Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.     

Distribution of Na+ channels and ankyrin in neuromuscular junctions is complementary to that of acetylcholine receptors and the 43 kd protein

We have used immunogold electron microscopy to study the organization of the acetylcholine receptor, 43 kd protein, voltage-sensitive Na+ channel, and ankyrin in the postsynaptic membrane of the rat neuromuscular junction. The acetylcholine receptor and the 43 kd protein are concentrated at the crests of the postsynaptic folds, coextensive with the subsynaptic density. In contrast, Na+ channels and ankyrin are concentrated in the membranes of the troughs and in perijunctional membranes, both characterized by discontinuous submembrane electron-dense plaques. This configuration of interspersed postsynaptic membrane domains enriched in either Na+ channels or acetylcholine receptors may facilitate the initiation of the muscle action potential. Furthermore, the results support the involvement of ankyrin in immobilizing Na+ channels in specific membrane domains, analogous to the proposed involvement of the 43 kd protein in acetylcholine receptor immobilization.     

THE SARCOPLASMIC RETICULUM, THE T SYSTEM, AND THE MOTOR TERMINALS OF SLOW AND TWITCH MUSCLE FIBERS IN THE GARTER SNAKE

Twitch and slow muscle fibers, identified morphologically in the garter snake, have been examined in the electron microscope. The transverse tubular system and the sarcoplasmic reticulum are separate entities distinct from each other. In twitch fibers, the tubular system and the dilated sacs of the sarcoplasmic reticulum form triads at the level of junction of A and I bands. In the slow fibers, the sarcoplasmic reticulum is severely depleted in amount and the transverse tubular system is completely absent. The junctional folds of the postsynaptic membrane of the muscle fiber under an "en grappe" ending of a slow fiber are not so frequent or regular in occurrence or so wide or so long as under the "en plaque" ending of a twitch fiber. Some physiological implications of these differences in fine structure of twitch and slow fibers are discussed. The absence of the transverse tubular system and reduction in amount of sarcoplasmic reticulum, along with the consequent disposition of the fibrils, the occurrence of multiple nerve terminals, and the degree of complexity of the post junctional folds of the sarcolemma appear to be the morphological basis for the physiological reaction of slow muscle fibers.     

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.     

Ultrastructural visualization of the transmembranous and cytomatrix-related part of nicotinic acetylcholine receptor of frog motor endplate by means of an immunochemical avidity of IgG for d-tubocurarine

In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR) was attempted, using d-tubocurarine (d-TC), a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG) for acetylcholine (ACh) and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA), a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC) to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB) precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest) of the postsynaptic membrane (between the junctional folds) which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm), while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex.     

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction

The distribution and turnover rate of acetylcholine receptors labeled with 125I-alpha-bungarotoxin were examined in innervated mouse sternomastoid muscle by electron microscope autoradiography using the "mask" analysis procedure. We compared the total population of receptors with receptors newly inserted at the junction 2 d after inactivation with nonradioactive alpha-bungarotoxin, both at the top (thickened) region of the postjunctional folds (pjm) and the nonthickened bottom folds. We found that the receptor site density was approximately 10 times greater on the thickened pjm than on the nonthickened bottom folds for both total and newly inserted receptors. This ratio does not change significantly during a 6-d period after labeling the new receptors. Furthermore, calculated values for turnover time of receptors show that both total and newly inserted receptors at both regions of the junctional folds have half-lives for degradation within the range given in the literature for slow junctional receptors. These data exclude a simple migration model whereby receptors are preferentially inserted in the nonthickened region of the junctional folds and then migrate into the thickened membrane at a rate equal to the turnover rate of the receptors.     

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.     

Structural development of myoneural junctions in the human embryo

Summary The structure of myoneural junctions in the tibialis anterior and intercostal muscles was studied after histochemical reaction for myoneural acetylcholinesterase (E.C. 3.1.1.7) in human embryos.Myoneural junctions of a primitive form were first seen in the intercostal muscle at the age of 8.6 weeks (crown-rump length of 3.2 cm), and in the tibialis anterior muscle at the age of 10 weeks (4.3 cm). The postsynaptic membrane was devoid of any junctional folds typical of adult synapses up to the age of about 19 weeks. At first small, the junctional folds gradually became deeper and more prominent during the following weeks, and some ramification of the previously coherent postsynaptic area took place. Myoneural morphogenesis was not completed at birth, although well developed postsynaptic Moldings were present.     

STUDIES OF EXCITABLE MEMBRANES : I. Macromolecular Specializations of the Neuromuscular Junction and the Nonjunctional Sarcolemma

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100–110-Å particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110–140-Å particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-Å particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol. 63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.     

Bridging structures spanning the junctioning gap at the triad of skeletal muscle

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.     

Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin

The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated alpha-syntrophin null mice by targeted gene disruption to test the function of this association. The alpha-Syn(-/)- mice show no evidence of myopathy, despite reduced levels of alpha-dystrobrevin-2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the alpha-Syn(-/)- mice, even where other syntrophin isoforms are present. alpha-Syn(-/)- neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, alpha-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.     

THE MOLECULAR ORGANIZATION OF CHLOROPLAST MEMBRANES

The presence of subunits in chloroplast membranes is suggested by polarization, fluorescence, and X-ray studies. Subunits (quantasomes) may be observed in the electron microscope on dried shadowed membranes and in replicas of membranes produced by the freeze-etching technique. Regular subunits are also observed with the electron microscope in thin sections of chloroplast membranes. Chemical considerations suggest that many membranes are composed of lipoprotein subunits. Thin sections reveal two types of chloroplast membranes, the fret membranes composed of one layer of subunits, and the partitions composed of two layers of subunits. Chloroplast membranes consist of about 45% protein and 55% lipid. Some 80% of the lipids are the highly surfactant glycolipids. In this paper the subunits are visualized as assymetric lipoproteins, probably having a protein core surrounded by components determined by the nature and environment of the membrane. Since the stroma, fret channels, and loculi contain aqueous materials, it is further postulated that the membranes bordering these spaces bind the highly surfactant glycolipids. The region between the two rows of subunits in the partition appears to be highly hydrophobic, rich in chlorophyll, and low in glycolipids. Some chlorophyll also may occur within the subunits both in the partitions and in the fret membranes. Since four subunits appear to comprise a quantasome, at least two types of forces, inter- and intra-quantasome forces, bind the subunits together in sheets. Chloroplast membranes thus differ from a “unit membrane” in two important respects: (1) they must be an aggregate of globular subunits, and (2) the lipoprotein subunits consist of a protein matrix which binds the chlorophylls and lipids by hydrophobic association with their hydrocarbon moieties.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Relationship of sertoli-sertoli tight junctions to ectoplasmic specialization in conventional and en face views

Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.     

Effect of castration and testosterone administration on the neuromuscular junction in the levator ani muscle of the rat

Summary The ultrastructure of the neuromuscular junction (n.m.j.) of the androgen-sensitive levator ani muscle was studied in normal adult male rats, in 8-month-old rats castrated at the age of one month and in castrated rats treated with testosterone propionate (TP). Castration does not result in significant changes of the n.m.j. The density of synaptic vesicles and the postsynaptic junctional folds remain practically normal in spite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in preand postsynaptic structures. There is slow progressive depletion of synaptic vesicles, appearance of cisternae and coated vesicles in axon terminals, and coalescence of coated vesicles with the plasma membrane. Coated vesicles are also found inside Schwann cells and among junctional folds. Dense core vesicles appear both in the axon terminals and in the postsynaptic area. Collateral sprouting of terminal axons with the formation of new immature junctions is observed. After 35 days of TP administration depletion of synaptic vesicles continues. Glycogen -particles, mostly freely dispersed, occasionally seen in axon terminals 7 days after TP administration, subsequently increase in number. In the endplate zone of the muscle fibre increased protein synthesis is indicated by a rapid increase in ribosomes and irregularly located myofilaments and myofibrils. The appearance of n.m.j. after testosterone administration resembles that described after nerve stimulation; the degree of change is however less pronounced.The authors wish to acknowledge the skillful technical assistance of Mrs. L. Vedralová     

Synaptic current between neuromuscular junction folds

Measurements of membrane infoldings of vertebrate subsynaptic membranes were taken to evaluate the possible electrophysiological implications. The shapes of standard interfolds of different neuromuscular junctions were established from micrographs available in the literature. Electrical properties were estimated using published fibre membrane and myoplasm electrical values. Models of synaptic current pathways were designed taking into account the small size of the postsynaptic patch activated by a transmitter quantum. This analysis reveals a resistance "in series" between the ACh-sensitive interfold crest and the remainder of the muscle fibre. The calculated cytoplasmic resistance of an interfold is between 0.2 and 3 Mohms which is in the same range as the fibre DC input resistance. The calculated interfold resistance appears to be dependent on the fibre type, the age and the pathology. Functional roles of junctional folds and dendritic spines are discussed.     

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system

The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.     

Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers

Mammalian fast and slow twitch skeletal muscles are compared by freeze-fracture, thick and thin sectioning, and histochemical techniques using conventional and high voltage electron microscopy. Despite gross morphological differences in endplate structure visualized at relatively low magnifications in this sections, rat extensor digitorum longus (EDL) (fast twitch) and soleus (slow twitch) fibers cannot be distinguished on the basis of size, number, or distribution of molecular specializations of the pre- and postsynaptic junctional membranes exposed by freeze fracturing. Specializations in the cortex of the juxtaneuronal portions of the junctional folds are revealed by high voltage electron stereomicroscopy as a branching, ladder-like filamentous network associated with the putative acetylcholline receptor complexes. These filaments are considered to be involved in restricting the mobility of receptor proteins to the perineuronal aspects of the postynaptic membrane. Although the junctional membranes of both EDL and soleus appear similar, a differential specialization of the secondary synaptic cleft was noted. The extracellular matrix in the bottom of soleus clefts was observed as an ordered system of filamentous "combs," These filamentous arrays have not been detected in EDL junctions. Examination of the extrajunctional sarcolemmas of EDL and soleus reveal additional differences which may be correlated with variations in electrical and contractile properties. For example, particle aggregates termed "square arrays" previously described in the sarcolemmas of some fibers of the rat diaphragm were observed in large numbers in sarcolemmas of EDL fibers but were seldom encountered in soleus fibers. These gross compositional differences in the membranes are discussed in the light of functional differences between fiber types.     

The thin limbs of Henle's loop in the rabbit

Summary The thin limbs of short and long loops of Henle of the rabbit kidney were studied by freeze fracture techniques. According to TEM studies of thin sections four segments are discernible: descending thin limbs of short loops, descending thin limbs of long loops, subdivided into an upper and a lower part, and ascending thin limbs (Kaissling and Kriz 1979). This division is supported by findings obtained with the freeze fracture technique and based on differences in the organization of the junctional complexes as well as on differences in the internal morphology of the cell membranes. The descending thin limbs of short loops have junctional complexes established by several closely arranged junctional strands and numerous desmosomes. The upper parts of the long descending thin limbs have tight junctions consisting of a variable number of strands; their outstanding characteristic after freeze fracture is a high density of intramembrane particles in both luminal and baso-lateral membranes. The tight junctions of the lower part of the long descending thin limbs consist of several anastomosing junctional strands, which are, in contrast, loosely arranged; the cell membranes contain only a sparse population of intramembrane particles. The ascending thin limbs are characterized by shallow tight junctions (frequently consisting of only one single junctional strand). Moreover, the epithelial cells of this segment are heavily interdigitated; thereby the tight junctions are correspondingly lengthened.In addition, this study presents further evidence that remarkable species differences occur among thin limb epithelia. The junctional complexes of the long descending thin limbs of the rabbit are organized quite differently from those of small rodents (e.g., rat, Psammomys).The data of this study support the concept that the tight junctions are the main determinant of ionic conductances of the paracellular pathway. However, with reference to recent findings from microperfusion studies, it becomes obvious that a correlation of the junctional morphology with the transepithelial water permeability is lacking, at least for the thin limbs.This investigation was supported by the Deutsche Forschungsgemeinschaft; project Kr 546 Henlesche Schleife     

Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.