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A principal components analysis was performed on a set (10) of acoustic, aerodynamic, perceptual and laryngoscopic data obtained from 87 dysphonic patients. Two principal components were clearly identified: the first represents in some way the glottal air leakage, resulting in turbulent noise, particularly obvious in higher spectral frequencies, and giving the perceptual impression of breathiness; the second accounts rather for the degree of aperiodicity in vocal fold oscillation, reflected in jitter measurements and with a perceptual correlate of harshness or roughness. Morphological changes of vocal folds correlate more closely with this second principal component. Among acoustic parameters, harmonics-to-noise ratio in the formant zone and magnitude of the dominant cepstrum peak seem to integrate to some extent the effects of both principal components.

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Medial frontal cortex (MFC) is crucial when actions have to be inhibited, reprogrammed, or selected under conflict, but the precise mechanism by which it operates is unclear. Importantly, how and when the MFC influences the primary motor cortex (M1) during action selection is unknown. Using paired-pulse transcranial magnetic stimulation, we investigated functional connectivity between the presupplementary motor area (pre-SMA) part of MFC and M1. We found that functional connectivity increased in a manner dependent on cognitive context: pre-SMA facilitated the motor evoked-potential elicited by M1 stimulation only during action reprogramming, but not when otherwise identical actions were made in the absence of conflict. The effect was anatomically specific to pre-SMA; it was not seen when adjacent brain regions were stimulated. We discuss implications for the anatomical pathways mediating the observed effects.

The segregation between cortical pathways for the identification and localization of objects is thought of as a general organizational principle in the brain. Yet, little is known about the unimodal versus multimodal nature of these processing streams. The main purpose of the present study was to test whether the auditory and tactile dual pathways converged into specialized multisensory brain areas. We used functional magnetic resonance imaging (fMRI) to compare directly in the same subjects the brain activation related to localization and identification of comparable auditory and vibrotactile stimuli. Results indicate that the right inferior frontal gyrus (IFG) and both left and right insula were more activated during identification conditions than during localization in both touch and audition. The reverse dissociation was found for the left and right inferior parietal lobules (IPL), the left superior parietal lobule (SPL) and the right precuneus-SPL, which were all more activated during localization conditions in the two modalities. We propose that specialized areas in the right IFG and the left and right insula are multisensory operators for the processing of stimulus identity whereas parts of the left and right IPL and SPL are specialized for the processing of spatial attributes independently of sensory modality.

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout ( ZnT8 −/− ) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8 −/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8 +/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8 −/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8 −/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

OBJECTIVE: We studied how glucose and ATP-sensitive K(+) (K(ATP)) channel modulators affect alpha-cell [Ca(2+)](c). RESEARCH DESIGN AND METHODS: GYY mice (expressing enhanced yellow fluorescent protein in alpha-cells) and NMRI mice were used. [Ca(2+)](c), the K(ATP) current (I(KATP), perforated mode) and cell metabolism [NAD(P)H fluorescence] were monitored in single alpha-cells and, for comparison, in single beta-cells. RESULTS: In 0.5 mmol/l glucose, [Ca(2+)](c) oscillated in some alpha-cells and was basal in the others. Increasing glucose to 15 mmol/l decreased [Ca(2+)](c) by approximately 30% in oscillating cells and was ineffective in the others. alpha-Cell I(KATP) was inhibited by tolbutamide and activated by diazoxide or the mitochondrial poison azide, as in beta-cells. Tolbutamide increased alpha-cell [Ca(2+)](c), whereas diazoxide and azide abolished [Ca(2+)](c) oscillations. Increasing glucose from 0.5 to 15 mmol/l did not change I(KATP) and NAD(P)H fluorescence in alpha-cells in contrast to beta-cells. The use of nimodipine showed that L-type Ca(2+) channels are the main conduits for Ca(2+) influx in alpha-cells. gamma-Aminobutyric acid and zinc did not decrease alpha-cell [Ca(2+)](c), and insulin, although lowering [Ca(2+)](c) very modestly, did not affect glucagon secretion. CONCLUSIONS: alpha-Cells display similarities with beta-cells: K(ATP) channels control Ca(2+) influx mainly through L-type Ca(2+) channels. However, alpha-cells have distinct features from beta-cells: Most K(ATP) channels are already closed at low glucose, glucose does not affect cell metabolism and I(KATP), and it slightly decreases [Ca(2+)](c). Hence, glucose and K(ATP) channel modulators exert distinct effects on alpha-cell [Ca(2+)](c). The direct small glucose-induced drop in alpha-cell [Ca(2+)](c) contributes likely only partly to the strong glucose-induced inhibition of glucagon secretion in islets.

AIMS/HYPOTHESIS: Survival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2 mmol/l glucose improves rodent beta cell survival and function, whereas glucose concentrations above 10 mmol/l are deleterious. METHODS: To identify the mechanisms of such beta cell plasticity, we tested the effects of 18 h culture at 2, 5, 10 and 30 mmol/l glucose on the transcriptome of rat islets pre-cultured for 1 week at 10 mmol/l glucose using Affymetrix Rat 230 2.0 arrays. RESULTS: Culture in either 2-5 or 30 mmol/l instead of 10 mmol/l glucose markedly impaired beta cell function, while little affecting cell survival. Of about 16,000 probe-sets reliably detected in islets, some 5,000 were significantly up- or downregulated at least 1.4-fold by glucose. Analysis of these probe-sets with GeneCluster software identified ten mRNA profiles with unidirectional up- or downregulation between 2 and 10, 2 and 30, 5 and 10, 5 and 30 or 10 and 30 mmol/l glucose. It also identified eight complex V-shaped or inverse V-shaped profiles with a nadir or peak level of expression in 5 or 10 mmol/l glucose. Analysis of genes belonging to these various clusters using Onto-express and GenMAPP software revealed several signalling and metabolic pathways that may contribute to induction of beta cell dysfunction and apoptosis after culture in low- or high- vs intermediate-glucose concentration. CONCLUSIONS/INTERPRETATION: We have identified 18 distinct mRNA profiles of glucose-induced changes in islet gene mRNA levels that should help understand the mechanisms by which glucose affects beta cell survival and function under states of chronic hypo- or hyperglycaemia.

OBJECTIVES. Precision grasping critically relies on the integrity of the corticospinal tract as evidenced in congenital hemiplegia by the correlation found between corticospinal dysgenesis and hand-movement deficits. Therefore, corticospinal dysgenesis could be used to anticipate upper-limb deficits in young infants with congenital hemiplegia. However, most studies have quantified corticospinal dysgenesis by measuring the cross-sectional area of cerebral peduncles on T1 MRI, a measure biased by other structures present in the peduncles. The purpose of this study was to evaluate the extent to which this may have hampered the conclusions of previous studies. We also aimed to investigate the relationship between upper-limb deficits and a more accurate measure of corticospinal dysgenesis to provide a tool for anticipating upper-limb deficits in infants with congenital hemiplegia. METHODS. To address this issue, we measured corticospinal tract areas in 12 patients with congenital hemiplegia and 12 matched control subjects by using the diffusion tensor imaging technique. Corticospinal dysgenesis was quantified by computing a symmetry index between the area of the contralateral and ipsilateral corticospinal tracts. This value was then compared with that resulting from the conventional MRI method. RESULTS. The symmetry indexes gathered with these 2 methods were highly correlated, although the diffusion tensor imaging symmetry indexes were significantly smaller. This indicates that, in patients with congenital hemiplegia, the conventional MRI measurement has led to a systematic underestimate of corticospinal dysgenesis. These 2 estimates of corticospinal dysgenesis were also correlated with upper-limb impairments and disabilities. Although the symmetry index computed from peduncle measurements was correlated solely with deficits in stereognosis, the diffusion tensor imaging index correlated with stereognosis, digital and manual dexterities, and ABILHAND-Kids, a measure of manual ability in daily life activities. CONCLUSIONS. The diffusion tensor imaging symmetry index provides a useful prognostic tool for anticipating upper-limb deficits and their consequences in daily life activities.

Context: Obesity in childhood is associated with an increased mortality due to cardiovascular (CV) diseases in adulthood, independent of adult weight. Recent studies in children indicate that the atherosclerosis process starts at an early age and is linked to obesity. Objective: The aim of the study was to investigate determinants of increased carotid intima-media thickness (IMT), an early marker of atherosclerosis, in obese children. Design: A total of 104 obese children [age, 12.7 ± 0.2 yr; body mass index (BMI)-z-score, 2.8 ± 0.7] underwent an oral glucose tolerance test. Fasting levels of glucose, insulin, C-reactive protein and adhesion molecules (sICAM, sVCAM, sE-selectin), lipid profile, adiponectin, and resistin were determined. IMT was measured by ultrasound. Insulin resistance was estimated by the homeostatic model assessment index. Baseline measurements of blood parameters were obtained from 93 nonobese children (age, 13.0 ± 0.2 yr; BMI-z-score, −0.2 ± 0.9), and IMT was measured in 23 other control children with similar characteristics. Results: Univariate analysis showed a significant positive correlation between IMT and relative BMI, the degree of systolic hypertension, fasting insulin levels, homeostatic model assessment-R index, and resistin concentrations, whereas an inverse correlation with adiponectin levels was found. No correlation was obtained between IMT and classical CV risk factors such as positive familial history of type 2 diabetes or precocious CV disease, visceral obesity, or the lipid profile. C-reactive protein and adhesion molecule levels were not associated with IMT in our obese population. When controlled for sex, Tanner stage, and relative BMI, only adiponectin levels remained an independent determinant of IMT. Conclusion: Adiponectin more than conventional CV risk factors and inflammation status may be related to early atherosclerosis in obese children.

AIMS/HYPOTHESIS: Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated. MATERIALS AND METHODS: We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca(2+) emptying with thapsigargin on the UPR (X-box binding protein [Xbp1] mRNA splicing and XBP1/activating transcription factor [ATF] 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets. RESULTS: Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca(2+) influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose. CONCLUSIONS/INTERPRETATION: Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.

Efficient behavior requires that internally specified motor plans be integrated with incoming sensory information. Motor preparation and visual signals converge in the intermediate and deep layers of the superior colliculus (SC) to influence saccade planning and execution; however, the mechanism by which these sometimes conflicting signals are combined remains unclear. We studied this issue by presenting visual distractors as monkeys prepared saccades toward an upcoming target whose timing and location were fully predictable. Monkeys made more distractor-directed errors when the spatial location of visual distractors more closely coincided with the saccadic goal. Concomitant pretarget activity of SC visuomotor neurons, whose response fields were centered on the saccadic goal, was similarly increased by the presentation of nearby distractors and inhibited by the presentation of distant distractors. Finally, subthreshold microstimulation of the SC shifted the pattern of distractor-directed errors away from the saccadic goal toward that specified by the site of stimulation. Together, our results suggest that the likelihood of saccade generation is influenced by the spatial register of internal motor preparation signals and external sensory signals across the topographically organized SC map.

A current model ascribes glucose-induced insulin secretion to the interaction of a triggering pathway (K(ATP) channel-dependent Ca(2+) influx and rise in cytosolic [Ca(2+)](c)) and an amplifying pathway (K(ATP) channel-independent augmentation of secretion without further increase of [Ca(2+)](c)). However, several studies of sulfonylurea receptor 1 null mice (Sur1KO) failed to measure significant effects of glucose in their islets lacking K(ATP) channels. We addressed this issue that challenges the model. Compared with controls, fresh Sur1KO islets showed slightly elevated basal [Ca(2+)](c) and insulin secretion. In 15 mm glucose, the absolute rate of secretion was approximately 3-fold lower in Sur1KO than control islets, with only poor increase above base line. Overnight culture of Sur1KO islets in 10 mm glucose (not in 5 mm) augmented basal insulin secretion and considerably improved the response to 15 mm glucose, which reached higher values than in control islets, in which culture had little impact. Glucose stimulation during KCl depolarization showed that the amplifying pathway is functional in fresh and cultured Sur1KO islets. The differences in insulin secretion between fresh and cultured Sur1KO islets and between Sur1KO and control islets were not attributable to differences in insulin content, glucose oxidation rate, or synchronization of [Ca(2+)](c) oscillations. The unmasking of glucose-induced insulin secretion in beta-cells lacking K(ATP) channels is paradoxically due to improvement in the production of a triggering signal (elevated [Ca(2+)](c)). The results show that K(ATP) channels are not the only transducer of glucose effects on [Ca(2+)](c) in beta-cells. They explain controversies in the literature and refute arguments raised against the model implicating an amplifying pathway in glucose-induced insulin secretion.

The finding that number processing activates a cortical network partly overlapping that recruited for hand movements has renewed interest in the relationship between number and finger representations. Further evidence about a possible link between fingers and numbers comes from developmental studies showing that finger movements play a crucial role in learning counting. However, increased activity in hand motor circuits during counting may unveil unspecific processes, such as shifting attention, reciting number names, or matching items with a number name. To address this issue, we used transcranial magnetic stimulation to measure changes in corticospinal (CS) excitability during a counting task performed silently and using either numbers or letters of the alphabet to enumerate items. We found an increased CS excitability of hand muscles during the counting task, irrespective of the use of numbers or letters, whereas it was unchanged in arm and foot muscles. Control tasks allowed us to rule out a possible influence of attention allocation or covert speech on CS excitability increase of hand muscles during counting. The present results support a specific involvement of hand motor circuits in counting because no CS changes were found in arm and foot muscles during the same task. However, the contribution of hand motor areas is not exclusively related to number processing because an increase in CS excitability was also found when letters were used to enumerate items. This finding suggests that hand motor circuits are involved whenever items have to be put in correspondence with the elements of any ordered series.

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.

The degree of commonality between the perceptual mechanisms involved in processing faces and objects of expertise is intensely debated. To clarify this issue, we recorded occipito-temporal event-related potentials in response to faces when concurrently processing visual objects of expertise. In car experts fixating pictures of cars, we observed a large decrease of an evoked potential elicited by face stimuli between 130 and 200 msec, the N170. This sensory suppression was much lower when the car and face stimuli were separated by a 200-msec blank interval. With and without this delay, there was a strong correlation between the face-evoked N170 amplitude decrease and the subject's level of car expertise as measured in an independent behavioral task. Together, these results show that neural representations of faces and nonface objects in a domain of expertise compete for visual processes in the occipito-temporal cortex as early as 130–200 msec following stimulus onset.

We previously showed that the stimulation of heme oxygenase-1 expression by high glucose and hydrogen peroxide (H2O2) in cultured rat islets is prevented by antioxidants and suggested that this effect of high glucose results from an oxidative stress. However, the role of oxidative stress in high-glucose-induced β-cell dysfunction is unclear. We therefore compared the preventative effects of N-acetyl-l-cysteine (NAC), a free radical scavenger, and manganese(III)tetrakis (4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase/catalase mimetic agent, on the alteration of stimulus-secretion coupling induced in rat islets by overnight exposure to hydrogen peroxide (H2O2-treated islets) or 1-wk culture in 30 vs. 10 mmol/l glucose (High-glucose vs. Control islets). The features of β-cell dysfunction differed between the two groups: reduced glucose-induced insulin secretion without changes in glucose sensitivity in H2O2-treated islets; increased sensitivity to glucose with parallel reductions in insulin content and maximal rate of glucose-induced insulin secretion in High-glucose islets. The latter alterations were accompanied by a decrease in preproinsulin without changes in pancreatic and duodenal homeobox gene 1 mRNA levels. The functional alterations induced by H2O2 were significantly prevented by addition of NAC or MnTBAP in the culture medium. In contrast, neither NAC nor MnTBAP affected the functional alterations induced by high glucose. These results suggest that β-cell dysfunction induced by 1-wk culture in high glucose does not result from an increase in oxidative stress.

Using functional magnetic resonance imaging, the brain activation related to unilateral sequential finger-to-thumb opposition was studied in six children with a right congenital hemiplegia of cortical origin. They were compared to six age-matched controls. In the control group, movements with either hand asymmetrically activated the sensorimotor cortex and premotor areas in both cerebral hemispheres with a typical contralateral predominance. By contrast, paretic finger movements activated both hemispheres in the hemiplegic patients, with a strong ipsilateral predominance favoring the undamaged hemisphere. The activation induced by nonparetic finger movements was restricted to the contralateral undamaged hemisphere. Furthermore, the level of activation in the undamaged cortex was partly related to residual finger dexterity, according to covariance analysis. These activation patterns indicate an adaptive reorganization of the cortical motor networks in this group of patients, with a prominent involvement of the undamaged hemisphere in the control of finger movements with either hand.

AIM/HYPOTHESIS: Increased expression of haeme-oxygenase 1 (HO1) and other antioxidant enzymes could improve pancreatic beta-cell survival under stressful conditions, including hyperglycaemia. However, how hyperglycaemia increases islet HO1 expression is not known. METHODS: Rat islets were pre-cultured for 1 week in RPMI medium containing 10 mmol x l(-1) glucose (G10), and further cultured overnight in G5-G30 plus various test substances. Islet HO1 mRNA and protein expression was measured by semiquantitative RT-PCR, western blot, and immunohistochemistry. RESULTS: Islet HO1 mRNA expression was minimal after overnight culture in G10, slightly increased in G5, and increased by five- to ten-fold in G30 in parallel with a heterogeneous increase in beta-cell HO1 protein expression. The effect of G30 was fully inhibited by agents decreasing cytosolic Ca2+ (diazoxide, nimodipine), but was only slightly reproduced by agents raising Ca2+ (tolbutamide, 30 mmol x l(-1) potassium). It was also suppressed by the alpha2-adrenoceptor agonist clonidine, whereas dibutyryl-cyclic-AMP largely increased beta-cell HO1 expression. The induction of HO1 mRNA expression by G30 was independent from changes in medium insulin concentration, but was completely inhibited by a cocktail of antioxidants. In contrast to HO1, islet mRNA expression of glutathione peroxidase and constitutive haeme-oxygenase 2 were not affected by G30, nor by dibutyryl-cyclic-AMP. CONCLUSION/INTERPRETATION: High glucose and dibutyryl-cyclic-AMP stimulate expression of HO1 in rat pancreatic beta cells. The inhibition of HO1 expression in G30 by nimodipine, clonidine, and antioxidants, suggests that Ca2+ influx and cyclic-AMP are necessary for the generation of oxidative stress by G30, or for the stimulation of beta-cell HO1 expression by increased oxidative stress.

The water channel aquaporin-1 (AQP1) is the molecular counterpart of the ultrasmall pore responsible for transcellular water permeability during peritoneal dialysis (PD). This water permeability accounts for up to 50% of ultrafiltration (UF) during a hypertonic dwell, and its loss can be a major clinical problem for PD patients. By analogy with the lung, the hypothesis was tested that corticosteroids may increase AQP1 expression in the peritoneal membrane (PM) and improve water permeability and UF in rats. First, the expression and distribution of the glucocorticoid receptor (GR) in the PM and capillary endothelium was documented. Time-course and dose-response analyses showed that a daily IM injection of dexamethasone (1 or 4 mg/kg) for 5 d induced an approximately twofold increase in the expression of AQP1 at the mRNA and protein levels. The GR antagonist RU-486 completely inhibited the dexamethasone effect. The functional counterpart of the increased AQP1 expression was a significant increase in sodium sieving and net UF across the PM, contrasting with a lack of effect on the osmotic gradient and permeability for small solutes. The latter observation reflected the lack of effect of corticosteroids on nitric oxide synthase (NOS) activity and endothelial NOS isoform expression in the PM. In conclusion, corticosteroids induce AQP1 expression in the capillary endothelium of the PM, which is reflected by increased transcellular water permeability and UF. These data emphasize the critical role of AQP1 during PD and suggest that pharmacologic regulation of AQP1 may provide a target for manipulating water permeability across the PM.

The role of TNF-α in muscle catabolism is well established, but little is known about the mechanisms of its catabolic action. One possibility could be that TNF-α impairs the production of local growth factors like IGF-I. The aim of this study was to investigate whether TNF-α can directly inhibit IGF-I gene and protein expression in muscle. First, we investigated whether the acute inflammation induced by endotoxin injection changes IGF-I and TNF-α mRNA in rat tibialis anterior muscle. Endotoxin rapidly increased TNF-α mRNA (7-fold at 1 h, P < 0.001) and later decreased IGF-I mRNA (−73% at 12 h, P < 0.001). Furthermore, in a model of C2C12 myotubes, TNF-α strongly inhibited IGF-I mRNA and protein (−73 and −47% after 72 h, P < 0.001 and P < 0.01, respectively). Other proinflammatory cytokines failed to inhibit IGF-I mRNA. The effect of TNF-α on IGF-I mRNA was not mediated by nitric oxide, and the activation of NF-κB was insufficient to inhibit IGF-I expression. Taken together, our data suggest that TNF-α induced in muscle after LPS injection can locally inhibit IGF-I expression. The inhibition of muscle IGF-I production could contribute to the catabolic effect of TNF-α.

We investigated the contribution of the lateral intraparietal area (LIP) to the selection of saccadic eye movement targets and to saccade execution using muscimol-induced reversible inactivation and compared those effects with inactivation of the adjacent ventral intraparietal area (VIP) and with sham injections of saline into LIP. Three types of tasks were used: saccades to single visual or memorized targets, saccades to synchronous and asynchronous bilateral targets, and visual search of a target among distractors. LIP inactivation failed to produce deficits in the latency or accuracy of saccades to single targets, but it dramatically reduced the frequency of contralateral saccades in the presence of bilateral targets, and it increased search time for a contralateral target during serial visual search. In the latter task, the observed deficits might reflect either an ispilateral bias in saccadic search strategy or an attentional impairment in locating a target among flanking distractors within the contralateral field. No effects were observed on any of these tasks after VIP inactivation. These results suggest that one important contribution of LIP to oculomotor behavior is the selection of targets for saccades in the context of competing visual stimuli.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.

Calcium is the most ubiquitous second messenger. Its concentration inside the cell is tightly regulated by a series of mechanisms, among which some have been extensively studied in nonmuscle cells. This is the case of the "store-operated entry of Ca(2+)", the uptake of Ca(2+) by mitochondria and the inositol 1,4,5-trisphosphate (IP(3)) cascade. These processes were recently found to be also present in skeletal muscle and are reviewed here. The "store-operated entry of Ca(2+)" allows the refilling of the stores after muscle fiber depolarization and is activated even after a partial depletion of the sarcoplasmic reticulum (SR). The uptake of Ca(2+) by mitochondria accelerates muscle relaxation and allows the adaptation of ATP supply to the increased energy demand. IP(3) receptors are found in the nuclear envelope and are involved in Ca(2+) waves propagating from one nucleus to another. This pathway is possibly involved in gene expression regulation. Finally, cytosolic Ca(2+) buffers like parvalbumins modify [Ca(2+)](i) transients and, therefore, muscle mechanics. The importance of these regulation mechanisms is also evaluated in Duchenne muscular dystrophy (DMD), a disease in which impairment of [Ca(2+)](i) homeostasis has been postulated but remains, however, controversial. This genetic disease is indeed characterized by the absence of a cytoskeletal protein called dystrophin, a situation leading to a disorganization of the cytoskeleton and to an abnormal influx of Ca(2+). How this increased entry of Ca(2+) affects the local concentration of Ca(2+) in subcellular compartments and whether this process is involved in the development of the disease are still unclear.

The adipose-derived hormone, adiponectin (ApN), has a role in fuel homoeostasis, insulin action and atherosclerosis. Regulation of ApN by catecholamines has scarcely been investigated. We examined the effects of beta-adrenergic agonists (and their second messenger, cAMP) on ApN gene expression, production and secretion in mouse in vitro and in vivo; their effects in human fat were also briefly studied in vitro. beta-Adrenergic agonists and cAMP inhibited ApN gene expression in human visceral adipose tissue. Likewise, cAMP down-regulated ApN mRNAs in cultured mouse explants from visceral and subcutaneous regions. The amount of ApN released into the medium decreased concomitantly. cAMP also caused qualitative changes in ApN secretion. Under basal conditions, ApN was secreted as a single 32 kDa species. In the presence of cAMP, an additional and probably immature (not modified post-translationally) 30 kDa species was also sorted. This altered secretion resulted from cAMP-induced quantitative and qualitative changes of ApN within the adipocyte. Under basal conditions, the 32 kDa form of ApN was mainly associated with high-density microsomes (HDMs), while the 30 kDa species was confined to a pool recovered with the cytosol fraction. cAMP depleted intracellular ApN at the expense of both HDM and cytosol fractions, and abnormally targeted ApN species to the different subcellular compartments as a result of impaired maturation. beta-Adrenergic agonists mimicked the inhibitory effects of cAMP on ApN mRNA and secretion, the beta(3)-agonist BRL37344 being the most potent. Administration of BRL37344 to mice reduced ApN mRNAs in both adipose regions, and ApN levels in plasma. In conclusion, beta-agonists inhibited ApN production and maturation, and thus exerted a dual (pre- and post-translational) negative effect on ApN secretion by cultured mouse adipose explants. ApN inhibition by beta-agonists was reproduced in mouse in vivo and in humans in vitro. ApN down-regulation may have an important role in fuel homoeostasis, insulin resistance and stress-induced atherosclerosis.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.

During visual tracking of a moving stimulus, primates orient their visual axis by combining two very different types of eye movements, smooth pursuit and saccades. The purpose of this paper was to investigate quantitatively the catch-up saccades occurring during sustained pursuit. We used a ramp-step-ramp paradigm to evoke catch-up saccades during sustained pursuit. In general, catch-up saccades followed the unexpected steps in position and velocity of the target. We observed catch-up saccades in the same direction as the smooth eye movement (forward saccades) as well as in the opposite direction (reverse saccades). We made a comparison of the main sequences of forward saccades, reverse saccades, and control saccades made to stationary targets. They were all three significantly different from each other and were fully compatible with the hypothesis that the smooth pursuit component is added to the saccadic component during catch-up saccades. A multiple linear regression analysis was performed on the saccadic component to find the parameters determining the amplitude of catch-up saccades. We found that both position error and retinal slip are taken into account in catch-up saccade programming to predict the future trajectory of the moving target. We also demonstrated that the saccadic system needs a minimum period of approximately 90 ms for taking into account changes in target trajectory. Finally, we reported a saturation (above 15°/s) in the contribution of retinal slip to the amplitude of catch-up saccades.

When tracking moving visual stimuli, primates orient their visual axis by combining two kinds of eye movements, smooth pursuit and saccades, that have very different dynamics. Yet, the mechanisms that govern the decision to switch from one type of eye movement to the other are still poorly understood, even though they could bring a significant contribution to the understanding of how the CNS combines different kinds of control strategies to achieve a common motor and sensory goal. In this study, we investigated the oculomotor responses to a large range of different combinations of position error and velocity error during visual tracking of moving stimuli in humans. We found that the oculomotor system uses a prediction of the time at which the eye trajectory will cross the target, defined as the “eye crossing time” ( T XE). The eye crossing time, which depends on both position error and velocity error, is the criterion used to switch between smooth and saccadic pursuit, i.e., to trigger catch-up saccades. On average, for T XEbetween 40 and 180 ms, no saccade is triggered and target tracking remains purely smooth. Conversely, when T XEbecomes smaller than 40 ms or larger than 180 ms, a saccade is triggered after a short latency (around 125 ms).

There has been considerable recent debate as to relative importance, in the primate, of propriospinal transmission of corticospinal excitation to upper limb motoneurons. Previous studies in the anesthetized macaque monkey suggested that, compared with the cat, the transmission of such excitation via a system of C3–C4 propriospinal neurons may be relatively weak. However, it is possible that in the anesthetized preparation, propriospinal transmission of cortical inputs to motoneurons may be depressed. To address this issue, the current study investigated the responses of single motor units (SMUs) to corticospinal inputs in either awake ( n = 1) or lightly sedated ( n = 3) macaque monkeys. Recordings in the awake state were made during performance of a precision grip task. The responses of spontaneously discharging SMUs to electrical stimulation of the pyramidal tract (PT) via chronically implanted electrodes were examined for evidence of non-monosynaptic, presumed propriospinal, effects. Single PT stimuli (up to 250 μA; duration, 0.2 ms, 2 Hz) were delivered during steady discharge of the SMU (10–30 imp/s). SMUs were recorded from muscles acting on the thumb (adductor pollicis and abductor pollicis brevis, n = 18), wrist (extensor carpi radialis, n = 29) and elbow (biceps, n = 9). In all SMUs, the poststimulus time histograms to PT stimulation consisted of a single peak at a fixed latency and with a brief duration [0.74 ± 0.25 (SD) ms, n = 56], consistent with the responses being mediated by monosynaptic action of cortico-motoneuronal (CM) impulses. Later peaks, indicating non-monosynaptic action, were not present even when the probability of the initial peak response was low and when there was no evidence for suppression of ongoing SMU activity following this peak ( n = 20 SMUs). Even when repetitive (double-pulse) PT stimuli were used to facilitate transmission through oligosynaptic linkages, no later peaks were observed (16 SMUs). In some thumb muscle SMUs ( n = 8), responses to PT stimulation were compared with those evoked by transcranial magnetic stimulation, using a figure-eight coil held over the motor cortex. Responses varied according the orientation of the coil: in the latero-medial position, single peak responses similar to those from the PT were obtained; their latencies confirmed direct excitation of CM cells, and there were no later peaks. In the posterio-anterior orientation, responses had longer latencies and consisted of two to three subpeaks. At least under the conditions that we have tested, the results provide no positive evidence for transmission of cortical excitation to upper limb motoneurons by non-monosynaptic pathways in the macaque monkey.

This study aimed to characterize the synchrony that occurs between cell discharges in the superior colliculus of the awake cat. We trained cats to perform a visual fixation in the presence of a visual moving stimulus and then recorded 686 pairs of neighboring cells in the superior colliculus during task performance. A new method to assess the significance of precise discharge synchronization is described, which permits analysis of nonstationary data. Of 181 pairs with sufficient data for quantitative analysis, 125 showed a cross-correlation histogram (CCH) with features assessed as significant using this approach. CCHs frequently showed an isolated central peak (41 of 125) or a peak flanked by one or two troughs (68 of 125), and in a few cases an oscillatory pattern of ∼65 Hz (16 of 125). This is in contrast to the oscillation frequency reported for the visual cortex and shows that oscillations in the superior colliculus probably arise from a cortex-independent mechanism.Our method also permits direct quantification of the correlation shift predictors, assessing precise time locking of spikes to the stimulus. Only 1 of 125 cross-correlation shift predictors had a significant central peak, meaning that most of the CCH features were not related to cell discharges time-locked to the stimulus presentation.Further investigation using a burst-jittering method showed that synchrony in the superior colliculus is attributable to precise synchronization of short bursts of spikes. Such synchrony could be related to the network dynamics and the common inhibitory feedback from local interneurons, which would act as temporal selectors of the cells with greatest or fastest response.

The motor cortex contains a distributed map of muscles, with a single muscle represented over a wide cortical area. We have searched for inter-connections between distant sites projecting to common muscles by delivering pairs of 20-microA single-pulse intracortical microstimuli (ICMS) to sites separated by 1.5-2 mm in the hand-area primary motor cortex of two macaque monkeys performing a precision grip task. The facilitation of hand- and forearm-muscle rectified EMG was measured. When stimuli were delivered simultaneously, responses were quantified using a technique to correct for non-linearities inherent in the use of averaged, rectified EMG. A spatial facilitation was seen for such simultaneous stimuli; however, it was of the same magnitude as that occurring when ICMS was paired with stimulation of corticospinal axons in the pyramidal tract (PT), so that it was likely to be spinal in origin. When two such distant sites were stimulated separated by a 10- or 20-ms delay, the second response scaled with the level of background EMG in the same way as a response to the PT stimulus. By contrast, when the same site was stimulated twice with these delays, the second response showed a facilitation compared with a similarly timed PT response. There would therefore appear to be a local facilitation of the cortical output at these intervals, which is not seen between distant sites. Antidromically identified pyramidal-tract neurones (PTNs) were recorded whilst stimuli were delivered to a cortical site, with a distance between stimulating and recording electrodes of also 1.5-2 mm. The most common response was a facilitation followed by a suppression. Six of eleven PTNs showed a facilitation in their discharge following this stimulation (maximum connection strength s=0.19), 8/11 showed a suppression (maximum s=0.16). It is concluded that powerful inter-connections do exist between distributed parts of the motor output and that there is widespread cortical activation after even a single ICMS pulse. However, these inter-connections do not lead to interactions between cortical outputs following stimulation, as assessed from the EMG. It is proposed that this is likely to reflect differences in the summation of output cells to local versus remote stimulation.

Molybdate (Mo) exerts insulinomimetic effects in vitro. In this study, we evaluated whether Mo can improve glucose homeostasis in genetically obese, insulin-resistant ob/ob mice. Oral administration of Mo (174 mg/kg molybdenum element) for 7 weeks did not affect body weight, but decreased the hyperglycaemia (approximately 20 mM) of obese mice to the levels of lean (L) (+/+) mice, and reduced the hyperinsulinaemia to one-sixth of pretreatment levels. Tolerance to oral glucose was improved: total glucose area was 30% lower in Mo-treated mice than in untreated ob/ob mice (O), while the total insulin area was halved. Hepatic glucokinase (GK) mRNA level and activity were unchanged in O mice compared with L mice, but the mRNA level and activity of L-type pyruvate kinase (L-PK) were increased in O mice by 3.5- and 1.7-fold respectively. Mo treatment increased GK mRNA levels and activity (by approximately 2.2-fold and 61% compared with O values), and had no, or only a mild, effect on the already increased L-PK variables. mRNA levels and activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK) were augmented in O liver (sixfold and by 57% respectively), and these were reduced by Mo treatment. Insulin binding to partially purified receptors from liver was reduced in O mice and restored by Mo treatment. Despite this correction, overall receptor tyrosine kinase activity was not improved in Mo mice. Moreover, the overexpression (by two- to fourfold) of the cytokine tumour necrosis factor alpha (TNF alpha) in white adipose tissue, which may have a determinant role in the insulin resistance of the O mice, was unaffected by Mo. Likewise, overexpression of the ob gene in white adipose tissue was unchanged by Mo. In conclusion, Mo markedly improved glucose homeostasis in the ob/ob mice by an insulin-like action which appeared to be exerted distal to the insulin receptor tyrosine kinase step. The blood glucose-lowering effect of Mo was unrelated to over-expression of the TNF alpha and ob genes in O mice, but resulted at least in part from attenuation of liver insulin resistance by the reversal of pre-translational regulatory defects in these mice.

The ob gene, specifically expressed in fat cells, encodes leptin, a hormone that induces satiety and increases energy expenditure. In this study, we investigated the interactions between glucocorticoids and insulin on ob gene expression in cultured explants of rat adipose tissue. Only low levels of ob mRNA were detected when adipose tissue from fasted rats was cultured for 12–24 h in minimal essential medium. However, the addition of dexamethasone to the medium increased ob gene expression in a concentration-dependent manner (EC50 10 nM). With 1 μM dexamethasone, ob mRNA levels were similar to those in fresh fat pads from fed rats, reaching a maximum after 12 h. The effect of dexamethasone was blocked by actinomycin D, which indicates an action on transcription. This effect was increased when a minimum amount of fuel (glucose or a mixture of lactate and pyruvate) was supplied in the medium. Unlike dexamethasone, insulin, even when combined with high glucose concentrations, did not induce ob expression, although it strongly increased the accumulation of mRNA species for fatty acid synthase (FAS), the insulin-sensitive glucose transporter GLUT4 and the γ isoform of peroxisome proliferator-activated receptor (PPARγ). Unexpectedly, insulin dose-dependently inhibited dexamethasone-induced ob mRNA accumulation. This effect was observed at low concentrations of insulin (IC50 1 nM) and was delayed in onset, beginning after 6–9 h of culture. It was mimicked by insulin-like growth factor 1 (IGF-1) (100 nM). The inhibition by insulin was only detectable when fuels were present and/or when a critical level of ob expression was reached. As this inhibitory effect was reversed by cycloheximide, this suggests that it required ongoing protein synthesis. In conclusion, unlike dexamethasone, insulin had no direct stimulatory effect on ob gene expression. On the other hand, insulin (and IGF-1) even inhibited the dexamethasone-induced accumulation of ob mRNA. The underlying mechanism involved ongoing synthesis of an inhibitory protein by insulin, which is in keeping with its delayed effect. Moreover, the expression of genes for FAS, GLUT4 and PPARγ may be inversely related to that of ob.

1. The effect of acetylcholine (ACh) on voltage-dependent Ca2+ currents in mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. 2. ACh (0.25-250 microM) reversibly and dose-dependently inhibited the Ca2+ current elicited by depolarizations from -80 mV to +10 mV. Maximal inhibition was observed at concentrations > 25 microM where it amounted to approximately 35%. The effect was voltage independent and prevented by atropine (10 microM) suggesting that it was mediated by muscarinic receptors. 3. The inhibitory action of ACh on the Ca2+ current was abolished when the cytoplasmic solution contained GDP beta S (2 mM) and became irreversible when the non-hydrolysable GTP analogue GTP gamma S (10 microM) was included in the pipette. This indicates the participation of G proteins in the inhibitory effect of ACh but pretreatment of the cells with either pertussis or cholera toxin failed to prevent the effect of ACh on the Ca2+ current. 4. ACh remained equally effective as an inhibitor of the whole-cell Ca2+ current in the presence of the L-type Ca2+ channel agonist (-)-Bay K 8644 and after partial inhibition of the current by nifedipine. Addition of omega-agatoxin IVA, omega-conotoxin GVIA or omega-conotoxin MVIIC neither affected the peak Ca2+ current amplitude nor the extent of inhibition produced by ACh. These pharmacological properties indicate that ACh acts by inhibiting L-type Ca2+ channels. 5. The inhibitory action of ACh on the B-cell Ca2+ current was not secondary to elevation of [Ca2+]i and ACh remained equally effective as an inhibitor when Ba2+ was used as the charge carrier, when [Ca2+]i was buffered to low concentrations using EGTA and under experimental conditions preventing the mobilization of Ca2+ from intracellular stores. 6. These results suggest that ACh reduces the whole-cell Ca2+ current in the B-cell through a G protein-regulated, voltage- and Ca(2+)-independent inhibition of L-type Ca2+ channels.

The ob gene encodes leptin, a hormone which induces satiety and increases energy expenditure. The peroxisome proliferator-activated receptor gamma 2 isoform (PPAR gamma 2) gene encodes a transcription factor which controls adipocyte differentiation and expression of fat-specific genes. We have studied the regulation of these two genes in white adipose tissue (WAT) during the suckling-weaning transition. Suckling rats ingest a high-fat diet (milk). Fat-pad weight barely varied during the last week of suckling. ob mRNA levels, which were very low in 15-day-old rats, rose approximately 6-fold until weaning at 21 days. When the rats were weaned on to a standard (high-carbohydrate) laboratory chow, epididymal WAT enlarged approximately 7-fold, and ob mRNA kept increasing progressively and doubled between 21 and 30 days. This evolution contrasted with that of fatty acid synthase (FAS) mRNA, which increased sharply, but only after weaning. To distinguish between the influence of developmental and nutritional factors on ob expression, a group of rats was weaned on to a high-fat diet. This prevented the rise in glycaemia and insulinaemia and the decrease in plasma non-esterified fatty acids which otherwise occurred at weaning. This also resulted in a slight (10-15%) decrease in food intake and body weight gain. Under this high-fat diet, the rise of ob mRNA in WAT was augmented (3.7-fold in 30- versus 21-day-old pups), whereas the normal rise in FAS mRNA levels was attenuated. Fat-pad weights and adipocyte cell size and number were roughly similar in high-carbohydrate- and high-fat-weaned pups. mRNA levels of PPAR gamma 2, like those of ob, were low in the WAT of 15-day-old suckling pups, doubled at 21 days, and reached a maximum as soon as 23 days. This evolution further differed from that of ob mRNA in not being influenced by diet composition. In conclusion, ob expression markedly increases during the suckling-weaning transition, and this effect is accentuated by a high-fat diet. Qualitative nutritional changes in ob mRNA were correlated with neither acute changes in adipose-tissue mass, nor cell size/number, nor variations in insulinaemia. PPAR gamma 2 also increased during suckling, but rapidly reached a plateau after weaning and no longer changed thereafter. Unlike ob, PPAR gamma 2 was not influenced by the diet composition.

Mouse pancreatic islets were used to investigate how muscarinic stimulation influences the cytoplasmic Ca2+ concentration ([Ca2+]i) in insulin-secreting B-cells. In the absence of extracellular Ca2+, acetylcholine (ACh) triggered a transient, concentration-dependent and thapsigargin-inhibited increase in [Ca2+]i. In the presence of extracellular Ca2+ and 15 mM glucose, ACh induced a biphasic rise in [Ca2+]i. The initial, transient phase increased with the concentration of ACh, whereas the second, sustained, phase was higher at low (0.1-1 microM) than at high (> or = 10 microM) concentrations of ACh. Thapsigargin attenuated (did not suppress) the first phase of the [Ca2+]i rise and did not affect the sustained response. This sustained rise was inhibited by omission of extracellular Na+ (which prevents the depolarizing action of ACh) and by D600 or diazoxide (which prevent activation of voltage-dependent Ca2+ channels). During steady-state stimulation, the Ca2+ action potentials in B-cells were stimulated by 1 microM ACh but inhibited by 100 microM ACh. When B-cells were depolarized by 45 mM K+, ACh induced a concentration-dependent, biphasic change in [Ca2+]i, consisting of a first peak rapidly followed by a decrease. Thapsigargin suppressed the peak without affecting the drop in [Ca2+]i. Measurements of 45Ca2+ efflux under similar conditions indicated that ACh decreases Ca2+ influx and slightly increases the efflux. All effects of ACh were blocked by atropine. In conclusion, three mechanisms at least are involved in the biphasic change in [Ca2+]i that muscarinic stimulation exerts in excitable pancreatic B-cells. A mobilization of Ca2+ from the endoplasmic reticulum contributes significantly to the first peak, but little to the steady-state rise in [Ca2+]i. This second phase results from an influx of Ca2+ through voltage-dependent Ca2+ channels activated by a Na(+)-dependent depolarization. However, when high concentrations of ACh are used, Ca2+ influx is attenuated.

1. The activity of thirty-one single motor units (SMUs) was recorded from forearm and hand muscles of three volunteers. The excitability of the rhythmically firing motoneurones supplying these SMUs was examined after voluntary discharge using transcranial magnetic stimulation (TMS). 2. The magnetic stimulus was delivered either at a fixed delay (range: 1-60 ms) after SMU discharge (triggered mode) or at random with respect to voluntary SMU discharge (random mode). Post-stimulus time histograms (PSTHs) of responses to 50-100 stimuli were constructed for each experimental condition. 3. In the triggered mode, the probability of response to TMS progressively decreased as the spike-to-stimulus interval was shortened. Shortening of the interval also resulted in redistribution of responses within the different subpeaks characterizing the short-latency response of motor units to TMS: the relative response probability of the first subpeak decreased with the shorter spike-to-stimulus intervals. 4. In the triggered mode, the probability of SMU responding to TMS was significantly higher when the firing rate of the motor unit was increased from a slow rate (< 10 impulses s-1) to a faster rate (> 12 impulses s-1), irrespective of the spike-to-stimulus interval. In contrast, in the random mode, the response probability was greater at the slower discharge rate. 5. The higher excitability of motoneurones at the fast rate in the triggered mode is consistent with findings in cat motoneurones suggesting a shallower after-hyperpolarization, but other mechanisms could contribute. Furthermore, our results suggest that there is an asymptotic recovery in the excitability of slow firing motoneurones towards the end of the interspike interval.

Recent experimental results show evidence for the corrective role of postsaccadic drifts. This paper addresses the modeling of these slow correcting gaze movements (SCMs). Classical arguments to explain drifts are presented, both in the head fixed condition (pulse-step mismatch) and in the head free condition (vestibulo-ocular reflex (VOR)). The most significant behavioral and electrophysiological experimental data related to SCMs are then briefly reported, with the conclusion that SCMs have a clear corrective role, incompatible with classical explanations of drifts. Based on these experimental data, existing models of the saccadic system are then compared. A theoretical comparison of the classical Robinson model with an alternative model is proposed. Two possible (slow and fast) pathways involved in the control of SCMs are examined, and simulation results are presented. Finally, the discussion addresses the observed species differences in SCMs. The link between natural SCMs and electrical SC stimulations, and the interactions between saccades, VOR, and SCMs are also discussed.

Glucose stimulation of insulin release involves metabolism of the sugar and elevation of cytoplasmic calcium (Ca2+i) in pancreatic B-cells. We compared the dynamic changes of metabolism (fluorescence of endogenous reduced pyridine nucleotides, NAD(P)H), membrane potential (intracellular microelectrodes), and Ca2+i (fura-2 technique), in intact mouse islets. Glucose (15 mM) sequentially triggered an increase in NAD(P)H fluorescence, a depolarization with electrical activity, and a rise in Ca2+i. The change in NAD(P)H was monophasic and regular, whereas the changes in membrane potential and Ca2+i were multiphasic, with steady-state regular oscillations of similar average frequencies (about 2.2/min). Digital image analysis revealed that Ca2+i oscillations were synchronous in all regions of the islets. Omission of extracellular Ca2+ abolished the rise in Ca2+i but not the increase in NAD(P)H. Both electrical and Ca2+i oscillations disappeared in low external Ca2+ (1 mM), and became larger but slower in high Ca2+ (10 mM). Sustained depolarization (by tolbutamide, arginine, or high K+) and hyperpolarization (by diazoxide) of B-cells caused sustained increases and decreases of Ca2+i, respectively. In conclusion, the changes in membrane potential induced by various secretagogues trigger synchronous changes in Ca2+i in all B-cells of the islets. The oscillatory pattern of the electrical and Ca2+i responses induced by glucose is not accompanied by and thus probably not due to similar oscillations of metabolism.

The sequence of several cDNAs encoding the amyloid protein precursor showed that two polyadenylation sites of the mRNA are utilized; RNA blot analysis with different riboprobes indicated that this explains the difference between the two major 3.2 and 3.4 kb mRNAs found in the human brain. These two mRNAs, which contain the whole sequence of the natural molecules, were synthesized by in vitro transcription and translated in Xenopus oocytes. The long mRNA using the second polyadenylation site produced more protein than the short mRNA. The sequence contained within the two polyadenylation sites used in the 3' untranslated region of the amyloid protein precursor mRNA was also able to increase the production of the chicken lysozyme or the chloramphenicol acetyl transferase, as demonstrated by in vivo translation of different chimeric mRNAs obtained by in vitro transcription. This difference in protein production was also observed when chimeric cDNA constructs were transfected into Chinese hamster ovary cells. Since long mRNAs are not more stable than short mRNAs, the sequence contained within the two polyadenylation sites of the amyloid protein precursor mRNA increases the translation.

We have recently identified several adipokines as oversecreted by omental adipose tissue (AT) of obese subjects: two chemokines (growth-related oncogene factor-alpha (GRO-alpha), macrophage inflammatory protein-1beta (MIP-1beta)), a tissue inhibitor of metalloproteinases-1 (TIMP-1), an interleukin-7 (IL-7) and a megakaryocytic growth-factor (thrombopoietin (TPO)). These adipokines are involved in insulin resistance and atherosclerosis. The objectives of this study were to determine whether the circulating levels of these adipokines were increased in obesity and to identify the responsible factors. A cross-sectional study including 32 lean (BMI (kg/m(2)) 40) age-matched women was carried out. Serum adipokine levels, insulin sensitivity, and substrate oxidation were measured by ELISA, euglycemic-hyperinsulinemic clamp, and indirect calorimetry, respectively. Circulating levels of GRO-alpha, TPO, and TIMP-1 were higher in obese and/or severely obese women than in lean ones (+30, 55, and 20%, respectively). Serum levels of these adipokines positively correlated with insulinemia or glycemia, and negatively with insulin sensitivity. TIMP-1 also positively correlated with blood pressure, and TPO with triglyceride levels. Multiple regression analysis showed that fat mass per se was an independent determinant of GRO-alpha, TPO, and TIMP-1 levels, suggesting that hypertrophied adipocytes and recruited macrophages in expanded AT mainly contribute to this hyperadipokinemia. Insulinemia, glycemia and resistance of glucose oxidation to insulin were additional predictors for TPO. Circulating GRO-alpha, TPO, and TIMP-1 levels are increased in obesity. This may be partially due to augmented adiposity per se and to hyperinsulinemia/insulin resistance. These high systemic levels may in turn worsen/promote insulin resistance and cardiovascular disease.

BACKGROUND: We have previously shown that gut microbial fermentation of prebiotics promotes satiety and lowers hunger and energy intake in humans. In rodents, these effects are associated with an increase in plasma gut peptide concentrations, which are involved in appetite regulation and glucose homeostasis. OBJECTIVE: Our aim was to examine the effects of prebiotic supplementation on satiety and related hormones during a test meal for human volunteers by using a noninvasive micromethod for blood sampling to measure plasma gut peptide concentrations. DESIGN: This study was a randomized, double-blind, parallel, placebo-controlled trial. A total of 10 healthy adults (5 men and 5 women) were randomly assigned to groups that received either 16 g prebiotics/d or 16 g dextrin maltose/d for 2 wk. Meal tolerance tests were performed in the morning to measure the following: hydrogen breath test, satiety, glucose homeostasis, and related hormone response. RESULTS: We show that the prebiotic treatment increased breath-hydrogen excretion (a marker of gut microbiota fermentation) by approximately 3-fold and lowered hunger rates. Prebiotics increased plasma glucagon-like peptide 1 and peptide YY concentrations, whereas postprandial plasma glucose responses decreased after the standardized meal. The areas under the curve for plasma glucagon-like peptide 1 and breath-hydrogen excretion measured after the meal (0-60 min) were significantly correlated (r = 0.85, P = 0.007). The glucose response was inversely correlated with the breath-hydrogen excretion areas under the curve (0-180 min; r = -0.73, P = 0.02). CONCLUSION: Prebiotic supplementation was associated with an increase in plasma gut peptide concentrations (glucagon-like peptide 1 and peptide YY), which may contribute in part to changes in appetite sensation and glucose excursion responses after a meal in healthy subjects.

Nociception begins when Adelta- and C-nociceptors are activated. However, the processing of nociceptive input by the cortex is required before pain can be consciously experienced from nociception. To characterize the cortical activity related to the emergence of this experience, we recorded, in humans, laser-evoked potentials elicited by physically identical nociceptive stimuli that were either perceived or unperceived. Infrared laser pulses, which selectively activate skin nociceptors, were delivered to the hand dorsum either as a pair of rapidly succeeding and spatially displaced stimuli (two-thirds of trials) or as a single stimulus (one-third of trials). After each trial, subjects reported whether one or two distinct painful pinprick sensations, associated with Adelta-nociceptor activation, had been perceived. The psychophysical feedback after each pair of stimuli was used to adjust the interstimulus interval (ISI) of the subsequent pair: when a single percept was reported, ISI was increased by 40 ms; when two distinct percepts were reported, ISI was decreased by 40 ms. This adaptive algorithm ensured that the probability of perceiving the second stimulus of the pair tended toward 0.5. We found that the magnitude of the early-latency N1 wave was similar between perceived and unperceived stimuli, whereas the magnitudes of the later N2 and P2 waves were reduced when stimuli were unperceived. These findings suggest that the N1 wave represents an early stage of sensory processing related to the ascending nociceptive input, whereas the N2 and P2 waves represent a later stage of processing related, directly or indirectly, to the perceptual outcome of this nociceptive input.

Behavior arises from a constant competition between potential actions. For example, movements performed unimanually require selecting one hand rather than the other. Corticospinal (CS) excitability of the nonselected hand is typically decreased prior to movement initiation, suggesting that response selection may involve mechanisms that inhibit nonselected candidate movements. To examine this hypothesis, participants performed a reaction time task, responding with the left, right, or both indexes. Transcranial magnetic stimulation was applied over the right primary motor cortex (M1) to induce motor-evoked potentials (MEPs) in a left hand muscle at various stages during response preparation. To vary the time of response selection, an imperative signal was preceded by a preparatory cue that was either informative or uninformative. Left MEPs decreased following the cue. Surprisingly, this decrease was greater when an informative cue indicated that the response might require the left hand than when it indicated a right hand response. In the uninformative condition, we did not observe additional attenuation of left MEP after an imperative indicating a right hand response. These results argue against the "deselection" hypothesis. Rather, CS suppression seems to arise from "impulse control" mechanisms that ensure that responses associated with potentially selected actions are not initiated prematurely.

Context: In obesity, adipocyte hypertrophy and macrophage infiltration lead to overproduction of proinflammatory adipokines, which play a crucial role in the metabolic syndrome. The molecular mechanisms underlying this overproduction are still unsettled. The role of TNF-alpha also remains controversial in human obesity. Objective: We revisited the contribution of TNF-alpha to adipokine dysregulation in central obesity. We more particularly assessed the involvement of TNF-alpha vs. other stromal-vascular cell (SVC)-secreted factors and searched for potential differential responses to TNF-alpha between adipocytes of lean and obese individuals. Design and participants: Primary cultures of omental adipocytes from obese and non-obese age- and sex-matched subjects were used. For some experiments, we generated media previously conditioned by SVC, which mimic adipocyte microenvironment. Results: Adipocytes of obese subjects mainly overexpressed adipokines, in comparison to those of lean ones, when cultured SVC-conditioned media. This was abrogated by immunoneutralization of TNF-alpha, indicating that among the numerous factors secreted by SVC, TNF-alpha is a crucial contributor to adipokine dysregulation. Accordingly, adipocytes of obese subjects overproduced adipokines in response to direct exposure of TNF-alpha. This hyper-responsiveness was mediated by TNF-alpha-receptor-1 and hyper-activation of the NF-kappaB pathway. Correspondingly, NF-kappaB activity was increased in adipocytes of obese subjects and correlated with adipocyte size, adipokine expression and in vivo insulin resistance. Eventually, adipokine overexpression in adipocytes of obese subjects was prevented by NF-kappaB inhibitors. Conclusions: In obesity, TNF-alpha that is, over other SVC-secreted factors, a crucial determinant of adipokine dysregulation acts on enlarged adipocytes, which are hyper-responsive to this triggering signal. This ultimately exacerbates adipokine production, inflammation and the metabolic syndrome.

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo.

Pancreatic beta-cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional beta-cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine-induced apoptosis and hypoxia. The glucose regulation of oxidative stress-response and integrated stress-response genes in cultured rat islets follows an asymmetric V-shaped profile parallel to that of beta-cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional beta-cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, beta-cell demise under glucotoxic conditions may also result from beta-cell hypoxia and, in vivo, from their exposure to inflammatory cytokines released locally by non-endocrine islet cells. A better understanding of the relative contribution of each type of stress to beta-cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional beta-cell mass in glucose intolerant and type 2 diabetic patients.

BACKGROUND AND AIMS: We have generated a transgenic (Tg) mouse model allowing persistent and moderate overexpression of native adiponectin (ApN) targeted to white adipose tissue [the transgene being placed under control of aP2 (fatty-acid binding protein) promoter]. Adiposity was reduced in aging mice fed a high-sucrose diet and this anti-obesity effect favored enhanced insulin sensitivity and improved lipid profile. APN appears to be a potent regulator of other adipokines. We took advantage of this unique in vivo model to study the influence of APN on adipokine secretion profiling. In order to investigate the early and specific effects of APN, mice were studied before any significant decrease of fat mass or glycemia and circulating lipids occurred […]

Synchronous calcium oscillations are observed in primary cultures of rat cortical neurons when mature networks are formed. This spontaneous neuronal activity needs an accurate control of calcium homeostasis. Alteration of intraneuronal calcium concentration is described in many neurodegenerative disorders, including Alzheimer disease (AD). Although processing of amyloid precursor protein (APP) that generates Aβ peptide has critical implications for AD pathogenesis, the neuronal function of APP remains unclear. Here, we report that expression of human APP (hAPP) in rat cortical neurons increases L-type calcium currents, which stimulate SK channels, calcium-dependent K+channels responsible for medium afterhyperpolarization (mAHP). In a neuronal network, increased mAHP in some neurons expressing hAPP leads to inhibition of calcium oscillations in all the cells of the network. This inhibition is independent of production and secretion of Aβ and other APP metabolites. In a neuronal network, reduction of endogenous APP expression using shRNA increases the frequency and reduces the amplitude of calcium oscillations. Altogether, these data support a key role for APP in the control of neuronal excitability.