Physiological Time Course Research Articles

A highly sensitive strategy for monitoring real-time proliferation of targeted cell types in vivo

Cell proliferation processes play pivotal roles in timely adaptation to many biological situations. Herein, we establish a highly sensitive and simple strategy by which time-series showing the proliferation of a targeted cell type can be quantitatively monitored in vivo in the same individuals. We generate mice expressing a secreted type of luciferase only in cells producing Cre under the control of the Ki67 promoter. Crossing these with tissue-specific Cre-expressing mice allows us to monitor the proliferation time course of pancreatic β-cells, which are few in number and weakly proliferative, by measuring plasma luciferase activity. Physiological time courses, during obesity development, pregnancy and juvenile growth, as well as diurnal variation, of β-cell proliferation, are clearly detected. Moreover, this strategy can be utilized for highly sensitive ex vivo screening for proliferative factors for targeted cells. Thus, these technologies may contribute to advancements in broad areas of biological and medical research.

High or hot-Perspectives on altitude camps and heat-acclimation training as preparation for prolonged stage races.

Adaptation to heat stress and hypoxia are relevant for athletes participating in Tour de France or similar cycling races taking place during the summertime in landscapes with varying altitude. Both to minimize detrimental performance effects associated with arterial desaturation occurring at moderate altitudes in elite athletes, respectively, reduce the risk of hyperthermia on hot days, but also as a pre-competition acclimatization strategy to boost blood volume in already highly adapted athletes. The hematological adaptations require weeks of exposure to manifest, but are attractive as an augmented hemoglobin mass may improve arterial oxygen delivery and hence benefit prolonged performances. Altitude training camps have in this context a long history in exercise physiology and are still common practice in elite cycling. However, heat-acclimation training provides an attractive alternative for some athletes either as a stand-alone approach or in combination with altitude. The present paper provides an update and practical perspectives on the potential to utilize hypoxia and heat exposure to optimize hematological adaptations. Furthermore, we will consider temporal aspects both in terms of onset and decay of the adaptations relevant for improved thermoregulatory capacity and respiratory adaptations to abate arterial desaturation during altitude exposure. From focus on involved physiological mechanisms, time course, and responsiveness in elite athletes, we will provide guidance based on our experience from practical implementation in cyclists preparing for prolonged stage races such as the Tour de France.

Unsupervised physiological noise correction of functional magnetic resonance imaging data using phase and magnitude information (PREPAIR).

Of the sources of noise affecting blood oxygen level-dependent functional magnetic resonance imaging (fMRI), respiration and cardiac fluctuations are responsible for the largest part of the variance, particularly at high and ultrahigh field. Existing approaches to removing physiological noise either use external recordings, which can be unwieldy and unreliable, or attempt to identify physiological noise from the magnitude fMRI data. Data-driven approaches are limited by sensitivity, temporal aliasing, and the need for user interaction. In the light of the sensitivity of the phase of the MR signal to local changes in the field stemming from physiological processes, we have developed an unsupervised physiological noise correction method using the information carried in the phase and the magnitude of echo-planar imaging data. Our technique, Physiological Regressor Estimation from Phase and mAgnItude, sub-tR (PREPAIR) derives time series signals sampled at the slice TR from both phase and magnitude images. It allows physiological noise to be captured without aliasing, and efficiently removes other sources of signal fluctuations not related to physiology, prior to regressor estimation. We demonstrate that the physiological signal time courses identified with PREPAIR agree well with those from external devices and retrieve challenging cardiac dynamics. The removal of physiological noise was as effective as that achieved with the most used approach based on external recordings, RETROICOR. In comparison with widely used recording-free physiological noise correction tools-PESTICA and FIX, both performed in unsupervised mode-PREPAIR removed significantly more respiratory and cardiac noise than PESTICA, and achieved a larger increase in temporal signal-to-noise-ratio at both 3 and 7T.

Oxygen Uptake Measurements and Rate of Perceived Exertion during a Marathon.

Although the marathon race has been democratized, it remains complex due to the famous “hitting the wall” phenomenon after the 25th km. To characterize this “wall” from a physiological and Rate of Perceived Exertion (RPE) perspective in recreational marathon runners, we report first continuous breath-by-breath gas exchange measurements during an actual marathon race. In order to test the hypothesis that RPE could be a candidate for controlling the marathon pace, this study examined the relationship between RPE and the physiological variables time course throughout a marathon. Only the respiratory frequency and heart rate increased progressively during the race in all the runners, while the oxygen uptake and ventilatory rate followed different kinetics according the individuals. However, the indexation of the physiological parameters and speed by RPE showed the same decreased tendency for all the runners. In conclusion, these results suggest that running a marathon must be self-paced with the RPE.

Charting the physiological time course of help-seeking during late childhood: Patterns of individual difference

In this study, we explored whether variability in children’s physiological reactivity—respiratory sinus arrhythmia and electrodermal activity—predict concurrent and subsequent levels of children’s observed help-seeking (HS) from their mothers during a failure task. In addition, we tested whether children’s perceptions of maternal positivity pre-task (CPMP) and children’s fearful temperament moderate these effects. In a community sample of 101 mother–child dyads, children (8–12 years of age) underwent a repeated failure task while their respiratory sinus arrythmia and electrodermal activity were monitored; their HS behaviors were later coded. Multilevel path analyses indicated that high-fearful children increased their HS at the same time as and following increased physiological reactivity regardless of CPMP pre-task. Low-fearful children showed increases in HS at the same time as and following increased physiological reactivity only when they perceived their mothers’ affect to be positive. This study demonstrates children’s individual differences in the physiological underpinning of time-linked HS behaviors.

Long-lasting actions of somatostatin on pyramidal cell excitability in the mouse cingulate cortex

Many neurological diseases are related to disturbances of somatostatin- (SOM-) expressing interneurons in the cingulate cortex. Therefore, their role within the circuitry of the cingulate cortex needs to be investigated. We describe here the physiological time course of SOM effects onto pyramidal cell excitability and action potential discharge pattern. Furthermore, we show that the GRK2 inhibitor Gallein had no effect on the reduced SOM-induced response following repetitive SOM applications.

Experience Affects Critical Period Plasticity in the Visual Cortex through an Epigenetic Regulation of Histone Post-Translational Modifications.

While it is known that an epigenetic remodeling of chromatin structure controls developmental plasticity in the visual cortex, three main questions have remained open. Which is the physiological time course of histone modifications? Is it possible, by manipulating the chromatin epigenetic state, to modulate plasticity levels during the critical period? How can we regulate histone acetylation in the adult brain in a noninvasive manner? We show that the early exposure of rat pups to enriching environmental conditions accelerates the critical period for plasticity in the primary visual cortex, linking this effect to increased histone acetylation, specifically at the BDNF gene level. Moreover, we report that the exposure of adult animals to environmental enrichment enhances histone acetylation and reopens juvenile-like plasticity.

Never seem to find the time: evaluating the physiological time course of visual word recognition with regression analysis of single-item event-related potentials

Visual word recognition is a process that, both hierarchically and in parallel, draws on different types of information ranging from perceptual to orthographic to semantic. A central question concerns when and how these different types of information come online and interact after a word form is initially perceived. Numerous studies addressing aspects of this question have been conducted with a variety of techniques [e.g., behaviour, eye-tracking, event-related potentials (ERPs)], and divergent theoretical models, suggesting different overall speeds of word processing, have coalesced around clusters of mostly method-specific results. Here, we examine the time course of influence of variables ranging from relatively perceptual (e.g., bigram frequency) to relatively semantic (e.g., the number of lexical associates) on ERP responses, analysed at the single-item level. Our results, in combination with a critical review of the literature, suggest methodological, analytic and theoretical factors that may have led to inconsistency in results of past studies; we will argue that consideration of these factors may lead to a reconciliation between divergent views of the speed of word recognition.

Absence of Endochondral Ossification and Craniosynostosis in Posterior Frontal Cranial Sutures of Axin2−/− Mice

During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2−/− mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2−/− PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2−/− PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2−/− PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2−/− PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2−/− PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.

Time course of 25(OH)D3 vitamin D3 as well as PTH (parathyroid hormone) during fracture healing of patients with normal and low bone mineral density (BMD)

BackgroundUntil now the exact biochemical processes during healing of metaphyseal fractures of healthy and osteoporotic bone remain unclear. Especially the physiological time courses of 25(OH)D3 (Vitamin D) as well as PTH (Parathyroid Hormone) the most important modulators of calcium and bone homeostasis are not yet examined sufficiently. The purpose of this study was to focus on the time course of these parameters during fracture healing.MethodsIn the presented study, we analyse the time course of 25(OH)D3 and PTH during fracture healing of low BMD level fractures versus normal BMD level fractures in a matched pair analysis. Between March 2007 and February 2009 30 patients older than 50 years of age who had suffered a metaphyseal fracture of the proximal humerus, the distal radius or the proximal femur were included in our study. Osteoporosis was verified by DEXA measuring. The time courses of 25(OH)D3 and PTH were examined over an eight week period. Friedmann test, the Wilcoxon signed rank test and the Mann-Withney U test were used as post-hoc tests. A p-value ≤ 0.05 was considered significant.ResultsSerum levels of 25(OH)D3 showed no differences in both groups. In the first phase of fracture healing PTH levels in the low BMD level group remained below those of the normal BMD group in absolute figures. Over all no significant differences between low BMD level bone and normal BMD level bone could be detected in our study.ConclusionsThe time course of 25(OH)D3 and PTH during fracture healing of patients with normal and low bone mineral density were examined for the first time in humans in this setting and allowing molecular biological insights into fracture healing in metaphyseal bones on a molecural level. There were no significant differences between patients with normal and low BMD levels. Hence further studies will be necessary to obtain more detailed insight into fracture healing in order to provide reliable decision criteria for therapy and the monitoring of fracture healing.

Maturation conditions and boar affect timing of cortical reaction in porcine oocytes

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.

Synchronous acquisition of hyperpolarised 3He and 1H MR images of the lungs – maximising mutual anatomical and functional information

The development of hybrid medical imaging scanners has allowed imaging with different detection modalities at the same time, providing different anatomical and functional information within the same physiological time course with the patient in the same position. Until now, the acquisition of proton MRI of lung anatomy and hyperpolarised gas MRI of lung function required separate breath-hold examinations, meaning that the images were not spatially registered or temporally synchronised. We demonstrate the spatially registered concurrent acquisition of lung images from two different nuclei in vivo. The temporal and spatial registration of these images is demonstrated by a high degree of mutual consistency that is impossible to achieve in separate scans and breath holds.

Different approaches for quantifying ventilation distribution and lung tissue properties by functional EIT

We investigated five different methods which can be applied to quantitatively construct functional tomograms of the lungs. The focus was on the sensitivity of functional tomograms to errors in acquired data. To quantify this sensitivity, theoretical, error-free data sets of well-known properties were artificially generated based on a ‘living thorax model’. Physiological time courses and a typical distribution of errors caused by a typical Goe-MF II EIT system were used for the calculations which encompassed a range up to 50 times greater than the initial error level (4 µVrms max–400 µVrms max). Additionally, low-pass filtering and principal component analysis (PCA) were used to quantify the effect of preprocessing the raw data. The results demonstrate that all methods based on fitting the local to the global time course were superior to the common functional tomograms utilizing standard deviation or maximum and minimum detection. Ventilation distribution was best quantified by the so-called VT methods. Filling capacity—a lung tissue property—was least dependent on increasing error levels. The errors introduced by filtering are significant with respect to a quantitative analysis of ventilation distribution. A preprocessing of raw data by applying a PCA performed well on the data sets which had been constructed but were, nonetheless, realistic. This approach appears to be highly promising for application on real data which is known to be erroneous.

Short Communication: Subcellular Motion Compensation for Minimally Invasive Microscopy, In Vivo

Rationale : In vivo microscopy seeks to observe dynamic subcellular processes in a physiologically relevant context. A primary limitation of optical microscopy in vivo is tissue motion, which prevents physiological time course observations or image averaging. Objective : To develop and demonstrate motion compensation methods that can automatically track image planes within biological tissues, including the tissue displacements associated with large changes in blood flow, and to evaluate the effect of global hypoxia on the regional kinetics and steady state levels of mitochondrial NAD(P)H. Methods and Results : A dynamic optical microscope, with real-time prospective tracking and retrospective image processing, was used collect high-resolution images through cellular responses to various perturbations. The subcellular metabolic response to hypoxia was examined in vivo. Mitochondria closest to the capillaries were significantly more oxidized at rest (67±3%) than the intrafibrillar mitochondria (83±3%; P <0.0001) in the same cell. Conclusions : These data are consistent with the hypothesis that a significant oxygen gradient from capillary to muscle core exists at rest, thereby reducing the oxidative load on the muscle cell.

The systemic angiogenic response during bone healing

Angiogenesis is known to be a critical and closely regulated step during bone formation and fracture healing driven by a complex interaction of various cytokines. Delays in bone healing or even nonunion might therefore be associated with altered concentrations of specific angiogenic factors. These alterations might in turn be reflected by changes in serum concentrations. To determine physiological time courses of angiogenic cytokines during fracture healing as well as possible changes associated with failed consolidation, we prospectively collected serum samples from patients who had sustained surgical treatment for a long bone fracture. Fifteen patients without fracture healing 4 months after surgery (nonunion group) were matched to a collective of 15 patients with successful healing (union group). Serum concentrations of angiogenin (ANG), angiopoietin 2 (Ang-2), basic fibroblast growth factor (bFGF), platelet derived growth factor AB (PDGF-AB), pleiotrophin (PTN) and vascular endothelial growth factor (VEGF) were measured using enzyme linked immunosorbent assays over a period of 24 weeks. Compared to reference values of healthy uninjured controls serum concentrations of VEGF, bFGF and PDGF were increased in both groups. Peak concentrations of these cytokines were reached during early fracture healing. Serum concentrations of bFGF and PDGF-AB were significantly higher in the union group at 2 and 4 weeks after the injury when compared to the nonunion group. Serum concentrations of ANG and Ang-2 declined steadily from the first measurement in normal healing fractures, while no significant changes over time could be detected for serum concentrations of these factures in nonunion patients. PTN serum levels increased asymptotically over the entire investigation in timely fracture healing while no such increase could be detected during delayed healing. We conclude that fracture healing in human subjects is accompanied by distinct changes in systemic levels of specific angiogenic factors. Significant alterations of these physiologic changes in patients developing a fracture nonunion over time could be detected as early as 2 (bFGF) and 4 weeks (PDGF-AB) after initial trauma surgery.

Investigation of spatial resolution, partial volume effects and smoothing in functional MRI using artificial 3D time series

This work addresses the balance between temporal signal-to-noise ratio (tSNR) and partial volume effects (PVE) in functional magnetic resonance imaging (fMRI) and investigates the impact of the choice of spatial resolution and smoothing. In fMRI, since physiological time courses are monitored, tSNR is of greater importance than image SNR. Improving SNR by an increase in voxel volume may be of negligible benefit when physiological fluctuations dominate the noise. Furthermore, at large voxel volumes, PVE are more pronounced, leading to an overall loss in performance. Artificial fMRI time series, based on high-resolution anatomical data, were used to simulate BOLD activation in a controlled manner. The performance was subsequently quantified as a measure of how well the resulted activation matched the simulated activation. The performance was highly dependent on the spatial resolution. At high contrast-to-noise ratio (CNR), the optimal voxel volume was small, i.e. in the region of 23 mm3. It was also shown that using a substantially larger voxel volume in this case could potentially negate the CNR benefits. The optimal smoothing kernel width was dependent on the CNR, being larger at poor CNR. At CNR >1, little or no smoothing proved advantageous. The use of artificial time series gave an opportunity to quantitatively investigate the effects of partial volume and smoothing in single subject fMRI. It was shown that a proper choice of spatial resolution and smoothing kernel width is important for fMRI performance.

Dendritic Enlightenment: Using Patterned Two-Photon Uncaging to Reveal the Secrets of the Brain's Smallest Dendrites

It has been a longstanding challenge for experimentalists to manipulate precisely the spatial and temporal patterns of synaptic input to the dendritic tree in order to mimic activity occurring in the intact brain and determine their importance for synaptic integration. In this issue of Neuron, Losonczy and Magee have used rapid multisite two-photon uncaging of glutamate to define patterns of synaptic input on a submillisecond and micron scale to investigate the rules for summation of synaptic inputs in the fine oblique dendrites of pyramidal neurons.

Differences in time course of ACh and GABA modulation of excitatory synaptic potentials in slices of rat hippocampus.

Activation of muscarinic receptors and GABA(B) receptors causes presynaptic inhibition of glutamatergic synaptic potentials at excitatory feedback connections in cortical structures. These effects may regulate dynamics in cortical structures, with presynaptic inhibition allowing extrinsic afferent input to dominate during encoding, while the absence of presynaptic inhibition allows stronger excitatory feedback during retrieval or consolidation. However, proposals for a functional role of such modulatory effects strongly depend on the time course of these modulatory effects; how rapidly can they turn off and on? In brain slice preparations of hippocampal region CA1, we have explored the time course of suppression of extracellularly recorded synaptic potentials after pressure pulse application of acetylcholine and GABA. Acetylcholine causes suppression of extracellular potentials with onset time constants between 1 and 2 s, and decay constants ranging between 10 and 20 s, even with very brief injection pulses. GABA causes suppression of extracellular potentials with onset time constants between 0.2 and 0.7 s, and decay time constants that decrease to values shorter than 2 s for very brief injection pulses. These techniques do not give an exact measure of the physiological time course in vivo, but they give a notion of the relative time course of the two modulators. The slow changes due to activation of muscarinic acetylcholine receptors may alter the dynamics of cortical circuits over longer intervals (e.g., between different stages of waking and sleep), setting dynamics appropriate for encoding versus consolidation processes. The faster changes in synaptic potentials caused by GABA could cause changes within each cycle of the theta rhythm, rapidly switching between encoding and retrieval dynamics during exploration.

Leptin production in human adipose tissue.

Following the initial confirmation of the existence of leptin in humans by Friedman and his colleagues (Zhang et al. I994), many publications rapidly appeared concerning all aspects of its regulation and action. However, only a small proportion of these have addressed the in vivo physiology of leptin in man. It is apparent that the main determinant of leptin concentrations in both animals and human subjects is whole-body fat mass (Lonnqvist et al. 1995; Maffei et al. 1995; Considine et al. 1996; Bray & York, 1997). The major target for leptin action in rodents is the hypothalamus (Campfield et al. 1995, 1996; Stephens et al. 1995; Tartaglia et al. 1995). The effects of leptin on the human hypothalamus may be inferred from on-going human clinical trials in which leptin is being administered therapeutically to obese subjects, who have either high leptin concentrations (Amgen Inc., Thousand Oaks, CA, USA), or the very rare leptin deficiency states (Montague et al. 1997~). However, many questions remain unanswered regarding the place of leptin in human energy regulation. These include the manner in which different endocrine and paracrine regulators interact in both the acute and longer-term regulation of leptin production, and the physiological time courses of both the regulation and actions of leptin. Furthermore, the extent to which leptin influences energy balance through actions at peripheral target organs requires further clarification (see Cawthome etal. 1998). In the present paper we have focused on the question of the short-term regulation of leptin production in human subjects. Rather than try to cover comprehensively areas of the leptin field that are discussed in detail elsewhere in the present symposium, or in other reviews (Trayhum, 1996; Bray & York, 1997; Halaas & Friedman, 1997), we will seek to focus on: (1) the effect of obesity on leptin production by human adipose tissue; (2) the effect of feeding on leptin production by human adipose tissue; (3) the periodicity of leptin production in human subjects; (4) the effects of sympathomimetic agents on circulating leptin concentrations; (5) the effect of thyroid status on circulating leptin concentrations. Finally, we discuss the possibility that the modulation of the shortterm regulation of leptin may offer therapeutic opportunities in the management of obesity. Before turning to these questions, however, we will first discuss some of the methodological issues concerning the study of human adipose tissue in vivo. Methods for studying human adipose tissue

Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile

A model culture system has been developed whereby individual, primary ovarian mouse follicles can be grown in vitro to the Graafian stage in the normal physiological time course, and then ovulated in response to luteinizing hormone. We report here on the successful fertilization and subsequent embryo development of the oocytes from such follicles. This is the first time that oocytes from in-vitro matured whole follicles have been fertilized and shown to produce viable offspring in host animals. The study demonstrates that the culture system mimics physiological conditions for normal follicle development.