerve terminals containing dense-core vesicles (80–110 nm in diam.) and muscle fibres. We suggest that the dense-core vesicle terminals contain a catecholamine, and this is supported by ultrahistochemical tests for monoamines. Two types of junctions are found: one in which the nerve terminal is embedded in the muscle cell (both species) and one in which protrusions from the muscle cell meet nerve terminals (Pacifastacus). Gammarus pulex, which has only circular muscles in the hindgut, has only catecholaminergic innervation, whereas Pacifastacus leniusculus has circular and longitudinal muscles both with at least two types of innervation.The investigation was supported by grants from the Swedish Natural Science Research Council (B 2760-009), the Hungarian Academy of Sciences, the Royal Swedish Academy of Sciences, and the Magnus Bergvall Foundation. We are also indebted to Mrs. Lena Sandell for her skilful technical assistance     

The ultrastructure of locust pleuroaxillary "steering" muscles in comparison to other skeletal muscles

The ultrastructure of locust muscles with different function is examined: the pleuroaxillary flight steering muscle is compared with a typical flight (power muscle) and a typical leg muscle, in particular with respect to sarcomere length, tracheation, mitochondria, and sarcoplasmatic reticulum. The pleuroaxillary muscle exhibits some features characteristic of flight muscles but most of the ultrastructure resembles that of leg muscles. This is in agreement with the innervation of this muscle by an octopaminergic neuron, which also innervates leg muscles but no other flight muscles. It also supports the hypothesis that octopaminergic neurons are important metabolic regulators and that the above muscle types exhibit important differences in energy metabolism.     

弓形革囊星虫组织结构和超微结构观察

弓形革囊星虫(Phascolosoma arcuatum)是近年兴起的水产养殖品种,目前已形成一定养殖规模,但是其生物学特性相关的研究只有零星报道。本文主要采用石蜡切片苏木精-伊红(H.E)染色和扫描电镜方法对弓形革囊星虫的整体形态和各主要器官的结构进行研究。结果表明,弓形革囊星虫主要由吻部和躯干部构成,躯干部有乳突分布;吻部由触手和项器构成,伸长可达躯干部的两倍,具有感光性。其消化系统由口、食道、肠、肛门四个部分构成,不同部分的肌肉组织存在明显差异;收吻肌一端与吻相连,另一端分成4支,分别连于背部两侧(背收吻肌)和腹部两侧(腹收吻肌);食道贴于收吻肌,后连接肠。肠分为肠下回环与肠上回环,肠上回环与肠下回环缠绕盘旋,肠上回环后接直肠。直肠具有盲囊,后连接肛门;两条肾管分布在腹侧。