Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Methyl-β-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids

Anti-angiogenic effect of high doses of ascorbic acid

Pharmaceutical doses of ascorbic acid (AA, vitamin C, or its salts) have been reported to exert anticancer activity in vitro and in vivo. One proposed mechanism involves direct cytotoxicity mediated by accumulation of ascorbic acid radicals and hydrogen peroxide in the extracellular environment of tumor cells. However, therapeutic effects have been reported at concentrations insufficient to induce direct tumor cell death. We hypothesized that AA may exert anti-angiogenic effects. To test this, we expanded endothelial progenitor cells (EPCs) from peripheral blood and assessed, whether or not high dose AA would inhibit EPC ability to migrate, change energy metabolism, and tube formation ability. We also evaluated the effects of high dose AA on angiogenic activities of HUVECs (human umbilical vein endothelial cells) and HUAECs (human umbilical arterial endothelial cells). According to our data, concentrations of AA higher than 100 mg/dl suppressed capillary-like tube formation on Matrigel for all cells tested and the effect was more pronounced for progenitor cells in comparison with mature cells. Co-culture of differentiated endothelial cells with progenitor cells showed that there was incorporation of EPCs in vessels formed by HUVECs and HUAECs. Cell migration was assessed using an in vitro wound healing model. The results of these experiments showed an inverse correlation between AA concentrations relative to both cell migration and gap filling capacity. Suppression of NO (nitric oxide) generation appeared to be one of the mechanisms by which AA mediated angiostatic effects. This study supports further investigation into non-cytotoxic antitumor activities of AA

Affective regulation of cognitive-control adjustments in remitted depressive patients after acute tryptophan depletion

Negative affect in healthy populations regulates the appraisal of demanding situations, which tunes subsequent effort mobilization and adjustments in cognitive control. In the present study, we hypothesized that dysphoria in depressed individuals similarly modulates this adaptation, possibly through a neural mechanism involving serotonergic regulation. We tested the effect of dysphoria induced by acute tryptophan depletion (ATD) in remitted depressed patients on conflict adaptation in a Simon task. ATD temporarily lowers the availability of the serotonin precursor L-Tryptophan and is known to increase depressive symptoms in approximately half of remitted depressed participants. We found that depressive symptoms induced by ATD were associated with increased conflict adaptation. Our finding extends recent observations implying an important role of affect in regulating conflict-driven cognitive control

Context specificity of post-error and post-conflict cognitive control adjustments

There has been accumulating evidence that cognitive control can be adaptively regulated by monitoring for processing conflict as an index of online control demands. However, it is not yet known whether top-down control mechanisms respond to processing conflict in a manner specific to the operative task context or confer a more generalized benefit. While previous studies have examined the taskset-specificity of conflict adaptation effects, yielding inconsistent results, controlrelated performance adjustments following errors have been largely overlooked. This gap in the literature underscores recent debate as to whether post-error performance represents a strategic, control-mediated mechanism or a nonstrategic consequence of attentional orienting. In the present study, evidence of generalized control following both high conflict correct trials and errors was explored in a task-switching paradigm. Conflict adaptation effects were not found to generalize across tasksets, despite a shared response set. In contrast, post-error slowing effects were found to extend to the inactive taskset and were predictive of enhanced post-error accuracy. In addition, post-error performance adjustments were found to persist for several trials and across multiple task switches, a finding inconsistent with attentional orienting accounts of post-error slowing. These findings indicate that error-related control adjustments confer a generalized performance benefit and suggest dissociable mechanisms of post-conflict and post-error control. © 2014 Forster, Cho

Acute effects of MDMA (3,4-methylenedioxymethamphetamine) on EEG oscillations: alone and in combination with ethanol or THC (delta-9-tetrahydrocannabinol)

Item does not contain fulltextRATIONALE: Typical users of 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") are polydrug users, combining MDMA with alcohol or cannabis [most active compound: delta-9-tetrahydrocannabinol (THC)]. OBJECTIVES: The aim of the present study was to investigate whether co-administration of alcohol or THC with MDMA differentially affects ongoing electroencephalogram (EEG) oscillations compared to the administration of each drug alone. METHODS: In two separate experiments, 16 volunteers received four different drug conditions: (1) MDMA (100 mg); (2) alcohol clamp (blood alcohol concentration = 0.6 per thousand) or THC (inhalation of 4, 6 and 6 mg, interval of 1.5 h); (3) MDMA in combination with alcohol or THC; and (4) placebo. Before and after drug administration, electroencephalography was recorded during an eyes closed resting state. RESULTS: Theta and alpha power increased after alcohol intake compared to placebo and reduced after MDMA intake. No interaction between alcohol and MDMA was found. Significant MDMA x THC effects for theta and lower-1-alpha power indicated that the power attenuation after the combined intake of MDMA and THC was less than the sum of each drug alone. For the lower-2-alpha band, the intake of MDMA or THC alone did not significantly affect power, but the intake of combined MDMA and THC significantly decreased lower-2-alpha power. CONCLUSIONS: The present findings indicate that the combined intake of MDMA and THC, but not of MDMA and alcohol, affects ongoing EEG oscillations differently than the sum of either one drug alone. Changes in ongoing EEG oscillations may be related to the impaired task performance that has often been reported after drug intake

The Dopamine Augmenter L-DOPA Does Not Affect Positive Mood in Healthy Human Volunteers

Dopamine neurotransmission influences approach toward rewards and reward-related cues. The best cited interpretation of this effect proposes that dopamine mediates the pleasure that commonly accompanies reward. This hypothesis has received support in some animal models and a few studies in humans. However, direct assessments of the effect of transiently increasing dopamine neurotransmission have been largely limited to the use of psychostimulant drugs, which elevate brain levels of multiple neurotransmitters in addition to dopamine. In the present study we tested the effect of more selectively elevating dopamine neurotransmission, as produced by administration of the immediate dopamine precursor, L-DOPA (0, 100/25, 200/50 mg, Sinemet), in healthy human volunteers. Neither dose altered positive mood. The results suggest that dopamine neurotransmission does not directly influence positive mood in humans

Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells

Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy

Spatio-Temporal Brain Mapping of Motion-Onset VEPs Combined with fMRI and Retinotopic Maps

Neuroimaging studies have identified several motion-sensitive visual areas in the human brain, but the time course of their activation cannot be measured with these techniques. In the present study, we combined electrophysiological and neuroimaging methods (including retinotopic brain mapping) to determine the spatio-temporal profile of motion-onset visual evoked potentials for slow and fast motion stimuli and to localize its neural generators. We found that cortical activity initiates in the primary visual area (V1) for slow stimuli, peaking 100 ms after the onset of motion. Subsequently, activity in the mid-temporal motion-sensitive areas, MT+, peaked at 120 ms, followed by peaks in activity in the more dorsal area, V3A, at 160 ms and the lateral occipital complex at 180 ms. Approximately 250 ms after stimulus onset, activity fast motion stimuli was predominant in area V6 along the parieto-occipital sulcus. Finally, at 350 ms (100 ms after the motion offset) brain activity was visible again in area V1. For fast motion stimuli, the spatio-temporal brain pattern was similar, except that the first activity was detected at 70 ms in area MT+. Comparing functional magnetic resonance data for slow vs. fast motion, we found signs of slow-fast motion stimulus topography along the posterior brain in at least three cortical regions (MT+, V3A and LOR)