Live-Cell Imaging of Single Neurotrophin Receptor Molecules on Human Neurons in Alzheimer's Disease.

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75NTR play a critical role in neuronal survival and their functions are altered in Alzheimer's disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored. Using live-cell single-molecule imaging, we examined the surface trafficking of TrkA and p75NTR molecules on live neurons that were derived from human-induced pluripotent stem cells (hiPSCs) of presenilin 1 (PSEN1) mutant familial AD (fAD) patients and non-demented control subjects. Our results show that the surface movement of TrkA and p75NTR and the activation of TrkA- and p75NTR-related phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) signaling pathways are altered in neurons that are derived from patients suffering from fAD compared to controls. These results provide evidence for altered surface movement of receptors in AD and highlight the importance of investigating receptor dynamics in disease conditions. Uncovering these mechanisms might enable novel therapies for AD.

Identification of the binding site between bovine serum albumin and ultrasmall SiC fluorescent biomarkers.

Ultrasmall silicon carbide nanoparticles (SiC USNPs) are very promising biomarkers for developing new applications in diagnostics, cell monitoring or drug delivery, even though their interaction with biological molecules such as different proteins has not yet been investigated in detail. In this study, the biological behaviour of SiC USNPs in a medium modeling a living organism was investigated in detail through the dependence of the fluorescence on interactions between bovine serum albumin (BSA) and SiC USNPs. The interaction shows transient nanoparticle-protein associations due to the restricted diffusion behaviour of the nanoparticles in the vicinity of a protein. The transient association manifests in a complex fluorescence quenching mechanism where the dynamic component was dominated by Förster resonance energy transfer. By studying SiC nanoparticles of different sizes, it can be concluded that the transient effect is an ultrasmall nanoparticle behaviour.

Maximum likelihood-based estimation of diffusion coefficient is quick and reliable method for analyzing estradiol actions on surface receptor movements.

The rapid effects of estradiol on membrane receptors are in the focus of the estradiol research field, however, the molecular mechanisms of these non-classical estradiol actions are poorly understood. Since the lateral diffusion of membrane receptors is an important indicator of their function, a deeper understanding of the underlying mechanisms of non-classical estradiol actions can be achieved by investigating receptor dynamics. Diffusion coefficient is a crucial and widely used parameter to characterize the movement of receptors in the cell membrane. The aim of this study was to investigate the differences between maximum likelihood-based estimation (MLE) and mean square displacement (MSD) based calculation of diffusion coefficients. In this work we applied both MSD and MLE to calculate diffusion coefficients. Single particle trajectories were extracted from simulation as well as from α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor tracking in live estradiol-treated differentiated PC12 (dPC12) cells. The comparison of the obtained diffusion coefficients revealed the superiority of MLE over the generally used MSD analysis. Our results suggest the use of the MLE of diffusion coefficients because as it has a better performance, especially for large localization errors or slow receptor movements.

17ß-estradiol does not have a direct effect on the function of striatal cholinergic interneurons in adult mice in vitro.

The striatum is an essential component of the basal ganglia that is involved in motor control, action selection and motor learning. The pathophysiological changes of the striatum are present in several neurological and psychiatric disorder including Parkinson's and Huntington's diseases. The striatal cholinergic neurons are the main regulators of striatal microcircuitry. It has been demonstrated that estrogen exerts various effects on neuronal functions in dopaminergic and medium spiny neurons (MSN), however little is known about how the activity of cholinergic interneurons are influenced by estrogens. In this study we examined the acute effect of 17ß-estradiol on the function of striatal cholinergic neurons in adult mice in vitro. We also tested the effect of estrus cycle and sex on the spontaneous activity of cholinergic interneurons in the striatum. Our RNAscope experiments showed that ERα, ERß, and GPER1 receptor mRNAs are expressed in some striatal cholinergic neurons at a very low level. In cell-attached patch clamp experiments, we found that a high dose of 17ß-estradiol (100 nM) affected the spontaneous firing rate of these neurons only in old males. Our findings did not demonstrate any acute effect of a low concentration of 17ß-estradiol (100 pM) or show any association of estrus cycle or sex with the activity of striatal cholinergic neurons. Although estrogen did not induce changes in the intrinsic properties of neurons, indirect effects via modulation of the synaptic inputs of striatal cholinergic interneurons cannot be excluded.

Prevention of bronchial hyperreactivity in a rat model of precapillary pulmonary hypertension.

BACKGROUND: The development of bronchial hyperreactivity (BHR) subsequent to precapillary pulmonary hypertension (PHT) was prevented by acting on the major signalling pathways (endothelin, nitric oxide, vasoactive intestine peptide (VIP) and prostacyclin) involved in the control of the pulmonary vascular and bronchial tones. METHODS: Five groups of rats underwent surgery to prepare an aorta-caval shunt (ACS) to induce sustained precapillary PHT for 4 weeks. During this period, no treatment was applied in one group (ACS controls), while the other groups were pretreated with VIP, iloprost, tezosentan via an intraperitoneally implemented osmotic pump, or by orally administered sildenafil. An additional group underwent sham surgery. Four weeks later, the lung responsiveness to increasing doses of an intravenous infusion of methacholine (2, 4, 8 12 and 24 µg/kg/min) was determined by using the forced oscillation technique to assess the airway resistance (Raw). RESULTS: BHR developed in the untreated rats, as reflected by a significant decrease in ED50, the equivalent dose of methacholine required to cause a 50% increase in Raw. All drugs tested prevented the development of BHR, iloprost being the most effective in reducing both the systolic pulmonary arterial pressure (Ppa; 28%, p = 0.035) and BHR (ED50 = 9.9 ± 1.7 vs. 43 ± 11 µg/kg in ACS control and iloprost-treated rats, respectively, p = 0.008). Significant correlations were found between the levels of Ppa and ED50 (R = -0.59, p = 0.016), indicating that mechanical interdependence is primarily responsible for the development of BHR. CONCLUSIONS: The efficiency of such treatment demonstrates that re-establishment of the balance of constrictor/dilator mediators via various signalling pathways involved in PHT is of potential benefit for the avoidance of the development of BHR.

Sevoflurane and desflurane protect cholinergic-induced bronchoconstriction of hyperreactive airways in rabbits.

PURPOSE: The potential of desflurane to alter respiratory mechanics in the presence of bronchial hyperresponsiveness (BHR) is still a subject of debate. Accordingly, we evaluated the bronchoprotective potential of desflurane compared with sevoflurane following cholinergic lung constriction in rabbits with normal and hyperreactive airways. METHODS: The input impedance of the respiratory system (Zrs) was measured during midazolam-based anesthesia before and during intravenous infusions of increasing doses of methacholine (MCh). The rabbits in the control group (Group C) were then randomized to receive either sevoflurane 1 MAC followed by desflurane 1 MAC or vice versa, whereas ovalbumin-sensitized rabbits received sevoflurane followed by desflurane (Group S-SD) or vice versa (Group S-DS). Baseline Zrs measurements and the MCh provocations were repeated under the maintenance of each volatile agent. Airway resistance (Raw), tissue damping (G), and elastance data were obtained from Zrs by model fitting. RESULTS: Similar bronchoprotective effects of sevoflurane and desflurane against MCh-induced bronchoconstriction were observed independently of the severity of the bronchospasm and the presence of BHR. With sevoflurane, the decreases in Raw ranged from 22 (8.8)% to 44 (12)%, and with desflurane, they ranged from 22 (8.7)% to 50 (12)%. The increases in G reflecting the enhanced ventilation heterogeneities in the lung periphery were not affected by the volatile agents. CONCLUSIONS: If the contractile stimulus is cholinergic in origin, sevoflurane and desflurane exert similar bronchoprotective potentials to act against lung constriction independent of the presence of BHR. These volatile anesthetics otherwise lack a potential to improve the enhanced ventilation heterogeneities that develop particularly in the presence of BHR.

Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons.

Gonadal steroid 17ß-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.

Mechanisms for lung function impairment and airway hyperresponsiveness following chronic hypoxia in rats.

Although chronic normobaric hypoxia (CH) alters lung function, its potential to induce bronchial hyperreactivity (BHR) is still controversial. Thus the effects of CH on airway and tissue mechanics separately and changes in lung responsiveness to methacholine (MCh) were investigated. To clarify the mechanisms, mechanical changes were related to end-expiratory lung volume (EELV), in vivo results were compared with those in vitro, and lung histology was assessed. EELV was measured plethysmographically in two groups of rats exposed to 21 days of CH (11% O(2)) or to normoxia. Total respiratory impedance was measured under baseline conditions and following intravenous MCh challenges (2-18 microg x kg(-1) x min(-1)). The lungs were then excised and perfused, and the pulmonary input impedance was measured, while MCh provocations were repeated under a pulmonary capillary pressure of 5, 10, and 15 mmHg. Airway resistance, tissue damping, and elastance were extracted from the respiratory impedance and pulmonary input impedance spectra. The increases in EELV following CH were associated with decreases in airway resistance, whereas tissue damping and elastance remained unaffected. CH led to the development of severe BHR to MCh (206 +/- 30 vs. 95 +/- 24%, P < 0.001), which was not detectable when the same lungs were studied in vitro at any pulmonary capillary pressure levels maintained. Histology revealed pulmonary arterial vascular remodeling with overexpression of alpha-smooth muscle actin antibody in the bronchial wall. These findings suggest that, despite the counterbalancing effect of the increased EELV, BHR develops following CH, only in the presence of intact autonomous nervous system. Thus neural control plays a major role in the changes in the basal lung mechanics and responsiveness following CH.

Acute cigarette smoke inhalation blunts lung responsiveness to methacholine and allergen in rabbit: differentiation of central and peripheral effects.

Despite the prevalence of active smoking in asthmatics, data on the short-term effect of acute mainstream tobacco smoke exposure on airway responsiveness are very scarce. The aim of this study was to assess the immediate effect of acute exposure to mainstream cigarette smoke on airway reactivity to subsequent nonspecific and allergenic challenges in healthy control (n = 5) and ovalbumin-sensitized rabbits (n = 6). We combined low-frequency forced oscillations and synchrotron radiation CT imaging to differentiate central airway and peripheral airway and lung parenchymal components of the response to airway provocation. Acute exposure to smoke generated by four successive cigarettes (CS) strongly inhibited the central airway response to subsequent IV methacholine (MCh) challenge. In the sensitized animals, although the response to ovalbumin was also inhibited in the central airways, mainstream CS did not blunt the peripheral airway response in this group. In additional groups of experiments, exposure to HEPA-filtered CS (n = 6) similarly inhibited the MCh response, whereas CO (10,000 ppm for 4 min, n = 6) or nitric oxide inhalation instead of CS (240 ppm, 4 x 7 min, n = 5) failed to blunt nonspecific airway responsiveness. Pretreatment with alpha-chymotrypsin to inhibit endogenous VIP before CS exposure had no effect (n = 4). Based on these observations, the gas phase of mainstream cigarette smoke may contain one or more short-term inhibitory components acting primarily on central airways and inhibiting the response to both specific and nonspecific airway provocation, but not on the lung periphery where both lung mechanical parameters, and synchrotron-imaging derived parameters, showed large changes in response to allergen challenge in sensitized animals.

Precapillary pulmonary hypertension leads to reversible bronchial hyperreactivity in rats.

Congenital heart disease with left-to-right shunt may lead to precapillary pulmonary hypertension (PREPHT) with potential lung function impairment. The authors investigated the effects of PREPHT on lung responsiveness in a rat model of PREPHT by creating and repairing an abdominal aortocaval shunt (ACS). Rats were studied 4 weeks after the induction of ACS, and 4 weeks after its surgical repair. Control rats underwent sham surgery. To assess bronchial hyperreactivity, airway resistance (Raw) was measured at baseline and after increasing doses of methacholine. Raw was estimated by model fitting of the mechanical impedance of the respiratory system generated by forced oscillation technique. Lung morphological changes were assessed by histology. The prolonged presence of the ACS led to only minor changes in the basal respiratory mechanics, whereas it induced marked bronchial hyperreactivity, the methacholine-induced elevations in Raw being 49% +/- 5% before and 232% +/- 32% (P <.001) after ACS. These alterations were not associated with any changes in lung histology and were completely reversible on closure of the shunt. These results suggest that the induction of chronic increases in pulmonary blood flow and pressure causes reversible bronchial hyperreactivity. This may be consequent to the altered mechanical interdependence between the pulmonary vasculature and the respiratory tract.

Methacholine and ovalbumin challenges assessed by forced oscillations and synchrotron lung imaging.

RATIONALE: Methacholine (Mch) is routinely used to assess bronchial hyperreactivity; however, little is known about the differences in the lung response pattern between this provocation and that observed with ovalbumin (Ova) after allergic sensitization. OBJECTIVES: To compare (1) the central versus peripheral effects of Mch and Ova within the lung by combining measurements of airway and tissue mechanics with synchrotron radiation (SR) imaging, and (2) to assess the extent to which mechanical and imaging parameters are correlated. METHODS: We used the low-frequency forced oscillation technique and SR imaging in control (n = 12) and ovalbumin-sensitized (n = 13) rabbits, at baseline, during intravenous Mch infusion (2.5 microg/kg/min, 5.0 microg/kg/min, or 10.0 microg/kg/min), after recovery from Mch, and after intravenous Ova injection (2.0 mg). We compared intravenous Mch challenge with inhaled Mch (125 mg/ml, 90 s) in a separate group of control animals (n = 5). MEASUREMENTS AND MAIN RESULTS: Airway conductance and tissue elastance were measured by low-frequency forced oscillation technique. The central airway cross-sectional area, the ventilated alveolar area, and the heterogeneity of specific ventilation were quantified by SR imaging. Mch infusion induced constriction predominantly in the central airways, whereas Ova provocation affected mainly the peripheral airways, leading to severe ventilation heterogeneities in sensitized animals. Mch inhalation affected both conducting and peripheral airways. The correlations between airway conductance and central airway cross-sectional area (R = 0.71) and between tissue elastance and ventilated alveolar area (R = -0.72) were strong. CONCLUSIONS: The pattern of lung response caused by intravenous Mch and Ova are fundamentally different. Although inhaled Mch induces a heterogeneous lung response similar to that observed with intravenous allergen, these similar patterns are due to different mechanisms.

Impact of elevated pulmonary blood flow and capillary pressure on lung responsiveness.

Since alterations in pulmonary hemodynamics may lead to airway hyperreactivity, the consequences of individual changes in pulmonary blood flow (Qp) and capillary pressure (Pc) on lung responsiveness were investigated. During maintenance of a steady-state Pc of 5, 10, or 15 mmHg (groups 1-3), acute increases of Qp were generated in isolated, perfused rat lungs by simultaneous pulmonary arterial pressure elevation and venous pressure lowering. Conversely, at constant low (groups 4 and 5) or high Qp (groups 6 and 7), Pc was lowered or elevated by changing, in parallel, the pulmonary arterial and venous pressures. Pulmonary input impedance was measured under baseline conditions and during methacholine provocation (2-18 microg*kg(-1)*min(-1)), whereas the pulmonary hemodynamics were altered in accordance with the group allocation. The airway resistance and constant-phase parenchymal model parameters were identified from the pulmonary input impedance spectra. Increases of Qp at constant Pc had no effect on the basal lung mechanics, whereas they enhanced the lung reactivity to methacholine, particularly when high Pc was maintained [peak airway resistance increases of 299 +/- 99% (SE) vs. 609 +/- 217% at Qp levels of 5 and 10 ml/min, respectively, P < 0.05]. In contrast, the change of Pc at constant Qp slightly deteriorated the basal parenchymal mechanics without affecting the lung responsiveness. These findings suggest that increases in Qp per se may lead to the development of airway hyperreactivity. This phenomenon may contribute to the airway susceptibility under conditions associated with simultaneous elevations in pulmonary vascular pressures and Qp, such as exercise-induced asthma and the situation in children with congenital heart diseases.

The bimodal quasi-static and dynamic elastance of the murine lung.

The double sigmoidal nature of the mouse pressure-volume (PV) curve is well recognized but largely ignored. This study systematically examined the effect of inflating the mouse lung to 40 cm H2O transrespiratory pressure (Prs) in vivo. Adult BALB/c mice were anesthetized, tracheostomized, and mechanically ventilated. Thoracic gas volume was calculated using plethysmography and electrical stimulation of the intercostal muscles. Lung mechanics were tracked during inflation-deflation maneuvers using a modification of the forced oscillation technique. Inflation beyond 20 cm H2O caused a shift in subsequent PV curves with an increase in slope of the inflation limb and an increase in lung volume at 20 cm H2O. There was an overall decrease in tissue elastance and a fundamental change in its volume dependence. This apparent "softening" of the lung could be recovered by partial degassing of the lung or applying a negative transrespiratory pressure such that lung volume decreased below functional residual capacity. Allowing the lung to spontaneously recover revealed that the lung required approximately 1 h of mechanical ventilation to return to the original state. We propose a number of possible mechanisms for these observations and suggest that they are most likely explained by the unfolding of alveolar septa and the subsequent redistribution of the fluid lining the alveoli at high transrespiratory pressure.

Methacholine responsiveness in mice from 2 to 8 wk of age.

Many chronic human lung diseases have their origin in early childhood, yet most murine models used to study them utilize adult mice. An important component of the asthma phenotype is exaggerated airway responses, frequently modelled by methacholine (MCh) challenge. The present study was undertaken to characterize MCh responses in mice from 2 to 8 wk of age measuring absolute lung volume and volume-corrected respiratory mechanics as outcome variables. Female BALB/c mice aged 2, 3, 4, 6, and 8 wk were studied during cumulative intravenous MCh challenge. Following each MCh dose, absolute lung volume was measured plethysmographically at functional residual volume and during a slow inflation to 20-hPa transrespiratory pressure. Respiratory system impedance was measured continuously during the inflation maneuver and partitioned into airway and constant-phase parenchymal components by model fitting. Volume-corrected (specific) estimates of respiratory mechanics were calculated. Intravenous MCh challenge induced a predominantly airway response with no evidence of airway closure in any age group. No changes in functional residual volume were seen in mice of any age during the MCh challenge. The specific airway resistance MCh dose response curves did not show significant differences between the age groups. The results from the present study do not show systematic differences in MCh responsiveness in mice from 2 to 8 wk of age.

Plethysmographic estimation of thoracic gas volume in apneic mice.

Electrical stimulation of intercostal muscles was employed to measure thoracic gas volume (TGV) during airway occlusion in the absence of respiratory effort at different levels of lung inflation. In 15 tracheostomized and mechanically ventilated CBA/Ca mice, the value of TGV obtained from the spontaneous breathing effort available in the early phase of the experiments (TGVsp) was compared with those resulting from muscle stimulation (TGVst) at transrespiratory pressures of 0, 10, and 20 cmH2O. A very strong correlation (r2= 0.97) was found, although with a systematically (approximately 16%) higher estimation of TGVst relative to TGVsp, attributable to the different durations of the stimulated (approximately 50 ms) and spontaneous (approximately 200 ms) contractions. Measurements of TGVst before and after injections of 0.2, 0.4, and 0.6 ml of nitrogen into the lungs in six mice resulted in good agreement between the change in TGVst and the injected volume (r2= 0.98). In four mice, TGVsp and TGVst were compared at end expiration with air or a helium-oxygen mixture to confirm the validity of isothermal compression in the alveolar gas. The TGVst values measured at zero transrespiratory pressure in all CBA/Ca mice [0.29 +/- 0.05 (SD) ml] and in C57BL/6 (N = 6; 0.34 +/- 0.08 ml) and BALB/c (N = 6; 0.28 +/- 0.06 ml) mice were in agreement with functional residual capacity values from previous studies in which different techniques were used. This method is particularly useful when TGV is to be determined in the absence of breathing activity, when it must be known at any level of lung inflation or under non-steady-state conditions, such as during pharmaceutical interventions.

Tracking of airway and tissue mechanics during TLC maneuvers in mice.

A tracking impedance estimation technique was developed to follow the changes in total respiratory impedance (Zrs) during slow total lung capacity maneuvers in six anesthetized and mechanically ventilated BALB/c mice. Zrs was measured with the wave-tube technique and pseudorandom forced oscillations at nine frequencies between 4 and 38 Hz during inflation from a transrespiratory pressure of 0-20 cmH2O and subsequent deflation, each lasting for approximately 20 s. Zrs was averaged for 0.125 s and fitted by a model featuring airway resistance (Raw) and inertance, and tissue damping and elastance (H). Lower airway conductance (Glaw) was linearly related to volume above functional residual capacity (V) between 0 and 75-95% maximum V, with a mean slope of dGlaw/dV = 13.6 +/- 4.6 cmH2O-1. s-1. The interdependence of Raw and H was characterized by two distinct and closely linear relationships for the low- and high-volume regions, separated at approximately 40% maximum V. Comparison of Raw with the highest-frequency resistance of the total respiratory system revealed a marked volume-dependent contribution of tissue resistance to total respiratory system resistance, resulting in the overestimation of Raw by 19 +/- 8 and 163 +/- 40% at functional residual capacity and total lung capacity, respectively, whereas the lowest frequency reactance was proportional to H; these findings indicate that single-frequency resistance values may become inappropriate as surrogates of Raw when tissue impedance is changing.

Sensitivity analysis of respiratory parameter estimates in the constant-phase model.

The constant-phase model is increasingly used to fit low-frequency respiratory input impedance (Zrs), highlighting the need for a better understanding of the use of the model. Of particular interest is the extent to which Zrs would be affected by changes in parameters of the model, and conversely, how reliable are parameters estimated from model fits to the measured Zrs. We performed sensitivity analysis on respiratory data from 6 adult mice, at functional residual capacity (FRC), total lung capacity (TLC), and during bronchoconstriction, obtained using a 1-25 Hz oscillatory signal. The partial derivatives of Zrs with respect to each parameter were first examined. The limits of the 95% confidence intervals, 2-dimensional pairwise and p-dimensional joint confidence regions were then calculated. It was found that airway resistance was better estimated at FRC, as determined by the confidence region limits, whereas tissue damping and elastance were better estimated at TLC. Airway inertance was poorly estimated at this frequency range, as expected. During methacholine-evoked pulmonary constriction, there was an increase in the uncertainty of airway resistance and tissue damping, but this can be compensated for by using the relative (weighted residuals) in preference over the absolute (unweighted residuals) fitting criterion. These results are consistent with experimental observation and physiological understanding.