Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors.

Long-term potentiation (LTP) of excitatory synaptic transmission has long been considered a cellular correlate for learning and memory. Early LTP (less than 1 h) had initially been explained either by presynaptic increases in glutamate release or by direct modification of postsynaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor function. Compelling models have more recently proposed that synaptic potentiation can occur by the recruitment of additional postsynaptic AMPA receptors (AMPARs), sourced either from an intracellular reserve pool by exocytosis or from nearby extra-synaptic receptors pre-existing on the neuronal surface. However, the exact mechanism through which synapses can rapidly recruit new AMPARs during early LTP remains unknown. In particular, direct evidence for a pivotal role of AMPAR surface diffusion as a trafficking mechanism in synaptic plasticity is still lacking. Here, using AMPAR immobilization approaches, we show that interfering with AMPAR surface diffusion markedly impairs synaptic potentiation of Schaffer collaterals and commissural inputs to the CA1 area of the mouse hippocampus in cultured slices, acute slices and in vivo. Our data also identify distinct contributions of various AMPAR trafficking routes to the temporal profile of synaptic potentiation. In addition, AMPAR immobilization in vivo in the dorsal hippocampus inhibited fear conditioning, indicating that AMPAR diffusion is important for the early phase of contextual learning. Therefore, our results provide a direct demonstration that the recruitment of new receptors to synapses by surface diffusion is a critical mechanism for the expression of LTP and hippocampal learning. Since AMPAR surface diffusion is dictated by weak Brownian forces that are readily perturbed by protein-protein interactions, we anticipate that this fundamental trafficking mechanism will be a key target for modulating synaptic potentiation and learning.

Regulation of AMPA receptor surface trafficking and synaptic plasticity by a cognitive enhancer and antidepressant molecule.

The plasticity of excitatory synapses is an essential brain process involved in cognitive functions, and dysfunctions of such adaptations have been linked to psychiatric disorders such as depression. Although the intracellular cascades that are altered in models of depression and stress-related disorders have been under considerable scrutiny, the molecular interplay between antidepressants and glutamatergic signaling remains elusive. Using a combination of electrophysiological and single nanoparticle tracking approaches, we here report that the cognitive enhancer and antidepressant tianeptine (S 1574, [3-chloro-6-methyl-5,5-dioxo-6,11-dihydro-(c,f)-dibenzo-(1,2-thiazepine)-11-yl) amino]-7 heptanoic acid, sodium salt) favors synaptic plasticity in hippocampal neurons both under basal conditions and after acute stress. Strikingly, tianeptine rapidly reduces the surface diffusion of AMPA receptor (AMPAR) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism that enhances the binding of AMPAR auxiliary subunit stargazin with PSD-95. This prevents corticosterone-induced AMPAR surface dispersal and restores long-term potentiation of acutely stressed mice. Collectively, these data provide the first evidence that a therapeutically used drug targets the surface diffusion of AMPAR through a CaMKII-stargazin-PSD-95 pathway, to promote long-term synaptic plasticity.

Wavelet analysis for single molecule localization microscopy.

Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

Surface trafficking of N-methyl-D-aspartate receptors: physiological and pathological perspectives.

The N-methyl-D-aspartate receptor (NMDAR) plays a crucial role in shaping the strength of synaptic connections. Over the last decades, extensive studies have defined the cellular and molecular mechanisms by which synaptic NMDARs control the maturation and plasticity of synaptic transmission, and how altered synaptic NMDAR signaling is implicated in neurodegenerative and psychiatric disorders. It is now clear that activation of synaptic or extrasynaptic NMDARs produces different signaling cascades and thus neuronal functions. Our current understanding of NMDAR surface distribution and trafficking is only emerging. Exchange of NMDARs between synaptic and extrasynaptic areas through surface diffusion is a highly dynamic and regulated process. The aim of this review is to describe the identified mechanisms that regulate surface NMDAR behaviors and discuss the impact of this new trafficking pathway on the well-established NMDAR-dependent physiological and pathophysiological processes.

Inspiratory muscle activity during incremental exercise in obese men.

OBJECTIVE: The aim of this study was to assess overall inspiratory muscle activity during incremental exercise in obese men and healthy controls using the non-invasive, inspiratory muscle tension-time index (T(T0.1)). We studied 17 obese subjects (mean age+/-s.d.; 49+/-13 years) and 14 control subjects (42+/-16) during an incremental, maximal exercise test. METHODS: Measurements included anthropometric parameters, spirometry, breathing patterns and inspiratory muscle activity. T(T0.1) was calculated using the equation T(T0.1)=P(0.1)/P(Imax) x T(I)/T(TOT) (where P(0.1) is mouth occlusion pressure, P(Imax) is maximal inspiratory pressure and T(I)/T(TOT) is the duty cycle). RESULTS: At same levels of maximal exercise (%W(max)) (20, 40, 60, 80, 100% W(max)), obese subjects showed higher P(0.1) (P<0.001) and P(0.1)/P(Imax) (P<0.001) values than controls. T(T0.1) was thus higher in obese subjects for each workload increment and at maximal exercise (P<0.001). CONCLUSIONS: During exercise, patients with obesity show alterations in inspiratory muscle activity as a result of both reduced inspiratory strength (as measured by maximal inspiratory pressure) and increased ventilatory drive (as reflected by mouth occlusion pressure), which prone obese subject to respiratory muscle weakness. Our results suggest that impaired respiratory muscle activity could contribute to a decrease in exercise capacity. T(T0.1) may be useful in our understanding concerning the benefits of endurance training.

Noninvasive assessment of the tension-time index of inspiratory muscles at rest in obese male subjects.

OBJECTIVE: The aim of this study was to investigate the effect of excessive mechanical load caused by obesity on the inspiratory muscle performance in obese men at rest. METHODS: We therefore measure at rest spirometric flows and the noninvasive tension time index of inspiratory muscle (TTmus = PI/PImax x TI/TTOT) in eight obese male subjects (body mass index (BMI) > 30) and 10 controls. RESULTS: Spirometric flow (FEV1% pred, FVC% pred) and maximal inspiratory pressure (PImax) were significantly lower in obese subjects compared to controls (P < 0.001). The mean TTmus was significantly higher in obese subjects than in controls (0.136 +/- 0.003 vs 0.045 +/- 0.01). The increase in TTmus was primarily due to an increase in the ratio of mean inspiratory pressure to maximal inspiratory pressure (PI/PImax) and the duty cycle (TI/TTOT). We found a significant negative relationship between PImax and BMI (r = -0.74, P < 0.001), a positive correlation between TTmus and BMI (r = 0.80, P < 0.001) and a negative correlation between TTmus and forced expiratory volume in 1 s (r = -0.85, P < 0.001). CONCLUSION: Excessive mechanical load caused by obesity imposes a great burden on the inspiratory muscle, which may predispose such subjects to respiratory muscle weakness at rest.

Single metallic nanoparticle imaging for protein detection in cells.

We performed a visualization of membrane proteins labeled with 10-nm gold nanoparticles in cells, using an all-optical method based on photothermal interference contrast. The high sensitivity of the method and the stability of the signals allows 3D imaging of individual nanoparticles without the drawbacks of photobleaching and blinking inherent to fluorescent markers. A simple analytical model is derived to account for the measurements of the signal amplitude and the spatial resolution. The photothermal interference contrast method provides an efficient, reproducible, and promising way to visualize low amounts of proteins in cells by optical means.

Aerobic capacity and peripheral skeletal muscle function in coronary artery disease male patients.

The aim of this study was to determine if the diminished aerobic capacity of coronary artery disease male (CAD) patients is accompanied by an impaired skeletal muscle function compared to healthy control subjects. Thirteen CAD patients and 9 healthy subjects performed both a maximal laboratory exercise testing and an assessment of the peripheral skeletal muscle function on an isokinetic apparatus. The cardiorespiratory and mechanical parameters were measured at ventilatory threshold and at maximal effort during a maximal exercise testing. The peripheral skeletal muscle function of the quadriceps was assessed from the maximal voluntary isometric force (MVIF) and from the static endurance time (SET) at an intensity of 50 % of the MVIF. The CAD patients showed a diminished aerobic capacity compared to healthy control subjects at maximal effort (maximal VO(2) uptake: p < 0.0001, maximal ventilation: p < 0.01; maximal heart rate: p < 0.0001, maximal power: p < 0.001) but also at VT (VO(2) uptake VT: p < 0.0001, Power VT: p < 0.001). No difference was found on the MVIF (p < 0.90) between the CAD patients and the control subjects whereas the SET was lower in the CAD patients (p < 0.01). The CAD patients had a lower aerobic capacity and an impaired skeletal muscle endurance compared to healthy subjects.

On the elastic properties of tetramethylrhodamine F-actin.

(Iodoacetamido)tetramethylrhodamine disrupts F-actin. At the 1:1 fluorophore to actin (as monomer) ratio approximately 80% of the protein becomes non-sedimentable. The fluorescent, non-sedimentable actin copolymerizes with G-actin to yield fluorescent filaments. The tensile strength of these filaments changes with the ratio of the fluorescent non-sedimentable actin to the G-actin, being 1.6 pN, 2.9 pN and 3.6 pN at the 1/4, 2/3 and 1/1 ratios, respectively. These tensile strengths are approximately two orders of magnitude lower than those obtained by decoration of F-actin with phalloidin.

Fast and reversible trapping of surface glycine receptors by gephyrin.

Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

Effects of aging on cardiorespiratory responses to brief and intense intermittent exercise in endurance-trained athletes.

The aim of this study was to investigate the effects of aging on athletes' cardiorespiratory responses to a brief intense intermittent effort, using the force-velocity test as an exercise model. Twelve young athletes (24.8 +/- 1.3 years) and twelve master athletes (65.1 +/- 1.2 years) with similar heights, body masses, and endurance training schedules participated in this study. They performed both a maximal graded exercise and the force-velocity tests. The force-velocity test consisted of the repetition of 6-second sprints against increasing braking forces with 5-minute recovery periods. None of the subjects presented abnormal electrocardiogram responses to the tests. During the force-velocity test, the heart rate magnitudes of response in all subjects were correlated to the corresponding sprint power output (p < .001), with higher values for the young athletes (p < .001). Both groups had similar systolic blood pressure peaks of response during the force-velocity test. Both groups had similar preexercise and end-of-recovery oxygen consumption (VO2), but the young athletes had higher peaks of response (p < .001). The VO2 magnitudes of response increased during the test (p < .01) in all subjects, with higher values for the young athletes (p < .001). There was a positive correlation between the VO2 magnitude of response and (1) the corresponding sprint power output (R = .58,p < .001) and (2) the corresponding number of sprint repetitions (R = .29, p < .02). The young athletes had higher end-of-recovery and peak carbon dioxide production (VCO2) responses than the master athletes (p < .001). Pulmonary ventilation (V(E)) peaks of response to the sprints were higher in the young athletes (p < .001). There was a positive relation between the V(E) and VCO2 peaks of response (R = 84,p < .001). In both groups the peak heart rate, VO2, VCO2, and V(E) values attained during the force-velocity test represented similar percentages of the maximal values reached at exhaustion of maximal graded exercise. These results showed that aging does not alter the percentage of the cardiorespiratory response to a brief intense intermittent exercise such as the force-velocity test. Moreover, the arterial blood pressure response is not significantly altered, whereas the vasodilatatory response is.

Rhodamine phalloidin F-actin: critical concentration versus tensile strength.

The mechanic and elastic properties of rhodamine phalloidin F-actin were investigated as a function of the ionic strength and in the absence of Mg2+. By increasing ionic strength from 3 to 19 mM, critical concentration decreased from 146 to 36 nM and the yield strength increased from 5.6 pN to 28.6 pN. At the ionic strength of 12-13 mM, the elastic modulus by stretching increased by 330-430 kP. nm-1 up to the break point, where it was 38-44.2 MP. The work required to break the filament, 403-439 kJ.M-1 provides an estimate of the free energy of annealing of rhodamine phalloidin F-actin, the annealing constant being 2.8 x 1074 M-1.

Cell migration as a five-step cycle.

The migration of cells over substrata is a fundamental and critical function that requires the co-ordination of several cellular processes which operate in a cycle. At the level of the light microscope, the cycle can be divided into five steps: (1) extension of the leading edge; (2) adhesion to matrix contacts; (3) contraction of the cytoplasm; (4) release from contact sites; and (5) recycling of membrane receptors from the rear to the front of the cell. Each step is dependent upon one or more cyclical biochemical processes. The development of many in vitro and subcellular assays for the fundamental biochemical processes involved has increased our understanding of each cycle dramatically in the last several years to include a definition of many of the protein and enzymic components, the role of the position of extracellular-matrix receptors on the cell, and the contribution of physical force. The next generation of questions are directed at resolving the roles of the many individual proteins in each step of the cell migration process. In this chapter we will examine each of the migration steps and discuss the biochemical mechanisms that may underlie them.

[Transesophageal echocardiography before electric cardioversion for supraventricular arrhythmia]. / Echocardiographie transoesophagienne avant cardioversion électrique pour trouble du rythme supra-ventriculaire.

OBJECTIVES: The aim of this prospective study was to assess the risks of electrical shock cardio-version in the treatment of supraventricular rhythm disorders when administered under effective-dose but short duration anticoagulation in patients with no intracavitary thrombus detectable by transesophageal echocardiography. PATIENTS AND METHODS: One hundred nineteen patients, mean age 66 years, with permanent arrhythmia due to atrial fibrillation (n = 102), atrial flutter (n = 16) or atrial tachycardia (n = 1) and taking no long-term anticoagulant therapy were treated by electrical shock cardioversion. The patients were given heparin at an effective dose 72 hours prior to cardioversion. A transthoracic and a transesophageal echocardiography were performed less than 24 hours prior to cardioversion. RESULTS: Twenty-one thrombi were evidenced in 16 patients (14.6%) including 18 in the left auricle, 1 in the left atrium and 2 in the right atrium. A spontaneous contrast was visualized in 38 patients (32%). Cardioversion was performed in 103 patients without thrombus and later in 9 of the 16 patients with thrombus after absorption under anticoagulant therapy as evidenced on the control transesophageal echocardiography. A sinus rhythm was obtained in 82% of the cases. All patients were given anti-vitamin K anticoagulants for one month. There were no clinical manifestation of ischemic vascular events during cardioversion nor during the one-month follow-up. CONCLUSION: Early use of electrical shock cardioversion in patients with supraventricular rhythm disorders can be proposed without long-term anticoagulation therapy if the absence of thrombi is demonstrated by transesophageal echocardiography and short-term heparin is given followed by oral anticoagulants for at least 4 weeks. A large-scale randomized prospective study comparing the conventional strategy with the protocol used in this study would be required to definitively validate this approach and determine its possible advantages.

Effects of interval training at the ventilatory threshold on clinical and cardiorespiratory responses in elderly humans.

This study assessed clinical and cardiorespiratory responses after an interval training programme in sedentary elderly adults using the ventilatory threshold (Vth) as the index of exercise training intensity. A selection of 22 subjects were randomized into two groups: 11 subjects served as the training group (TG) and the others as controls (CG). Maximal exercise tests were performed on a treadmill before (T0), each month (T1, T2) and after the 3-month interval training programme period (T3). The TG subjects were individually trained at the heart rate corresponding to Vth measured at T0, T1 and T2 as the breakpoint in the oxygen uptake-carbon dioxide production relationship. Their training programme consisted of walking/jogging sessions on a running track twice a week. The sessions consisted of varying durations of exercise alternating with active recovery in such a way that the subjects slowly increased their total exercise time from an initial duration of 30 min to a final duration of 1 h. During training the heart rate was continuously monitored by a cardiofrequency meter. Compared with the daily activities of the controls, no training programme-related injuries were observed in TG. Moreover, programme adherence (73%) and attendance (97.3%) were high. The maximal oxygen uptake and Vth were increased in TG, by 20% (P<0.05) and 26% (P<0.01), respectively. Interval training at Vth also significantly increased maximal O2 pulse (P<0.05) and maximal ventilation (P<0.01). A significant decrease in submaximal ventilation (P<0.05) and heart rate (P<0.01) was also noted. These results would suggest that for untrained elderly adults, an interval training programme at the intensity of Vth may be well-tolerated clinically and may significantly improve both maximal aerobic power and submaximal exercise tolerance.

Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.

To move forward, migrating cells must generate traction forces through surface receptors bound to extracellular matrix molecules coupled to a rigid structure. We investigated whether cells sample and respond to the rigidity of the anchoring matrix. Movement of beads coated with fibronectin or an anti-integrin antibody was restrained with an optical trap on fibroblasts to mimic extracellular attachment sites of different resistance. Cells precisely sense the restraining force on fibronectin beads and respond by a localized, proportional strengthening of the cytoskeleton linkages, allowing stronger force to be exerted on the integrins. This strengthening was absent or transient with antibody beads, but restored with soluble fibronectin. Hence, ligand binding site occupancy was required. Finally, phenylarsine oxide inhibited strengthening of cytoskeletal linkages, indicating a role for dephosphorylation. Thus, the strength of integrin-cytoskeleton linkages is dependent on matrix rigidity and on its biochemical composition. Matrix rigidity may, therefore, serve as a guidance cue in a process of mechanotaxis.

Ligand binding regulates the directed movement of beta1 integrins on fibroblasts.

To enable cells to crawl, adhesion receptors such as integrins must bind to extracellular molecules and simultaneously interact with force-generating components of the cytoskeleton. We show here that the binding of extracellular ligand in living cells induces the attachment of beta1 integrins to the retrograde-moving cytoskeleton. Unliganded integrins are not associated with the rearward-moving cytoskeleton: gold particles attached to beta1 integrin by a monoclonal antibody diffuse in the membrane. However, addition of soluble RGD peptide (single-letter amino-acid code) or the use of fibronectin-coated gold particles causes the attachment of integrins to the rearward-moving cytoskeleton. Deletion of the beta1 cytoplasmic tail blocks cytoskeletal attachment. The directed movement of integrins in response to ligand indicates that ligand binding is the critical step in regulating organized receptor movement on the cell surface and the migration of adherent cells.

Regulation of ITAM signaling by specific sequences in Ig-beta B cell antigen receptor subunit.

B cell antigen receptors (BCR) are composed of an antigen binding subunit, the membrane Ig, and Ig-alpha/Ig-beta heterodimers, that contain a transducing motif named ITAM for "immuno-receptor tyrosine-based activation motif." Ig-alpha and Ig-beta ITAMs only differ by four amino acids located before the second conserved tyrosine (DCSM in Ig-alpha and QTAT in Ig-beta), which determine the in vitro association of Ig-alpha with the src kinase fyn. We have previously shown that Ig-alpha and Ig-beta BCR subunits activate different signaling pathways by expressing, in B cells, FcgammaRII chimeras containing the cytoplasmic tails of Ig-alpha or Ig-beta. We report here that the signaling capacity of Ig-beta ITAM is regulated by peptide sequences located inside (QTAT region) or outside the ITAM (flanking sequences). Furthermore, when isolated, Ig-alpha and Ig-beta ITAM have similar abilities as the entire Ig-alpha tail and the whole BCR in triggering tyrosine kinase activation, an increase of intracellular calcium concentration as well as late events of cell activation as assessed by cytokine secretion. These data show that alterations that modify the ability of Ig-alpha and Ig-beta to interact in vitro with the src kinase fyn (switch between QTAT and DCSM) also determine signal transduction capabilities of these molecules expressed in B cells.

Clinical significance of intracavitary spontaneous echo contrast in patients with dilated cardiomyopathy.

To assess the occurrence rate and major determinants of spontaneous echo contrast and to examine its impact on thromboembolic events and mortality in patients with dilated cardiomyopathy, 86 hospitalized patients (73 men and 13 women, mean age 63 +/- 11 years) with dilated cardiomyopathy who underwent transthoracic and transesophageal echocardiographic examinations were followed up for a mean of 20 +/- 13 months. Spontaneous echo contrast was observed in 36 patients (42%) and was detected only with the transesophageal approach. It was seen in the left atrium in 33 patients, in both right and left atria in 1 patient, in both left atrium and left ventricle in 1 patient, and in the descending aorta in 1 patient. Spontaneous echo contrast was more frequent in the presence of atrial fibrillation (p < 0.05), left atrial enlargement (p < 0.02) and severely depressed left ventricular function (p < 0.01), but was less common in patients with moderate to severe mitral regurgitation (p < 0.05). This imaging phenomenon was the only significant independent predictor of intracardiac thrombus formation and previous and subsequent thromboembolic events. During follow-up, there were 26 deaths, and survival in patients with spontaneous echo contrast was significantly lower than in those without it (p < 0.02). A spontaneous echo contrast is commonly detected with transesophageal echocardiography in patients with dilated cardiomyopathy especially in the presence of atrial fibrillation, left atrial enlargement and severe left ventricular dysfunction. This imaging phenomenon represents an important marker for thromboembolic risk and may influence the treatment and clinical outcome of these patients.