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Apoptosis was detected in different muscular diseases, including severe dystrophin deficiency, but apoptotic mechanisms are not completely described in adult skeletal muscle. Studying patients affected by Duchenne muscular dystrophy (DMD) and by facio-scapulo-humeral dystrophy (FSHD) we showed an increase of apoptotic myonuclei, bax, and bcl-2-positive myofibers. Positive correlation was detected between apoptotic nuclei and bax expression (p < 0.01). Expression of caspases was analyzed by RNase protection. Caspase transcript was not detected in normal skeletal muscles. DMD muscles expressed caspase 8, 3, 5, 2, 7 and Granzyme B mRNAs. Low levels of caspase 6, 3, and Granzyme B transcripts were detected in FSHD patients. Tissue levels of caspase 3 protein significantly correlated with apoptotic myonuclei (p < 0.05) and with bax expression (p < 0.01). In all DMD cases the activity of caspase 3 was increased, while the FSHD samples were heterogeneous. These data indicate that human skeletal muscle fibers. during the dystrophic process, modulate the expression of caspases and that caspase 3 is involved in myofiber cell death. opening new perspective in the pharmacological treatments of muscular dystrophies, such as the use of caspase inhibitors.

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Macrophage-muscle cell interactions are complex, and the majority is unknown. The persistence of inflammatory cells in skeletal muscle could be critical for myofiber viability. In the present paper, we show that FasL plays a role in the resolution of muscle inflammation. We analyzed inflamed muscles of normal mice treated from day 3 to day 8 with a FasL inhibitor (Fas-Ig) or with control Ig. Treated muscles were collected at 3, 5, and 10 days. The treatment with recombinant Fas-Ig protein induced a severe persistence of inflammatory cells at 5 days (115,000+/-27,838 vs. 41,661+/-6848, p<0.01) and 10 days from injury (145,500+/-40,850 vs. 5000+/-1000, p<0.001). Myofiber regeneration was highly impaired (37+/-14 vs. 252+/-28, p<0.01). Apoptosis of phagocytic cells was absent during Fas-Ig treatment (0.9+/-0.6 vs. 1300+/-150, p<0.0001), but apoptotic, mononucleated cells appeared at day 10, 2 days after the suspension of Fas-Ig administration. The time course of FasL expression during muscle inflammation, at mRNA and protein level, reveals a peak during myoblast proliferation. The peak of FasL expression coincides with the peak of apoptosis of phagocytic cells. In situ hybridization shows the co-expression of FasL and MyoD mRNA in mononucleated cells, i.e., myoblasts. Experiments on the myoblast cell culture confirmed the expression of FasL in myoblasts. The findings shown here indicate one of the pathways to control myoblast-macrophage interaction and might be relevant for the control of inflammatory cells in muscle tissue. Perhaps altering FasL expression with recombinant proteins could ameliorate inflammation in degenerative myopathies and up-regulate muscle regeneration.

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During early cardiac development the atrial myocardium is continuous with the ventricular myocardium throughout the atrioventricular canal. The atrioventricular canal undergoes complex remodelling involving septation, formation of atrioventricular valves and insulation between atria and ventricles except at the level of the atrioventricular node. Understanding of these processes has been hampered by the lack of markers specific for this heart region. We have generated transgenic mice expressing beta-galactosidase under the control of the cardiac troponin I gene that show transgene expression mainly confined to the atrioventricular canal myocardium during early embryonic development. With further development beta-galactosidase positive cells are observed in the atrioventricular node and in the lower rim of both right and left atria, supporting the view that atrioventricular canal myocardium contributes to the atrioventricular node and is in part incorporated into the lower rim of the atria. These results identify the atrioventricular canal myocardium as a distinct transcriptional domain.

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The SH3BGR gene has been recently isolated and mapped to chromosome 21 within the Down syndrome (DS) congenital heart disease (CHD) minimal region. As a first step to evaluate the possible involvement of SH3BGR in CHD that affect 40% of DS patients, we have analyzed by in situ hybridization the expression pattern of the mouse homolog gene (Sh3bgr), during development. Our results show that Sh3bgr is already expressed at embryonic day 7.75 (E7.75) in the precardiogenic mesoderm and that from E8.5 to E10.5 its expression is restricted to the heart. In subsequent developmental stages, Sh3bgr transcripts are also detected in skeletal muscle and in some visceral smooth muscles including urinary bladder and gut wall, but not in vascular smooth muscle. Our results, demonstrating that Sh3bgr is expressed in earliest stages of mouse heart development, support a possible role of this gene in heart morphogenesis and, consequently, in the pathogenesis of CHD in DS.

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We studied by in situ hybridization the expression of IIx myosin heavy chain (MHC) transcripts in Duchenne Muscular Dystrophy (DMD) muscle. In normal muscle fibers IIx MHC transcripts were only expressed in type 2b or very fast fibers. Our results indicate that muscle fibers expressing IIx MHC transcripts disappear early in DMD muscle. This may be due to the transformation of type 2b fibers into type 2a fibers and/or to selective degeneration of the type 2b fibers. The very fast 2b fibers may be subjected to a greater mechanical stress during muscle contraction and dystrophin deficiency may render them more susceptible to mechanical damage.

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The effects of a 37-day period of bed rest on myosin heavy chain (MHC) expression on both mRNA and protein level in human skeletal muscle fibers were studied. Muscle biopsies from vastus lateralis muscle were obtained from seven healthy young male subjects before and after the bed-rest period. Combined in situ hybridization, immunocytochemistry, and ATPase histochemistry analysis of serial sections of the muscle biopsies demonstrated that fibers showing a mismatch between MHC isoforms at the mRNA and protein level increased significantly after the bed-rest period, suggesting an increase in the amount of muscle fibers in a transitional state. Accordingly, fibers showing a match in expression of MHC-1 and of MHC-2A at the mRNA and protein level decreased, whereas fibers showing a match between MHC-2X mRNA and protein increased after bed rest. Overall, there was an increase in fibers in a transitional state from phenotypic type 1 --> 2A and 2A --> 2X. Furthermore, a number of fibers with unusual MHC mRNA and isoprotein combinations were observed after bed rest (e.g., type 1 fibers with only mRNA for 2X and type 1 fibers negative for mRNA for MHC-beta/slow, 2A, and 2X). In contrast, no changes were revealed after an examination at the protein level alone. These data suggest that the reduced load-bearing activity imposed on the skeletal muscles through bed rest will alter MHC gene expression, resulting in combinations of mRNA and MHC isoforms normally not (or only rarely) observed in muscles subjected to load-bearing activity. On the other hand, the present data also show that 37 days of bed rest are not a sufficient stimulus to induce a similar change at the protein level, as was observed at the gene level.

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Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.

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The association between caregiver's stress and depressive symptoms, and sociodemographic and clinical characteristics of Alzheimer's patients and caregivers was evaluated in 63 primary caregivers consecutively recruited at the Alzheimer's Dementia Research and Care Unit of Brescia, northern Italy. Family caregivers' informal support was also evaluated. Patients' behavioral disturbances, and the number of persons who lived in the household were the main correlates of caregivers' stress. The greater the number of persons, the lower the report of caregiver stress. Lower frequency of visits of friends or relatives, caregivers' poor health and higher age, and the presence of patients' behavioral disturbances were the main determinants of caregivers' depressive symptoms. Cognitive impairment of patients was not correlated to caregivers' distress. Our study underlines that the imperatives of dementia and caregiving appear to be so significant that they have similar consequences in the Italian population as well as in other very different cultures. Factors related to primary caregivers, other than patients' behavior, such as informal supports or caregivers' health and age, should be taken into account in the evaluation of caregivers' burden.

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This study was made to identify the inhibitory interneurons belonging to the spinal circuitry activated by the Mauthner axons in the tench (Tinca tinca L.). The histological investigations were focused on a segmental pair of commissural interneurons that were reconstructed in toto from their distinguishing topographical and ultrastructural features. These features are: (a) the adendritic soma located 100-150 microns dorsal to the central canal; (b) the first node of Ranvier which is precommissural and connected to the ipsilateral Mauthner axon via gap junction; (c) the second node of Ranvier, from which two first-order branches arise postcommissurally each supplying roughly the rostral and caudal half of the contralateral spinal cord segment; (d) their second-order branches, which arise at intervals that correspond closely to those of the Mauthner axon collaterals; (e) the postsynaptic targets of the second-order branches, which are exclusively all the motoneurons and interneurons innervated by the contralateral Mauthner axon; (f) the axon terminals of these branches, which contain F-type vesicles, form Gray type-2 synapses, and abut either on the initial segment or on the first node of Ranvier of the target neurons. Thus, it appears that this segmental interneuron has all the appropriate features that could provide the structural basis for the reciprocal fast-acting inhibitory coupling underlying the startle reflex elicited by the Mauthner neurons in response to auditory stimuli.

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The electromotor system of the electric eel, Electrophorus electricus, was studied by injection of horseradish peroxidase as a retrograde tracer. The electromotor neurons, which innervate the electrocytes, comprise a midline nucleus, largely dorsal to the spinal canal. Spinal motoneurons lie ventrolaterally. The electromotor and skeletal motor neuron populations correspond to the acetylcholinesterase-negative and -positive cells previously described. The medullary relay neurons were labeled following HRP injection into the spinal cord at a level where electromotor neurons occurred, but not after injection into the cord in the abdominal region rostral to these cells. Other medullary neurons, presumably bulbospinal motor fibers, were labeled after both levels of spinal cord injection. The results suggest that these axosomatic synapses, which are electrically transmitting but morphologically mixed, take up retrograde tracers in a manner similar to chemical synapses and that tracer uptake is at least largely at terminal regions.

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Sternarchus emits low voltage biphasic pulses at about 700-900/s. These signals (and changes in them caused by external objects) are detected by the tuberous or phasic electroreceptors. We used electron microscopy to examine extracellular compartments in the current pathway to the receptor cells, which are delineated by cells joined by tight junctions. Highly specialized accessory cells were found to separate the receptor cells from the extracellular space continuous with the exterior. Except for synaptic specializations complements of intramembrane particles of cell membranes were unremarkable and did not correlate with presumed high and low resistivity. We propose an equivalent electrical circuit that is consistent with the morphological and physiological observations.

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In order to investigate the transglial pathways in the Schwann sheath of squid giant axons, an electron microscopic study of thin sections and freeze-fracture replicas was carried out. Hitherto the mesaxonal clefts between Schwann cells were regarded as the only pathway between the extracellular space and the periaxonal space which, like the clefts, is about 10 nm in width. The clefts were now found to be obstructed by a putative single-stranded tight junction between neighbouring Schwann cells along the entire border near the axon. The Schwann cells were found to be penetrated like a sponge by a three-dimensional tubular transglial lattice that is confluent with the periaxonal space, the mesaxonal clefts and the extracellular space. The transglial channel system (TGCS) would, therefore, serve as an alternative diffusional pathway, provided that the tubular lumen was permeable. The diameter of the tubules is about 40 nm. In freeze-fracture replicas the density of tubular openings towards the axon was estimated to be 3.3 +/- 0.72 per micron 2. In relation to the periaxonal cell surface, this constitutes a relative opening area of 0.42% as compared to the 0.15% of the mesaxonal clefts (neglecting their tight junctions). Therefore, the TGCS would provide a ubiquitous access for ionic flow between axolemma and extracellular space. The fact that the TGCS has only recently been observed in squid, but has been described for some time in the giant nerve fibres of crayfish and lobster, can be explained by the use of different fixation methods. The TGCS system is preserved in aldehyde fixation as used in the present study, whereas osmium tetroxide was applied in earlier work on squid. The comparison with the results obtained in other species suggests strongly that the TGCS is permeable and constitutes a transglial pathway for rapid ionic flow.

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Using ultrastructural features as a natural tracer we identified a commissural interneuron in the tench (Tinca tinca L.) spinal cord with the following characteristics: (1) the unipolar cell soma is located 100-150 micron dorsal from the central canal, the frequency of its occurrence being one cell on each side per spinal cord segment; (2) the first node of Ranvier of its axon is connected to a presynaptic branch of the ipsilateral Mauthner axon via gap junctions; (3) its postsynaptic targets are contralateral moto- and interneurons; (4) its terminal boutons contain f-type vesicles and form Gray-type-2 synapses abut on the initial segment or node of Ranvier of these target neurons distal to the input site of monosynaptic excitatory links from the contralateral Mauthner axon. Thus, it appears that this segmental interneuron may provide the structural basis for the well-known mutual crossed inhibition within the spinal circuit of Mauthner axons.

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The freeze-etching technique was applied to the tench spinal cord. Replicas of Mauthner axons revealed that the non-junctional axolemma at the tip of axon collaterals alone exhibits intramembranous E-face particles whose size, distribution and overall density are comparable with those at Ranvier nodes. Since typical Ranvier nodes are lacking along the stem of the heavily myelinated Mauthner giant axon, this finding contributes further evidence to our earlier observations suggesting that the spiny Mauthner axon collaterals represent true nodal equivalents and most likely the long-sought substrate of impulse propagation.

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In a light microscopic study the ferric ion-ferrocyanide staining of the squid giant axon was investigated. In other neurons this stain is known to indicate excitable membranes with high sodium channel density, such as the node of Ranvier or the initial segment. In the squid the stain reacted in small patches of the axolemmal, suggesting an aggregation of sodium channels. These axolemma patches were always adjacent to the nuclei of special Schwann cells, suggesting a glial-axonal interaction.

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Impulse generation and propagation was previously shown to occur in skin epithelium of newt (Cynops orientalis) embryos during certain stages of development and to be correlated with morphological changes of gap junctions. These properties are not detected in embryonic epithelia explanted and grown in culture. However, early explants when transplanted to a host embryo develop conductivity, and relatively large gap junctions with loose arrangement of connexons occur as soon as the host embryo reaches the stage when conductivity is at its maximum. In contrast, morphological and physiological characteristics of impulse propagation are lost when the transplanted epithelium is extirpated from the host embryo and returned to in-vitro conditions. Therefore, it appears that impulse propagation is dependent not solely on the differentiation of epithelial cells but upon signals from non-epithelial (possibly mesodermal) tissue as well.

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The spiny collaterals of the Mauthner axon were reinvestigated in the tench (Tinca tinca L.) with the electron microscope and special staining procedures. These collaterals, as demonstrated by intraaxonal labelling with lucifer yellow, are more or less regularly spaced (100-300 micrometers) and make synaptic contacts with processes of spinal motoneurons and interneurons. The unmyelinated tips of the collaterals are further characterized by the following structural features: (1) an electron-dense undercoating of the axolemma, (2) a positive Prussian blue reaction of the inner surface of the axolemma following ferric ion-ferrocyanide staining (Waxman and Quick, '78a), (3) expanded extracellular spaces which react specifically to inorganic phosphate, metallic ions, and diaminobenzidine. All these properties are known to be shared by the axolemma of central and peripheral nodes of Ranvier. Previous studies from this laboratory have shown that the nerve impulse is propagated along the Mauthner axon in a saltatory mode. Since classical nodal gaps could not be identified within the myelin sheath of this giant fiber, it is concluded on the basis of the present findings that the unmyelinated tips of the spiny collaterals represent nodal equivalents, and thus provide the morphological substrate for the saltatory propagation of the nerve impulse along the Mauthner axon. The typical latency steps, as demonstrated in the latency plot of the longitudinal current signals (Greeff and Yasargil, '80), and the distances between the identified membrane specializations at the axon collaterals are consistent with this conclusion.

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Epithelium of amphibian embryos (Cynops orientalis, Xenopus laevis) was found in preceding experiments to generate and conduct impulses during a limited stage (26-37) of development . In order to elucidate the structural basis of impulse propagation, epithelial cells of four stages were examined by the freeze-etching method: (I) before and (II) during acquisition of conductivity; (III) when propagation was fully established, and (IV) when it was no longer present. Only few gap junctions (GJ) of small size were found in groups I and IV. GJ in epithelia of group III were increased in number and size, and appeared morphologically "coupled", i.e., with more loosely arranged connexons. the size of gap-junctional particles did not differ significantly between coupled and uncoupled stages. Zonulae occludentes seemed "leaky" in stage *, and "tight" in stages II-IV. Thus, the morphological characteristics of specialized junctions between "non excitable cells" correlated with the opening and closing of low resistance intercellular current pathways during embryonic development. Gap junctions in particular seem to form an essential link in the non-neural stimulus-response system, which may facilitate the mobility of the embryo during early phases of aquatic life before the reflex pathways have been established. Coupling and uncoupling of gap junctions may also play an important role in the regulation of cell differentiation and morphogenetic movement. The experimental model used in this study provides a useful tool for further investigations of structural correlates of gap junctional permeability under physiological conditions.

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Axo-axonic and dendro-dendritic synapses of the rat substantia gelatinosa Rolandi (SGR) have been studied in 14 adult rats by means of thin section and freeze-etch electron microscopy. Out of 6045 synaptic contacts we identified 54 between axon terminals, 10 between dendritic processes. Aside from vesicle shape the following parameters were used in a morphometric study; width of synaptic clefts, depth and length of postsynaptic densities. There are two main types of axo-axonic synapses, those with agranular vesicles (AA-type) and those with an increased population of large granular vesicles (AAgr-type). The former is prevailing and can be subdivided into two subgroups: AA1 and AA2. The pre- and postsynaptic terminals of AA1 contain spherical vesicles, the depth and length of postsynaptic densities as well as the width of the junctional clefts being significantly larger than that of AA2. Presynaptic terminals of AA2 synapses contain predominantly flattened vesicles, while spherical vesicles were found exclusively in the postsynaptic boutons. AAgr-type is rarely encountered; it is characterized by large granular vesicles (65-110 nm) which accumulate postsynaptically and occasionally in both pre- and postsynaptic boutons. Two types of dendro-dendritic synapses (DD1 and DD2) constitute another SGR feature. The difference between DD1 and DD2 is analogous to that between AA1 and AA2 except that pleomorphic synaptic vesicles appear in both groups. The width of subsynaptic membrane appositions turned out to be the most consistent criterion by which the two groups could be differentiated.

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The medullary electromotor nucleus (EMN) of Sternarchus albifrons was studied at the light and electron microscopic levels. The EMN consists of a dense meshwork of myelinated axons and glial elements with interposed large neurons; it is provided with an abundant supply of capillaries. Two types of essentially adrendritic nerve cells were distinguished on the basis of size: giant neurons (approx. 70 micrometers in diameter) and large neurons (approx. 30 micrometers in diameter). Their population ratio is 1:4. Only giant cells are labelled following the injection of retrograde tracer into the spinal cord; they are therefore identified with the so-called "relay cells" of other gymnotids. Tracer experiments further suggest that the descending axons of these relay cells give off collateral branches throughout the elongated spinal electromotor nucleus. In contrast, the large cells remain unlabelled and therefore lack spinal projections; they most likely correspond to "pacemaker cells." The perikaryal surface, including axon hillock and proximal part of initial segment of both types of EMN cells, is contacted by clusters of synaptic terminals and astrocytic processes. Two main varieties of synaptic terminals occur: (1) large endings and (2) ordinary end feet with standard size (S-type) and variable size (Sv-type) clear, spherical vesicles. The junction between large endings and EMN cells is characterized by the combination of gap junctions and surrounding intermediate junctions whose freeze-fracture characteristics were morphometrically analyzed. The large endings were formed by nodes of Ranvier as well as by fiber terminations, and synchronization within the EMN may be achieved by presynaptic fibers. Some of the contacts occur directly on the initial segment, which could allow activity to bypass the soma. It is concluded that the elctromotor system of Sternarchus is comprised of a rapid conduction pathway where medullary pacemaker and relay cells as well as spinal electromotor neurons are coupled by synapses with gap junctions. In contrast to the spinal electromotor neurons, the medullary EMN cells receive synapses with morphological characteristics of chemical transmission, and the S-type and SV-type terminals may possibly correspond to Gray's Type I and Type II synapses, respectively. These synapses may be involved in modulation of the electric organ discharge frequency.

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