Structural and molecular adaptations to dexamethasone and unacylated ghrelin administration in skeletal muscle of the mice.

The central goal of this study was to identify the primary mechanisms triggering steroid atrophy. Adaptations of soleus (Sol) and vastus lateralis (VL) muscles of C57BL/6 female mice were studied following 3, 7 and 15 days of daily intraperitoneal injection (5 mg kg-1 day-1) of dexamethasone (dEx) (chronic treatment) and 1, 3 and 10 hours after a single dEx injection (acute treatment). In the chronic treatment, analyses were performed 24 hours after the last injection. Gene expression of major components of the intracellular signalling pathways controlling mass and metabolism were assessed. Analyses were repeated following dEx and unacylated ghrelin (uAG) (100 µg kg-1day-1), co-administration. We found a significant VL fibres atrophy after 7 (13%) and 15 (28%) days and a Sol fibres atrophy (23%) after 15 days of dEx treatment. The acute treatment showed, in both muscles, several responses in most signalling pathways, among which the enhanced gene expression of Murf-1 (6-fold change in VL and 3-fold in Sol) and myostatin (6-fold change in VL and 20-fold in Sol). In Sol, uAG administration was able to fully counteract muscle atrophy and Murf-1 upregulation, but not the upregulation of myostatin, suggesting a causal relationship between muscle atrophy and Murf-1. Results indicate that: a) the primary mechanism triggering steroid atrophy is an early transient activation of Murf-1; b) uAG inhibits Murf-1 induction counteracting steroid atrophy. The present work contributes to the understanding of the complexity of the muscle response to glucocorticoids.

Actin sliding velocity on pure myosin isoforms from hindlimb unloaded mice.

AIM: Notwithstanding the widely accepted idea that following disuse skeletal muscles become faster, an increase in shortening velocity was previously observed mostly in fibres containing type 1 myosin, whereas a decrease was generally found in fibres containing type 2B myosin. In this study, unloaded shortening velocity of pure type 1 and 2B fibres from hindlimb unloaded mice was determined and a decrease in type 2B fibres was found. METHODS: To clarify whether the decrease in shortening velocity could depend on alterations of myosin motor function, an in vitro motility assay approach was applied to study pure type 1 and pure type 2B myosin from hindlimb unloaded mice. The latter approach, assessing actin sliding velocity on isolated myosin in the absence of other myofibrillar proteins, enabled to directly investigate myosin motor function. RESULTS: Actin sliding velocity was significantly lower on type 2B myosin following unloading (2.70 ± 0.32 µm s(-1)) than in control conditions (4.11 ± 0.35 µm s(-1)), whereas actin sliding velocity of type 1 myosin was not different following unloading (0.89 ± 0.04 µm s(-1)) compared with control conditions (0.84 ± 0.17 µm s(-1)). Myosin light chain (MLC) isoform composition of type 2B myosin from hindlimb unloaded and control mice was not different. No oxidation of either type 1 or 2B myosin was observed. Higher phosphorylation of regulatory MLC in type 2B myosin after unloading was found. CONCLUSION: Results suggest that the observed lower shortening velocity of type 2B fibres following unloading could be related to slowing of acto-myosin kinetics in the presence of MLC phosphorylation.

Light sources and cameras for standard in vitro membrane potential and high-speed ion imaging.

Membrane potential and fast ion imaging are now standard optical techniques routinely used to record dynamic physiological signals in several preparations in vitro. Although detailed resolution of optical signals can be improved by confocal or two-photon microscopy, high spatial and temporal resolution can be obtained using conventional microscopy and affordable light sources and cameras. Thus, standard wide-field imaging methods are still the most common in research laboratories and can often produce measurements with a signal-to-noise ratio that is superior to other optical approaches. This paper seeks to review the most important instrumentation used in these experiments, with particular reference to recent technological advances. We analyse in detail the optical constraints dictating the type of signals that are obtained with voltage and ion imaging and we discuss how to use this information to choose the optimal apparatus. Then, we discuss the available light sources with specific attention to light emitting diodes and solid state lasers. We then address the current state-of-the-art of available charge coupled device, electron multiplying charge coupled device and complementary metal oxide semiconductor cameras and we analyse the characteristics that need to be taken into account for the choice of optimal detector. Finally, we conclude by discussing prospective future developments that are likely to further improve the quality of the signals expanding the capability of the techniques and opening the gate to novel applications.

Muscle injuries: ultrasound evaluation in the acute phase.

Muscle injuries can be classified as extrinsic or intrinsic injuries as well as contusions and lacerations, and clinical assessment is composed of the history and physical examination. Diagnostic imaging, particularly ultrasound (US) examination, is essential to a correct assessment of the severity of the injury and to exclude important complications as these two elements influence treatment decisions, prognosis and time to return to unrestricted physical activity. This paper presents the main clinical and US features of acute muscle injuries.

Baker's cyst in pediatric patients: Ultrasonographic characteristics.

OBJECTIVE: Evaluate incidence, etiology, and sonographic features of Baker's cyst in children. MATERIALS AND METHODS: We examined 16 pediatric patients, with the clinical diagnosis of Baker's cyst. The possibility to confirm or to exclude the presence of the lesion, assess the structure, presence of bilateralism and joint effusion were considered. Three subjects had known juvenile arthritis, 2 hemophilia, 11 a popliteal swelling in the absence of concomitant diseases. RESULTS: In all patients it was possible to confirm (11) or to exclude (5) the presence of Baker's cyst. The idiopathic forms (6) exhibited anechoic structure; in patients with arthritis (3) there was hypertrophic synovium; in hemophilic patients at the presentation (2) anechoic structure with layering (serum and red blood cells); in chronic hemophilia synovial hypertrophy was seen. Joint effusion was constantly present in children with hemophilia and arthritis and in 1 case of idiopathic cyst. CONCLUSION: Baker's cysts in children are rare. Ultrasound is able to confirm or to exclude the presence of the lesion and it is able to evaluate characteristics, bilateralism and association with joint effusion.

Skeletal muscle fibre diversity and the underlying mechanisms.

The review first briefly summarizes how myosin isoforms have been identified as the major determinant of the functional variability among skeletal muscle fibres. The latter feature is a major characteristic of muscle fibres and a major basis of skeletal muscle heterogeneity and plasticity in vivo. Then, evidence is reported, which indicates that the properties of muscle fibres can vary with no change in the myosin isoform they express. Moreover, the physiological and pathological conditions (ageing, disuse, exercise training, muscular dystrophy) in which such myosin isoform independent change in functional properties occurs and the possible underlying mechanisms are considered. Finally, the known molecular bases of the functional differences among slow and fast isoforms are briefly dealt with.

Single muscle fiber properties in aging and disuse.

Since the middle of the 1980s, it was understood that myosin, the motor of contraction, can be expressed in several isoforms. The isoforms of the myosin heavy-chain (MHC) portion of the molecule were found to be mostly responsible for the diversity in the contractile and energetic properties of muscle fibers. In humans, three MHC isoforms are expressed in limb muscles (MHC-1, MHC-2A and MHC-2X) and they generate three pure fiber types (types 1, 2A and 2X) and two hybrid types (types 1-2A and -2AX). Type 1, 2A and 2X fibers widely differ with respect to most of their contractile and energetic properties, and a change in their relative distribution within muscles is known to modulate their functional properties in vivo through a "qualitative" mechanism. On the basis of the MHC regulation of muscle fibers properties, it is expected that a given fiber type develops the same force and shortens at the same speed regardless of the physiologic and pathologic conditions under which the muscle works. Surprisingly, several evidences have been accumulating to show that in aging and disuse, the properties of a muscle fiber type can change with no change in its myosin isoform content. This short review considers the latter phenomenon and the possible underlying mechanisms.

Neuregulin signaling is dispensable for NMDA- and GABA(A)-receptor expression in the cerebellum in vivo.

Neuregulin-1s (NRG-1s) are a family of growth and differentiation factors with multiple roles in the development and function in different organs including the nervous system. Among the proposed functions of NRG-1s in the nervous system is the regulation of genes encoding certain neurotransmitter receptors during synapse formation as well as of other aspects of synaptic function. Here, we have examined, in granule cells of the cerebellum in vivo, the role of NRGs in the induction of NMDA receptor (NMDA-R) and GABA(A) receptor (GABA(A)-R), which are thought to be induced by NRG-1 secreted by the synaptic inputs. To this end, we used the Cre/loxP system to genetically ablate the NRG receptors ErbB2 and ErbB4 selectively in these cells, thus eliminating all NRG-mediated signaling to them. Unlike previous reports using cultured granule cells to address the same question, we found that the developmental expression patterns of the mRNAs encoding the NR2C subunit of the NMDA-R and the beta2-subunit of the GABA(A)-R is normal in mice lacking the NRG receptors ErbB2 and ErbB4. Likewise, no alterations in cerebellar morphology nor in certain aspects of cerebellar wiring were resolved in these mutants. We conclude that NRG/ErbB signaling to the granule cells is dispensable for the normal development of their synaptic inputs.

New techniques in linear and non-linear laser optics in muscle research.

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

Two independent mechanical events in the interaction cycle of skeletal muscle myosin with actin.

During skeletal muscle contraction, regular arrays of actin and myosin filaments slide past each other driven by the cyclic ATP-dependent interaction of the motor protein myosin II (the cross-bridge) with actin. The rate of the cross-bridge cycle and its load-dependence, defining shortening velocity and energy consumption at the molecular level, vary widely among different isoforms of myosin II. However, the underlying mechanisms remain poorly understood. We have addressed this question by applying a single-molecule approach to rapidly ( approximately 300 mus) and precisely ( approximately 0.1 nm) detect acto-myosin interactions of two myosin isoforms having large differences in shortening velocity. We show that skeletal myosin propels actin filaments, performing its conformational change (working stroke) in two steps. The first step ( approximately 3.4-5.2 nm) occurs immediately after myosin binding and is followed by a smaller step ( approximately 1.0-1.3 nm), which occurs much faster in the fast myosin isoform than in the slow one, independently of ATP concentration. On the other hand, the rate of the second phase of the working stroke, from development of the latter step to dissociation of the acto-myosin complex, is very similar in the two isoforms and depends linearly on ATP concentration. The finding of a second mechanical event in the working stroke of skeletal muscle myosin provides the molecular basis for a simple model of actomyosin interaction. This model can account for the variation, in different fiber types, of the rate of the cross-bridge cycle and provides a common scheme for the chemo-mechanical transduction within the myosin family.

Temperature dependence of speed of actin filaments propelled by slow and fast skeletal myosin isoforms.

It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiological (10-25 degrees C) than at temperatures closer to physiological (25-35 degrees C) and is higher in slow than fast muscles. However, because intact muscles invariably express several myosin isoforms, they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibers cannot be unequivocally attributed to a modulation of myosin function itself, as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay approach in which the impact of temperature on velocity can be studied at a molecular level, as in such assays acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A, and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35 degrees C) because isolated myosin is stable up to physiological temperature. The data show that, at the molecular level, the temperature sensitivity is higher at lower (10-25 degrees C) than at higher (25-35 degrees C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms, contrary to what was observed in intact slow and fast muscles.

Effects of resistance training on myosin function studied by the in vitro motility assay in young and older men.

It is generally believed that the maximum shortening velocity (V(o)) of a skeletal muscle fiber type does not vary unless a change in myosin heavy chain (MHC) isoform composition occurs. However, recent findings have shown that V(o) of a given fiber type can change after training, suggesting the hypothesis that the function of myosin can vary without a change in isoform. The present study addressed the latter hypothesis by studying the function of isolated myosin isoforms by the use of the in vitro motility assay (IVMA) technique. Four young (age 23-29 yr, YO) and four elderly men (age 68-82 yr, EL) underwent a 12-wk progressive resistance training program of the knee extensor muscles and to one pre- and one posttraining biopsy of the vastus lateralis muscle. The significant increase in one-repetition maximum posttraining in both YO and EL indicated that training was effective. After training, MHC isoform composition showed a shift from MHC(2X) toward MHC(2A) in YO and no shift in EL. The velocity of sliding (V(f)) of actin filaments on pure myosin isoforms extracted from single fibers was studied in IVMA. One hundred sixty IVMA samples were prepared from 480 single fibers, and at least 50 filaments were analyzed in each experiment. Whereas no training-induced change was observed in V(f) of myosin isoform 1 either in YO or in EL, a significant increase in V(f) of myosin isoform 2A after training was observed in both YO (18%) and EL (19%). The results indicate that resistance training can change the velocity of the myosin molecule.

Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles.

Maximum shortening velocity (V(0)) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V(0) were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V(0) were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V(0) decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V(0) of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation (V(f)) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V(0) and V(f) determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres.

Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters.

Reagents capable of rapid and efficient release of neuroactive amino acids (L-glutamate, GABA and glycine) upon flash photolysis of thermally stable, inert precursors have been elusive. 7-Nitroindolinyl (NI)-caged and 4-methoxy-7-nitroindolinyl (MNI)-caged compounds that fulfil these criteria are evaluated here. These caged precursors are highly resistant to hydrolysis. Photolysis is fast (half time< or =0.26 ms) and the conversion achieved with a xenon flashlamp is about 15% for the NI-caged L-glutamate and about 35% for the MNI-caged L-glutamate. A procedure is described for calibration of photolysis in a microscope-based experimental apparatus. NI-caged L-glutamate itself showed no agonist or antagonist effects on AMPA and NMDA receptors in cultured neurones, and had no effect on climbing fibre activation of Purkinje neurones. A control compound with identical photochemistry that generated an inert phosphate upon photolysis was used to confirm that the intermediates and by-products of photolysis have no deleterious effects. MNI-caged L-glutamate is as stable and fast as NI-caged L-glutamate and similarly inert at glutamate receptors, but about 2.5 times more efficient. However, NI-caged GABA is an antagonist at GABA(A) receptors and NI-glycine an antagonist at glycine receptors. The results show the utility and limitations of these fast and stable caged neurotransmitters in the investigation of synaptic processes.

alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles.

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.

Characterization of the variability of glutamatergic synaptic responses to presynaptic trains in rat hippocampal pyramidal neurons.

Excitatory postsynaptic currents from CA3 hippocampal neurons, elicited by trains of presynaptic action potentials either in mossy fibres or associative commissural fibres, have been analysed, by using a quantal analysis approach, in order to characterize their variability and the correlation among successive responses. As quantal parameters may change during the train according to the previous release events, correlation within consecutive EPSCs is expected. We tested simple hypotheses on how quantal parameters p and N may change on the basis of correlation detection in EPSCs. The statistical significance of these tests has been evaluated. The tests showed that, although simple binomial distributions can give a good description of synaptic responses at the level of single spikes, only stochastic chains can always account for correlations observed within the train. A systematic model fitting procedure has been developed and applied to extract information on the dynamics of synaptic transmission. As an application of this novel type of analysis, a measure of transmitted information to be associated with synaptic variability, a quantity that allows an estimate of the capability of the synapse to transmit reliable information in time, is proposed. We showed that this transmitted information depends on short-term plasticity and that the change in the type of short-term plasticity from facilitating to depressing obtained by increasing the extracellular calcium concentration results in a change of the related transmitted information.

The conductance underlying the parallel fibre slow EPSP in rat cerebellar Purkinje neurones studied with photolytic release of L-glutamate.

1. Tetanic stimulation of parallel fibres (PFs) produces a slow EPSP (sEPSP) or slow EPSC (sEPSC) in Purkinje neurones (PNs), mediated by type 1 metabotropic glutamate receptors (mGluR1). The conductance change underlying the sEPSP was investigated with rapid photolytic release of L-glutamate from nitroindolinyl (NI)-caged glutamate with ionotropic glutamate receptors blocked, and showed a slow mGluR1-activated cation channel. 2. In cerebellar slices rapid photolytic release (t (1/2) < 0.7 ms) of 7--70 microM L-glutamate on PNs voltage clamped at -65 mV activated first a transient inward current, peaking in 8 ms, followed by a slow inward current with time course similar to the PF sEPSP, peaking at -1 nA in 700 ms. 3. The initial current was inhibited by 300 microM threo-hydroxyaspartate (THA) and did not reverse as the potential was made positive up to +50 mV, suggesting activation of electrogenic glutamate uptake. 4. The slow current was inhibited reversibly by 1 mM (R,S)-MCPG or the non-competitive mGluR1 antagonist CPCCOEt (20 microM), indicating activation of metabotropic type 1 glutamate receptors. The mGluR current was associated with increases of input conductance and membrane current noise, and reversed close to 0 mV, indicating activation of channels permeant to Na(+) and K(+). 5. The sEPSC was not blocked by Cd(2+), Co(2+), Mg(2+) or Gd(3+) ions, by the inhibitor of hyperpolarisation-activated current (I(H)) ZD7288, or by the purinoceptor inhibitor PPADS. Activation was not affected by inhibitors of phospholipase C (PLC) or protein kinase C (PKC), nor mimicked by photorelease of InsP(3) or Ca(2+). The results show that mGluR1 in PNs produces a slow activation of cation-permeable ion channels which is not mediated by PLC activation, Ca(2+) release from stores, or via the activation of PKC.

Functional diversity between orthologous myosins with minimal sequence diversity.

To define the structural differences that are responsible for the functional diversity between orthologous sarcomeric myosins, we compared the rat and human beta/slow myosins. Functional comparison showed that rat beta/slow myosin has higher ATPase activity and moves actin filaments at higher speed in in vitro motility assay than human beta/slow myosin. Sequence analysis shows that the loop regions at the junctions of the 25 and 50 kDa domains (loop 1) and the 50 and 20 kDa domains (loop 2), which have been implicated in determining functional diversity of myosin heavy chains, are essentially identical in the two orthologs. There are only 14 non-conservative substitutions in the two myosin heavy chains, three of which are located in the secondary actin-binding loop and flanking regions and others correspond to residues so far not assigned a functional role, including two residues in the proximal S2 domain. Interestingly, in some of these positions the rat beta/slow myosin heavy chain has the same residues found in human cardiac alpha myosin, a fast-type myosin, and fast skeletal myosins. These observations indicate that functional and structural analysis of myosin orthologs with limited sequence diversity can provide useful clues to identify amino acid residues involved in modulating myosin function.

GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging.

In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAA antagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area.