Freeze-fracture studies of muscle plasma membrane in Fukuyama-type congenital muscular dystrophy

We used freeze-fracture to study muscle plasma membrane in six patients with Fukuyama-type congenital muscular dystrophy and six control children. In the patients, there was significantly fewer intramembranous particles (IMPs) and orthogonal arrays in the P face, with less conspicuous depletion of IMPs in E face. However, the density of caveolae was not affected.     

Freeze-fracture analysis of plasma membrane cholesterol in Duchenne muscle

We estimated cholesterol in Duchenne muscle plasma membrane using the sterol-specific ligand digitonin. In measuring the extent of digitonin-cholesterol complex formation with freeze-fracture electron microscopy, we found a significantly greater proportion of surface area taken up by complexes in Duchenne muscle (60% +/- 9%) than in controls (36% +/- 4%). This finding suggests that a population of Duchenne muscle fibers has a higher concentration of cholesterol in the plasma membrane. Membrane cholesterol is known to affect membrane deformability and the activity of membrane-bound enzymes and transport proteins; thus a difference in membrane cholesterol might account for structural and functional abnormalities in Duchenne plasma membrane.     

Freeze-fracture of developing neuronal plasma membrane in postnatal cerebellum

The structure of the plasma membrane of two neuronal cell types was quantitatively assessed in freeze-fracture replicas of rat cerebellar cortex from 2 to 73 postnatal days. The measurement of the number and size of intramembrane particles (IMP) in different regions of the Purkinje cell and of the granule cell showed that between 2 and 21 postnatal days there was a differential increase in the number of IMP; several regions of the cell characterized by a different pattern of synaptic interconnections showed differences in particle size and in particle number: neuronal processes had lower numbers of IMP than perikarya, whereas dendritic spines contained fewer IMP than dendritic trunks. These differences in IMP content appeared at the time at which specific synaptic contacts are established in these various regions of the cells.     

Freeze-fracture studies of myofiber plasma membrane in X chromosome-linked muscular dystrophy (mdx) mice

Summary The structure of the muscle plasma membrane of extensor digitorum longus muscles of X chromosome-linked muscular dystrophy (mdx) mice was studied by freeze-fracture technique at several time points after birth. The common denominator of the abnormalities was the decreased density of orthogonal arrays throughout all the time points examined. The results demonstrated that the ultrastructural features of the muscle plasma membrane alterations in mdx mice were similar to those in Duchenne dystrophy.Supported by grant (62A-2-20) from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Freeze-fracture analysis of the muscle fiber plasma membrane in Duchenne dystrophy

The intramembrane particle density in freeze-fractured muscle fiber plasma membranes did not differ in 4 patients with Duchenne dystrophy and 4 normal subjects. However, we confirmed reported loss of orthogonal arrays and increased density of caveolae in dystrophic muscle.     

Freeze-fracture cytochemistry of cholesterol content in neuronal plasma membrane following cerebral ischaemia

Minor changes on the neuronal perikaryon cytoplasm and large alterations in neuronal processes have been demonstrated with transmission electron microscopy after 60 minutes of focal and selective cerebral ischaemia. The distribution of cholesterol in neuronal plasma membrane and perikarya plasmalemma of normal and ischaemic caudate nucleus was investigated with the polyene antibiotic filipin, a morphological probe for membrane cholesterol domains in freeze-fracture replicas. After filipin incubation of prefixed vibratome slices, filipin-cholesterol complexes appeared as 20-30 proturberances and pits on P- and E-faces. Distinct patterns of filipin-cholesterol complexes were found in non-ischaemic and ischaemic neuronal membrane. The filipin-treated specimens showed a 35-40% drop in cholesterol content in the neuronal plasmalemma one hour after cerebral ischaemia.     

Freeze-fracture analysis of cholesterol in muscle plasma membrane of Fukuyama-type congenital muscular dystrophy

Summary We used digitonin in freeze-fracture analysis of the muscle plasma membrane cholesterol content in six patients with Fukuyama-type congenital muscular dystrophy and six control children. A significantly greater proportion of surface area was taken up by digitonin-cholesterol complexes in the patients (51.2%±4.7%) than in controls (31.9%±2.9%) (P<0.01). Since membrane cholesterol has a dynamic regulatory function in affecting the activity of membrane-bound proteins, the increased cholesterol content in the patients suggests a functional abnormality of the muscle plasma membrane in this disease.Supported by grant No. 86-02-34 from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Freeze fracture studies of muscle plasma membrane in human muscular dystrophy

Summary Freeze fracture analysis of intramembranous particle density in skeletal muscle plasma membrane from 7 patients with Duchenne muscular dystrophy (DMD), 5 patients with facioscapulohumeral muscular dystrophy (FSH) and 5 patients with myotonic dystrophy (MyD) were carried out. Marked deplction of intramembranous particles including orthogonal arrays were significantly decreased in FSH. No abnormalities were noted in MyD.This work was supported by a Research Center Grant from the Muscular Dystrophy Association of America and by grants NS-08075, NS-14471 and 5 M01 RR00040 from the U.S. Public Health Service     

Altered muscle polyamine levels in human neuromuscular diseases

Intracellular polyamine levels were measured in 17 patients with neuromuscular diseases and 13 normal controls and were correlated with morphological changes in muscle biopsies. Elevated polyamine levels were found in muscles with marked morphological changes, regardless of the nature of the disease process.     

Electron spin resonance studies of erythrocyte membrane in spinocerebellar degeneration

Erythrocyte membrane fluidity was examined by electron spin resonance spectra using nitroxide fatty acid spin labels in spinocerebellar degeneration (SCD). Subjects with SCD, motor neuron disease (MND) and controls did not differ in fluidity of the deep site (hydrophobic region) of the erythrocyte membrane. However, the fluidity of the shallow site (hydrophilic region) in the erythrocyte membrane was significantly less fluid in SCD than in controls and MND (outer hyperfine splitting of 5-nitroxide stearic acid: SCD 54.70 +/- 0.43 G, controls 53.57 +/- 0.41 G, MND 53.54 +/- 0.35 G, P less than 0.001). Serum HDL-cholesterol and membrane fluidity correlated significantly in controls, but not in SCD. A significant negative correlation between age and membrane fluidity was found in SCD, but not in controls. These data suggest that membrane abnormality exists in SCD and may be concerned with aging.     

Interpretation of electroneuromyographic studies in diseases of neuromuscular junction and myopathies

Electroneuromyography (ENMG) also called electrodiagnosis or at times simply (and erroneously) electromyography (EMG), has been used for the diagnosis of neuromuscular and muscle diseases. It consists of tests done serially and assessed collectively, to arrive at a diagnosis of neuromuscular weakness. The test should be treated as an extension of the clinical examination and not a replacement. When done adequately ENMG is the only test which gives information about the function of the peripheral nervous system and hence co-relates well with the clinical signs. This article reviews the role of ENMG in the evaluation of neuromuscular and muscle disease. We hope this will meet the requirements of both practicing neurologists and dedicated electromyographers.     

Freeze-fracture studies of human blood platelets activated by thrombin using rapid freezing

In this study the influence of thrombin activation on human blood platelets has been followed by freeze-fracturing electron microscopy using rapid freezing in order to catch the initial changes in shape and the morphological alterations during the process of exocytosis of secretory granules. We found that isolation of the platelets by itself leads to some degree of shape change, which made it impossible to study the resting discoid platelet by rapid freezing. Activation of the platelets by thrombin induced dilation of the "surface connecting system (SCS)" with formation of large vacuoles as a result of fusion of the secretory granules with SCS. No intermediary fusion stages or structures were observed even using rapid freezing. Volcano-like protrusions and the corresponding complementary pits were seen at the SCS. These structures were interpreted by us as fractures through protoplasmic channels crossing the SCS. These channels originate during the swelling of the SCS as a result of the fusion of secretory granules with the SCS.     

Electrophysiologic properties of intercostal muscle fibers in human neuromuscular diseases

Electrophysiologic properties of biopsied normal and diseased intercostal muscle fibers were examined using intracellular microelectrode techniques. The resting potentials of all diseased muscle fibers were found to be depolarized. Those from Duchenne dystrophy patients showed the largest depolarization, followed by those from patients with myotonic muscular dystrophy, myotonia congenita, and motor neuron disease. All of the diseased fibers except those from myotonia congenita patients demonstrated an impaired ability to generate action potentials. In the latter fibers, the higher-than-normal membrane resistance was associated with hyperexcitability. When the membrane was hyperpolarized to the normal range, however, action potential characteristics in all fibers were near normal, except in motor neuron disease. All action potentials were blocked by tetrodotoxin. These findings—i.e., that all fibers were capable of generating action potentials when hyperpolarized, and that all action potentials were blocked by tetrodotoxin—suggest the relative intactness, in the disease studied here, of the regenerative sodium conductance mechanism.     

Refractory period studies in a human neuromuscular preparation.

A subtraction technique has been employed to study refractoriness in the median nerve and thenar muscles in man. The absolutely refractory periods of the distal motor nerve branches and of the muscle fibres were found to be similar, the majority being within the 2.5--3.0 ms range; the values were distributed in a unimodal manner. The relatively refractory periods of the motor nerve terminals were about 5 ms. In some experiments it was possible to record non-propagated potentials at the endplate zones of the muscle fibres.     

Hyperestrogenemia in neuromuscular diseases

In order to elucidate the relationship between certain neuromuscular diseases and gonadal hormones, we measured the levels of serum estrogens and other sex-related hormones. The values were compared with those for age-matched controls. The cases, comprising bulbospinal muscular disease of the Kennedy-Alter-Sung type, Kugelberg-Welander disease, amyotrophic lateral sclerosis, and Duchenne muscular dystrophy, were all euthyroid males. The baseline levels of serum estrone were significantly higher in all of the patients than in age-matched normal subjects. Serum baseline testosterone, LH and FSH levels were all essentially normal, except low FSH levels in Duchenne muscular dystrophy. Since our patients had no overweight, liver or glandular abnormalities, we presume that the elevated serum estrone levels have resulted from increased peripheral androgen-to-estrogen conversion.     

Freeze-fracture characterization of isolated myelin and axolemma membrane fractions

The macromolecular organization of membranes isolated from the rabbit optic nerve and tract was analyzed using the freese-fracture technique. A myelin fraction and two axolemma-enriched fractions were prepared from a preparation of myelinated axons isolated by flotation in a buffered salt-sucrose medium.In the myelinated axon preparation, axolemma and myelin membranes were easily identified. Large areas of the axon membrane and myelin membrane totally lacked intramembronous particles. The particles remaining on the myelin membrane formed patches of evenly distributed elongated and globular particles. In contrast, the particles remaining on the axolemma were globular in shape and tightly clustered. Particle clustering and particle-free areas were not characteristic of either the axolemma or myelin membrane of whole nerves fixed in situ and processed for freeze-fracture.The isolated myelin membrane fraction contained a large number of vesicles completely lacking intramembronous particles. Of the remaining membrane vesicles, profiles with dispersed elongated and globular particles predominated. A small percentage of vesicles displayed intramembranous particles of the same size, shape and clustering pattern as that seen on the axolemma of the myelinated axon preparation. The two axolemma fractions were enriched in membrane containing tightly clustered globular particles. Particle-free vesicles as well as some myelin membrane vesicles were also seen in the axolemma fractions.     

Freeze-fracture studies on the synapses in the organ of corti

Receptoneural junctions and synapses in the organ of Corti of the chinchilla have been examined with the freeze-fracture technique. The presynaptic membranes at the receptoneural junctions of inner and outer hair cells have many structural features in common with membranes found at chemical synapses outside the organ of Corti. However, the membranes of the postsynaptic afferent terminals are quite different depending on whether they are part of an inner or outer hair cell synapse. These differences in the distribution of intramembrane particles suggest that the transmitters, or transmitter actions, may be different at these two synapses. The distribution of particles in the postsynaptic membrane at efferent synapses with outer hair cell differs from that in the postsynaptic membrane at efferent synapses with afferent terminals or fibers, suggesting that transmitter actions at these locations could also differ.