, 2005). The detailed dissociation method is described in Supplemental Experimental Procedures. Dissociated E14.5 retinal neurons (1 × 105 cells) were plated on top of a confluent monolayer of control HEK293 cells, or a stable HEK293 cell line expressing either Sema5A or Sema5B, in 24-well plates and then cultured for 48 hr in culture medium (containing B-27 supplement, 2 mM L-Glutamine, 10 ng/ml ciliary neurotrophic factor [CNTF] [R&D Systems], 50 ng/ml brain-derived

neurotrophic factor [BDNF], 5 mM forskolin, 5 mg/ml insulin, 50 U/ml penicillin, and 50 μg/ml streptomycin), fixed in 4% PFA for 15 min, incubated with anti-βIII-tubulin (Promega; at 1:1000), followed by incubation with goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen; at 1:500), and then imaged for neurite outgrowth analysis. Neurite lengths of retinal neurons were quantified selleck kinase inhibitor using ImageJ plugin. ERG measurements were performed as previously described (Samuels et al., 2010). The amplitude of the a-wave was measured at 8 ms after flash presentation from the prestimulus

baseline. The amplitude of the b-wave was measured to the b-wave peak from the a-wave trough or, if no a-wave was present, from the baseline. The amplitude of individual oscillatory potentials was measured from the negative trough to the subsequent peak. The implicit times of the b-wave and individual oscillatory potentials were measured at the positive peak. RGC responses to a variety of light stimuli were recorded using a Multielectrode Array System (Multi Channel Systems; ALA Scientific Instruments, www.selleckchem.com/products/DAPT-GSI-IX.html Farmingdale,

NY, USA). Dissections and recording conditions were previously described (Meister et al., 1994 and Ye et al., 2009). Visual stimuli were generated and presented using PAK6 MATLAB software (Natick, MA, USA; http://www.mathworks.com/), and the Psychophysics Toolbox extensions (Brainard, 1997 and Pelli, 1997). See Supplemental Experimental Procedures for a detailed description of the recordings and data analysis. OKR measurements were performed as previously described (Cahill and Nathans, 2008). The statistical significance of the differences between mean values among two or more groups was determined using Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s HSD test, respectively. The criterion for statistical significance was set at p < 0.05. Error bars are SEM. We thank K.-W. Yau and T. Xue for the Opn4Tau-LacZ/Tau-LacZ mice, M. Tessier-Lavigne for the PlexA3−/− mice, B. Howell for the Dab-1 antibody, and F. Haeseleer for the CaBP5 antibody. We also thank D. Kantor, S. Kozlov, C. Hawkins, and K. Takamiya for their assistance with the generation of the Sema5A−/− and Sema5B−/− mice. We thank M. Riccomagno, K. Mandai, S.-H. Wang, Y. Duan, and T. Tran for helpful suggestions and discussions throughout this project, D. Johnson for assistance with mouse experiments, and members of the Kolodkin laboratory for assistance.

How may connectivity rearrangements promote long-term learning and memory in enriched mice? We suggest that environmental enrichment may facilitate synapse turnover and de novo synaptogenesis upon learning and that those learning-related changes in connectivity may mediate long-term Selleckchem Ruxolitinib retention of specific

memories. Such a scenario implies that LTP at existing synapses may be but one synaptic mechanism to mediate learning and memory, that parallel pathways triggered by experience and involving structural rearrangements of connectivity can complement or even bypass a requirement for LTP, and that these pathways are augmented upon enriched environment (Figure 8; see also Ivanco et al.,

2000 and Rampon et al., 2000). In support of the notion that connectivity rearrangements Protein Tyrosine Kinase inhibitor underlie enhanced learning upon enrichment, learning was already enhanced at 2 weeks of enrichment, when remodeling was increased but no obvious net increases in synapse numbers were yet detectable. Accordingly, environmental enrichment may persistently elevate signals that promote the disassembly of labile synapses and induce filopodial and spine growth, thus facilitating long-term learning and memory and bypassing a requirement to induce these signals through LTP-related mechanisms. In conclusion, we have provided evidence that circuit remodeling and de novo synaptogenesis processes in the adult have important roles in learning and memory and that β-Adducin is critically important to establish new synapses under conditions of enhanced plasticity. Future studies will aim at elucidating how experience enhances synapse

turnover and synaptogenesis, how this potentiates memory processes, and how impairment of these processes may produce memory losses in disease. Transgenic mice expressing membrane-targeted GFP in a small subset of neurons (Thy1-mGFPSi1) were as described ( De Paola et al., 2003 and Galimberti et al., 2010). β-Adducin−/− mice (B6.129-β-Adducintm1Feb/Ibcm; Gilligan et al., 1999) were Rebamipide generously provided by Luanne Peters (Jackson Labs). Rab3a−/− mice (B6;129S-Rab3atm1Sud/J) were obtained from the Jackson Laboratory. Both β-Adducin−/− and Rab3a−/− mice were backcrossed into Thy1-mGFPSi1 mice. Enriched environment (EE) procedures were as described (Gogolla et al., 2009). Organotypic slice cultures were based on the Stoppini method (Stoppini et al., 1991), as described (De Paola et al., 2003). All procedures were approved by the Cantonal Veterinary Office of Basel, Switzerland. Lentiviral constructs were a generous gift from Pavel Osten (Cold Spring Harbor Laboratories; Dittgen et al., 2004); cytosolic GFP was replaced in the expression cassette by the mGFP or the GFP-β-Adducin sequence.

g., insight) by providing content-specific cortical regions (e.g., LO) with modulatory signals about the importance of these events. Sixty-five participants took part in this study: 37 in Experiment 1 (ages 21–29 years, mean 24 years, 26 females), 17 in Experiment 2 (aged 19–38, mean 25 years, 6 females), and 11 in Experiment 3 (aged 22–29, mean 25 years, 6 females). All participants selleck kinase inhibitor had normal or corrected to normal

vision. Participants in Experiments 2 and 3, which included an fMRI scan, were all right-handed. Unless otherwise indicated, participants were paid for their time. The stimuli used in these experiments were 40 camouflage images that were experimentally screened out of a large collection of degraded real-world pictures that portrayed a clear, nameable

object or scene. The chosen images were those in which the embedded object was not likely to be spontaneously identified, yet once the solution (the original, nondegraded image) was presented, the object embedded in the camouflage image was usually vividly perceived (i.e., the object was perceived BIBW2992 chemical structure as whole and created an impression of depth, and no spurious solutions—false alarms—were perceived in the image). For the full description of the generation and prescreening of the images, see the Generation and prescreening of camouflage images section in the Supplemental Experimental Procedures. Of the 40 images in the final set, 17 were images of animals, before 8 of human figures, 3 of human faces, 3 of insects, and 5 of inanimate objects, and 4 contained a more complex scene that combined, for example, a human figure and an object. (For an example of an image from the set and its solution, see Figure 1 and Figure 2.) Behavioral sessions took place in a quiet dark room, where

participants were seated in front of a 19” monitor (100 Hz refresh rate). Images were presented on a medium gray background in the center of the screen and subtended a mean height of 17.5° and a mean width of 21.26° visual angle. Participants responded using the keyboard number buttons. In the sessions performed in the fMRI scanner, the visual display was fed into an LCD projector. The projected image appeared on a plastic rear-projection screen, and participants viewed the stimuli through a mirror mounted on the head coil. In Experiment 2 the images subtended a mean height of 13.12° and a mean width of 16° visual angle; responses were collected on a five-button RIS-418 RURB button box (Rowland Institute, Cambridge, MA). In Experiment 3 the images subtended a mean height of 7.3° and a mean width of 10.9° visual angle; responses were collected using a response box that is part of a fORP system that includes an eight-button handheld response box manufactured by Current Designs Inc. (Philadelphia, PA).

Our electrophysiological analysis of Tor mutant larvae showed a mild but statistically significant reduction in the average amplitude of mEJCs ( Figures 3A and 3B). Postsynaptic receptors at the NMJ are non-NMDA

type ionotropic glutamate screening assay receptors. Genetic and electrophysiological findings have identified five GluR subunits: IIA, IIB, IIC (or III), IID, and IIE, of which IIC, IID, and IIE are essential, while IIA or IIB can replace each other to form a functional tetrameric receptor with the other three subunits ( Marrus et al., 2004, Petersen et al., 1997 and Qin et al., 2005). Loss of GluRIIA leads to a strong decrease in the single-channel mean open time, giving rise to smaller mEJCs and mEJPs ( DiAntonio et al., 1999). Consistent with the reduction in mEJC amplitude in Tor mutants, we found a mild but statistically significant reduction in the immunofluorescence associated with GluRIIA at postsynaptic sites in TorE161K/TorΔP larvae ( Figures 3C–3E), supporting a link between GluRIIA expression and local synaptic translation as previously shown ( Sigrist et al., 2000). The EJCs recorded from Tor mutants were also proportionally smaller than those in wild-type larvae resulting in statistically unchanged values for QC measurements ( Figures 3A and 3B). Since hemizygous male eIF2αG0272 mutants, described above, showed a similar reduction in postsynaptic muscle

growth as Tor mutants, these results prompted us to test whether eIF2αG0272 mutants also display a reduction in GluRIIA NVP-AUY922 chemical structure levels. Indeed, we found that eIF2α mutation led to a much larger reduction in GluRIIA

than Tor mutation did ( Figures 3F–3H). Consistently, eIF2αG0272 mutant larvae also showed a larger reduction in mEJCs ( Figures 3I and 3J). However, in contrast Thymidine kinase to TorE161K/TorΔP mutant larvae, these larvae had normal EJCs, reflecting a strong increase in QC ( Figures 3I and 3J). This suggested that the reduction in mEJCs in eIF2α mutants had triggered a retrograde compensation leading to increased neurotransmitter release, again demonstrating that the homeostatic response in eIF2α mutants was intact. These results further demonstrate that while a significant reduction in translation in eIF2αG0272 and TorE161K/TorΔP mutant larvae leads to qualitatively similar changes in muscle growth and GluRIIA levels, it has vastly different effects on retrograde signaling and neurotransmitter release. To address the tissue requirement of TOR, we took advantage of the UAS-Gal4 tissue specific expression system (Brand and Perrimon, 1993) and found that postsynaptic rather than presynaptic TOR activity is required for the homeostatic response. Transgenic overexpression of TOR in all muscles restored QC in GluRIIA−/−; TOR−/− double mutants, while neuronal expression of the same transgene had no effect ( Figures 4A and 4B).

At excitatory glutamatergic synapses, presynaptic beta-neurexin recruits postsynaptic neuroligin1 from a diffuse surface pool within minutes following initial contact (Barrow et al., 2009 and Krueger et al., 2012). Neuroligin in turn recruits the postsynaptic scaffolding protein PSD-95,

which is accumulated at sites of neurexin-neuroligin interactions within 1–4 hr after initial contact (Barrow et al., 2009, Heine et al., 2008b and Mondin et al., 2011). During this process, PSD-95 molecules are—at least partly—disassembled from preexisting synapses and recruited to nascent sites of neurexin-neuroligin contact, creating direct competition between earlier and newly formed synapses (Mondin et al., 2011). Following recruitment of PSD-95, functional membrane-diffusible AMPARs are trapped within 2–4 hr. This presumably involves their interaction with neuroligin–PSD-95 Metformin ic50 complexes through auxiliary subunits such as stargazin (Heine et al., 2008b and Mondin et al., 2011). A similar process involving neuroligin2 recruiting gephyrin likely occurs for the formation of inhibitory synapses. Whereas excitatory and inhibitory synapses coexist within microns on the same dendritic shaft, they exhibit different shapes and molecular compositions (Figure 1). Indeed, several elements of both synapse types are

identical or very similar, such as actin or adhesion proteins like neuroligins. Recent work indicates that ligand-dependent phosphorylation of Selleck Cobimetinib neuroligin subtypes could regulate their binding to specific scaffolds such as gephyrin or PSD-95 (Giannone et al., 2013 and Poulopoulos et al., 2009). In conclusion, postsynapse formation depends heavily not only on diffusion-trapping rates, but

also on the availability of the components and their respective aminophylline affinity that is regulated by posttranslational modifications. Hence, equilibrium between diffusion-reaction rates of molecular interactions is at the heart of synapse formation. The plasticity of mature synapses is a hallmark of learning and memory. It must comply with the paradoxical long-term stability necessary to store memories and high dynamics necessary for their fast encoding. As presented above, a major paradigm shift in the last decade has been the emergence that synapses maintain global stability while their components are in a dynamic equilibrium between subcellular compartments, hence shifting the concept of stability toward that of metastability (Figure 3). Activity-, development-, or environment-dependent changes in the efficacy of synaptic transmission are related to the modification of both synapse composition and biophysical properties of their individual elements. At the presynaptic level, modifications in the properties of neurotransmitter release mostly underlie plasticity.

, 1996). Future experiments on the ultrastructural localization of neuropeptide receptors may show similar sites of expression at specific regions of the plasma membrane. The classic view that neuropeptide-containing neurons represented an unusual type of neuron is giving way to the perspective that many, perhaps most neurons in the brain, probably contain some neuropeptide(s) or other neuromodulator in addition to fast-acting amino acid neurotransmitters. In an examination of individual

sections containing synaptic boutons with electron microscopy, with the boutons fixed to preserve the dense core of vesicles, some boutons appeared to contain only clear vesicles, others contained clear and DCVs. However, serial ultrathin section reconstruction of GABA-immunogold-labeled presynaptic boutons Everolimus in vitro from the paraventricular nucleus demonstrated that every bouton contained at least NVP-BKM120 solubility dmso a few dense core vesicles, suggesting that in addition to a fast amino acid transmitter, most if not all GABAergic axons here also contained some neuromodulator (Decavel and van den Pol, 1990). Release and actions of these neuromodulators remains to be demonstrated. Furthermore, because the axons studied contained GABA which is not found in magnocellular neurons, the profiles could not arise from the local

oxytocin or vasopressin neurosecretory cells. Similarly, presynaptic boutons showing no immunogold GABA labeling, many of which were probably glutamatergic, also showed a similar

frequency of DCVs in boutons, interspersed with small clear vesicles. A complication to the detection of DCVs with electron microscopy is that the dense core can be lost by suboptimal fixation pH, duration, chemistry, and osmotic pressure (Morris and Cannata, 1973), complicating detection in some studies and biasing results toward Tryptophan synthase a false-negative lack of detectable DCVs. A related question is whether all peptidergic axons also contain a fast amino acid transmitter. Most evidence, including that based on immunocytochemistry, calcium digital imaging, and electrophysiology supports the perspective that the great majority of peptidergic cells also employ fast amino acid transmitters (van den Pol, 1991, 2003; van den Pol et al., 1990; van den Pol and Trombley, 1993; Freund and Buzsáki, 1996). Whereas hypothalamic neurons have long been recognized as utilizing a large number of peptides, other regions of the brain are now being seen as not substantively different in this regard. For instance, in the hippocampus, a region with a rich history in the study of fast GABA and glutamate transmission, a plethora of neuropeptides are synthesized, particularly by GABAergic inhibitory interneurons, including neuropeptide Y, somatostatin, vasoactive intestinal polypeptide, cholecystokinin, dynorphin, enkephalin, neurokinin B, and substance P (Acsády et al., 1996, 2000; Billova et al., 2007; Antonucci et al.

Each aperture contained 500 white dots that moved radially with 80% coherence either toward fixation or away DNA Synthesis inhibitor from fixation. Dots moved continuously throughout the adaptor, disappeared during the blank and reappeared during the test. Test stimuli moved either in the same (adapted trials) or opposite direction (un-adapted trials) to the adaptor. In the auditory experiment, identical stimuli were presented to both ears through the Siemens headphones. The adaptor consisted of eleven 150 ms pure tone beeps (either 400 or 600 Hz) interleaved with 150 ms blanks, followed by 200 ms of blank and a test

composed of 3 tones at either the same (adapted trials) or different pitch (unadapted trials). In the somatosensory experiment, air puffs were presented at two alternative spatial NLG919 ic50 locations on the back of the left hand (about 5 cm apart). Air puffs were delivered through a manifold connected to a set of hoses (similar to Huang and Sereno, 2007). The manifold was controlled by a computer to achieve accurate stimulation timing. The adaptor

and test puffs followed the same timing as in the auditory experiment. Test puffs were presented either the same (adapted trials) or different location on the back of the left hand (unadapted trials). During all three experiments, subjects performed a demanding letter repetition-detection task at fixation. Capital letters presented within the fixation point changed every 500 ms, and subjects pressed a button with their right hand every time they detected a consecutive letter repeat (1-back). Subjects had 1 s to respond. Correct and incorrect responses were indicated by a change in the fixation spot background to green or red, respectively.

In the resting-state experiment, subjects were instructed to lay still with their eyes closed, and the MRI room lights and projector were turned off for the duration of this scan (8 min). We performed a statistical parameter mapping (SPM) analysis (Friston et al., 1994) to assess brain activation associated with each experimental condition. Response amplitudes Farnesyltransferase were computed separately for each voxel in each subject and then a “random-effects” analysis (Friston et al., 1999) was used (t test across subjects) to test the significance of response across all subjects of each group. We used a single functional run of each experiment to define bilateral regions of interest (ROIs) in visual, auditory, and secondary somatosensory cortices individually in each subject, based on the SPM analysis. The ROIs were defined using an automated procedure implemented in Matlab that selected 200 adjacent voxels in each hemisphere, which exhibited the most significant activation to the stimulus (Figure S2).

, 2007), but little is known about how olfactory inputs are transformed from PNs to higher-order lateral horn neurons (Ruta et al., 2010). Thus far, most physiological and behavioral studies of Drosophila PNs have focused on the uniglomerular ePNs, which reside dorsal and lateral to the antennal lobe and whose axons form the inner antenno-cerebral buy NVP-BKM120 tract (iACT), innervating both the mushroom body and the lateral horn ( Figure 1A). However, a separate group of PNs reside ventral to the antennal lobe. Individual ventral PNs send dendrites to either single or multiple glomeruli and project their axons through the middle antenno-cerebral tract (mACT) to terminate only in the lateral horn,

bypassing the mushroom body altogether Paclitaxel ( Jefferis et al., 2007, Lai et al., 2008, Okada et al., 2009 and Stocker et al., 1990). In this study, we use the olfactory response of a specific set of higher-order neurons to show that these ventral PNs provide GABAergic inhibition in the lateral horn to route selective inputs to specific higher-order neurons.

Ventral PNs of the antennal lobe have previously been characterized using two GAL4 lines. GH146-GAL4 labels ∼6 ventral PNs ( Jefferis et al., 2001), all of which are GABAergic ( Jefferis et al., 2007), and all are uniglomerular except one that innervates all glomeruli ( Marin et al., 2002). Mz699-GAL4 labels >45 ventral PNs that are mostly complementary to those labeled by GH146-GAL4 ( Lai et al., 2008). Most Mz699-GAL4-positive (Mz699+ hereafter) ventral PNs project to multiple glomeruli ( Lai et al., 2008) and more than 80% are GABAergic ( Okada et al., 2009). Mz699-GAL4 also labels neurons in the ventrolateral protocerebrum (vlpr) that send processes into the lateral medroxyprogesterone horn ( Okada et al., 2009;

Figure 1B). To further characterize neurons labeled by Mz699-GAL4, we used mosaic analysis with a repressible cell marker (MARCM)-based clonal analysis ( Lee and Luo, 1999). Consistent with a previous study ( Lai et al., 2008), we found that Mz699+ ventral PNs were derived from a single neuroblast ( Figure 1C; Figure S1A). Most single-cell clones innervated a few glomeruli ( Figure S1B; n = 38 out of 39), which collectively covered the majority of glomeruli. We also introduced synaptotagmin-hemagglutinin (Syt-HA) as a synaptic vesicle marker in these MARCM clones and found that Syt-HA was highly enriched in the lateral horn but was largely absent from the antennal lobe in neuroblast and single-cell clones ( Figures S1A–S1C). This is consistent with a previous report based on the labeling of all Mz699+ neurons ( Okada et al., 2009). With single-cell resolution, we observed that the majority of the ventral PN (vPN) neural processes in the antennal lobe had fine terminal branches without Syt-HA signal ( Figure S1B), whereas Syt-HA puncta, likely representing presynaptic terminals of en passant synapses, were distributed throughout the branches in the lateral horn ( Figure S1C).

, 2006), consistent with a prion-like template-dependent aggregation. Furthermore, both TDP-43 and FUS/TLS contain prion-like domains, which may facilitate seeding and aggregation (Figure 6). Indeed, a

recent study reported that intracellular aggregation of TDP-43 can be triggered in cultured cells by transduction of fibrillar aggregates prepared in vitro (Furukawa et al., 2011). More provocatively, insoluble TDP-43 isolated from brains of ALS or FTLD-TDP patients can trigger prion-like templating Anti-diabetic Compound Library and aggregation of transfected TDP-43 in cultured cells (Nonaka et al., 2013). In addition, disease-linked mutations in prion-like domains in hnRNP-A2B1 and hnRNP-A1 increase their propensity to form self-seeding fibrils and cross-assemble with wild-type counterparts (Kim et al., 2013). Altogether, along with recognition that the initial symptoms of ALS

are typically confined to a particular region, followed by an orderly spread that might be predicted for prion-like propagation, the evidence suggests that a prion-like seeding and spreading mechanism could underlie TDP-43 and FUS/TLS-mediated disease. One of the most devastating features of ALS is the relentless progression and spread of degeneration. We attempt here to provide a molecular basis for this phenomenon. The recent discovery of how RNA granules can form through a low-complexity/prion-like domain in TDP-43, FUS/TLS, and hnRNP Epigenetics Compound Library datasheet A2/B1 (Han et al., 2012b and Kato et al., 2012) has fueled an attractive hypothesis in which prion-like spreading of aggregated SOD1, TDP-43, or FUS/TLS could contribute not to ALS pathogenesis (Polymenidou and Cleveland, 2011). Both TDP-43 and FUS/TLS are intrinsically aggregation prone in vitro (Johnson et al., 2009 and Sun et al., 2011), which may predispose them to formation of pathological inclusions through their prion-like domains (Kato et al., 2012, Han et al., 2012b and Kim et al., 2013), independent of any proposed progression from an initiating stress granule complex (Dewey et al., 2012). Not surprisingly, both ubiquitin-proteasome and

autophagy pathways are used for TDP-43 clearance (Brady et al., 2011, Urushitani et al., 2010 and Wang et al., 2010). Mutations or disruption of many of ALS-linked genes involved in protein homeostasis pathways (VCP, ubiquilin-2, p62, and CHMP2B) lead to TDP-43 aggregation. Downregulation of VCP or expression of disease-linked mutations of VCP generate cytosolic TDP-43 aggregations (Gitcho et al., 2009, Ju et al., 2009 and Ritson et al., 2010), autophagy defects (Ju et al., 2009), and decreased proteasomal activity (Gitcho et al., 2009). Similarly, reduction of CHMP2B and expression of FTD-linked mutations in CHMP2B inhibit the maturation of autolysosomes, which in turn lead to accumulation of cytosolic TDP-43 aggregates (Filimonenko et al., 2007).

Other investigators, who remained blinded to treatment allocations, measured maximal inspiratory and expiratory pressures and the rapid shallow breathing

index twice a day until the end of the weaning period. The weaning period was defined as from the end of controlled ventilation (ie, the commencement of pressure-support ventilation) until extubation. A daily awakening trial with a minimum level of sedation identified which patients would be transitioned from controlled Selleck AZD6738 mechanical ventilation to pressure-support ventilation. The time of extubation was decided by the treating physicians, who were blinded to the treatment allocations. Patients were included in this study if they were aged 18 years or more, had undergone mechanical ventilation for more than 48 hours in a controlled mode, and were considered ready for weaning with pressure-support ventilation between 12 cmH2O and 15 cmH2O and positive end-expiratory pressure between 5 cmH2O and 7 cmH2O. They had to be haemodynamically stable without the aid of vasoactive drugs (dopamine, dobutamine or norepinephrine) or sedative agents. This study excluded patients with hypotension (systolic blood pressure < 100 mmHg or mean blood pressure < 70 mmHg), severe intracranial disease with inadequate consciousness level

(Glasgow Coma Scale ≤11), barotrauma, tracheostomy, or neuromuscular disease. In the experimental group, inspiratory muscle training began when the participants were changed from controlled to pressure-support ventilation. The patients were BMS 387032 ventilated using one of three mechanical ventilatorsa. Before each training session, the patients were positioned in 45-deg Fowler’s position and cardiorespiratory variables (respiratory rate, heart rate, systolic and diastolic blood pressures, and oxyhaemoglobin saturation) were recorded to ensure that participants did not undertake training if they were haemodynamically unstable, defined as: respiratory Sodium butyrate rate > 30 breaths/min, oxyhaemoglobin saturation < 90%, systolic blood pressure > 180 mmHg or < 90 mmHg, paradoxical breathing, agitation,

tachycardia, haemoptysis, arrhythmia, or diaphoresis (Caruso et al 2005). The pressure of the endotracheal tube cuff was maintained at 30 mmHg during the training session (Lewis et al 1978). The experimental group was trained using an inspiratory threshold deviceb with a load equal to 40% of the participant’s maximal inspiratory pressure. Each training session consisted of 5 sets with 10 breaths, twice a day, seven days a week. Supplementary oxygen was added if necessary during a training session (Martin et al 2002). The training session was interrupted in the presence of haemodynamic instability, as defined above. In the event of haemodynamic instability, the participant was returned to pressure-support ventilation.