From Pinocytosis to Methuosis-Fluid Consumption as a Risk Factor for Cell Death.

The volumes of a cell [cell volume (CV)] and its organelles are adjusted by osmoregulatory processes. During pinocytosis, extracellular fluid volume equivalent to its CV is incorporated within an hour and membrane area equivalent to the cell's surface within 30 min. Since neither fluid uptake nor membrane consumption leads to swelling or shrinkage, cells must be equipped with potent volume regulatory mechanisms. Normally, cells respond to outwardly or inwardly directed osmotic gradients by a volume decrease and increase, respectively, i.e., they shrink or swell but then try to recover their CV. However, when a cell death (CD) pathway is triggered, CV persistently decreases in isotonic conditions in apoptosis and it increases in necrosis. One type of CD associated with cell swelling is due to a dysfunctional pinocytosis. Methuosis, a non-apoptotic CD phenotype, occurs when cells accumulate too much fluid by macropinocytosis. In contrast to functional pinocytosis, in methuosis, macropinosomes neither recycle nor fuse with lysosomes but with each other to form giant vacuoles, which finally cause rupture of the plasma membrane (PM). Understanding methuosis longs for the understanding of the ionic mechanisms of cell volume regulation (CVR) and vesicular volume regulation (VVR). In nascent macropinosomes, ion channels and transporters are derived from the PM. Along trafficking from the PM to the perinuclear area, the equipment of channels and transporters of the vesicle membrane changes by retrieval, addition, and recycling from and back to the PM, causing profound changes in vesicular ion concentrations, acidification, and-most importantly-shrinkage of the macropinosome, which is indispensable for its proper targeting and cargo processing. In this review, we discuss ion and water transport mechanisms with respect to CVR and VVR and with special emphasis on pinocytosis and methuosis. We describe various aspects of the complex mutual interplay between extracellular and intracellular ions and ion gradients, the PM and vesicular membrane, phosphoinositides, monomeric G proteins and their targets, as well as the submembranous cytoskeleton. Our aim is to highlight important cellular mechanisms, components, and processes that may lead to methuotic CD upon their derangement.

Trypan Blue - Adapting a Dye Used for Labelling Dead Cells to Visualize Pinocytosis in Viable Cells.

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

Effect of Glycine on BV-2 Microglial Cells Treated with Interferon-γ and Lipopolysaccharide.

Microglia are first-line defense antigen-presenting phagocytes in the central nervous system. Activated microglial cells release pro-inflammatory cytokines and can trigger an oxidative burst. The amino acid glycine exerts anti-inflammatory, immunomodulatory and cytoprotective effects and influences cell volume regulation. This study aimed to investigate the role of glycine in the modulation of inflammatory processes in mouse BV-2 microglial cells. Inflammatory stress was induced by lipopolysaccharide/interferon-γ (LPS/IFN-γ) treatment for 24 h in the absence or presence of 1 or 5 mM glycine. Cells were analyzed by flow cytometry for cell volume, side scatter, apoptosis/necrosis and expression of activation-specific surface markers. Apoptosis progression was monitored by life cell imaging. Reduced glutathione/oxidized glutathione (GSH/GSSG) ratios and release of the pro-inflammatory cytokines IL-6 and TNF-α were measured using luminescence-based assays and ELISA, respectively. We found that LPS/IFN-γ-induced apoptosis was decreased and the fraction of living cells was increased by glycine. Expression of the surface markers CD11b, CD54 and CD80 was dose-dependently increased, while IL-6 and TNF-α release was not altered compared to LPS/IFN-γ-treated cells. We showed that in BV-2 microglial cells glycine improves viability and counteracts deleterious responses to LPS/IFN-γ, which might be relevant in neurodegenerative processes associated with inflammation, like Alzheimer's or Parkinson's disease.

A Swelling-Activated Chloride Current in Microglial Cells is Suppressed by Epac and Facilitated by PKA - Impact on Phagocytosis.

BACKGROUND/AIMS: Volume-regulated anion channels (VRACs) are of particular importance in regulating the cell volume (CV) and give rise to the swelling-activated Cl- current (ICl,swell), a main component driving global regulatory volume decrease (RVD) during cell swelling. Because ICl,swell affects numerous CV-regulated processes like migration, we assume that its role is also indispensable for phagocytosis which requires local cell swelling. Noradrenaline (NA) modulates phagocytosis in macrophages and microglial cells, macrophage-related cells in the central nervous system. Therefore we evaluated whether NA modulates ICl,swell and phagocytosis in microglia. METHODS: Experiments were performed in murine microglial BV-2 and primary mouse microglial cells. Patch clamp experiments were performed in BV-2 cells using the amphotericin-perforated method to minimize cytosolic disturbances. Phagocytosis was quantified by scanning electron microscopy. RESULTS: Following activation of ICl,swell by a hypotonic bath solution, noradrenaline, as well as the ß-adrenergic agonist isoproterenol, evoked a transient decrease of ICl,swell. Repeated application of adrenergic agonists caused a decline of this electrical response. Application of the agonist of exchange protein directly activated by cAMP (Epac), 8-pCPT-2-O-Me-cAMP, or the protein kinase A inhibitor H89 caused a persistent suppression of ICl,swell. When isoproterenol was added concomitantly with the hypotonic saline, ICl,swell developed more rapidly compared to control conditions. Uptake of IgG-coated beads was suppressed by NA or H89 when quantified after 15 min of exposure. CONCLUSION: The activation of ß-adrenergic receptors in microglial cells triggers a cAMP-Epac-dependent and a cAMP-PKA-dependent cascade which affects phagocytosis via modulation of the swelling-activated Cl- current ICl,swell.

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling.

BACKGROUND/AIMS: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. METHODS: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (Vmem) and swelling-dependent chloride (Cl-) currents (IClswell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. RESULTS: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl- current under isotonic conditions resembling features of the swelling-activated Cl- current (IClswell). The cell membrane potential (Vmem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na+-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. CONCLUSION: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Sex, age, and sex hormones affect recall of words in a directed forgetting paradigm.

During the course of serious discussion, an unexpected interruption may induce forgetting of the original topic of a conversation. Sex, age, and sex hormone levels may affect frequency and extension of forgetting. In a list-method directed forgetting paradigm, subjects have to learn two word lists. After learning list 1, subjects receive either a forget or a remember list 1 cue. When the participants had learned list 2 and completed a distraction task, they were asked to write down as many recalled items as possible, starting either with list 1 or list 2 items. In the present study, 96 naturally cycling women, 60 oral contraceptive users, 56 postmenopausal women, and 41 young men were assigned to one of these different experimental conditions. Forget-cued young subjects recall fewer list 1 items (list 1 forgetting) but more list 2 items (list 2 enhancement) compared with remember-cued subjects. However, forget-cued postmenopausal women showed reduced list 1 forgetting but enhanced list 2 retention. Remember-cued naturally cycling women recalled more list 1 items than oral contraceptive users, young men, and postmenopausal women. In forget-cued follicular women, salivary progesterone correlated positively with recalled list 2 items. Salivary 17ß-estradiol did not correlate with recalled list 1 or list 2 items in either remember- or forget-cued young women. However, salivary 17ß-estradiol correlated with item recall in remember-cued postmenopausal women. Our findings suggest that sex hormones do not globally modulate verbal memory or forgetting, but selectively affect cue-specific processing. © 2016 Wiley Periodicals, Inc.

Components of Mathematics Anxiety: Factor Modeling of the MARS30-Brief.

Mathematics anxiety involves feelings of tension, discomfort, high arousal, and physiological reactivity interfering with number manipulation and mathematical problem solving. Several factor analytic models indicate that mathematics anxiety is rather a multidimensional than unique construct. However, the factor structure of mathematics anxiety has not been fully clarified by now. This issue shall be addressed in the current study. The Mathematics Anxiety Rating Scale (MARS) is a reliable measure of mathematics anxiety (Richardson and Suinn, 1972), for which several reduced forms have been developed. Most recently, a shortened version of the MARS (MARS30-brief) with comparable reliability was published. Different studies suggest that mathematics anxiety involves up to seven different factors. Here we examined the factor structure of the MARS30-brief by means of confirmatory factor analysis. The best model fit was obtained by a six-factor model, dismembering the known two general factors "Mathematical Test Anxiety" (MTA) and "Numerical Anxiety" (NA) in three factors each. However, a more parsimonious 5-factor model with two sub-factors for MTA and three for NA fitted the data comparably well. Factors were differentially susceptible to sex differences and differences between majors. Measurement invariance for sex was established.

Behavioural evidence for sex differences in the overlap between subtraction and multiplication.

The present study aims to identify factors that may influence the dissociability of number magnitude processing and arithmetic fact retrieval at the behavioural level. To that end, we assessed both subtraction and multiplication performance in a within-subject approach and evaluated the interdependence of unit-decade integration measures on the one hand as well as sex differences in the interdependence of performance measures on the other hand. We found that subtraction items requiring borrowing (e.g. 53-29 = 24, 3 < 9) are more error prone than subtraction items not requiring borrowing (e.g. 59-23 = 34, 9 > 3), thereby demonstrating a borrowing effect, which has been suggested as a measure of unit-decade integration in subtraction. Furthermore, we observed that multiplication items with decade-consistent distractors (e.g. 6 × 4 = 28 instead of 24) are more error prone that multiplication items with decade-inconsistent distractors (e.g. 6 × 4 = 30 instead of 24), thereby demonstrating a decade-consistency effect, which has been suggested as a measure of unit-decade integration in simple multiplication. However, the borrowing effect in subtraction was not correlated with the effect of decade consistency in simple multiplication in either men or women. This indicates that unit-decade integration arises from different systems in subtraction and multiplication. Nevertheless, men outperformed women not only in subtraction, but also in multiplication. Furthermore, subtraction and multiplication performance on correct solution probes were correlated in women, but unrelated in men. Thus, the view of differential systems for number magnitude processing and arithmetic fact retrieval may not be universal across sexes.

Intentional updating in episodic memory: Low testosterone associates with enhanced memory updating.

OBJECTIVES: Although there is consensus that sex hormones modulate memory, we have an incomplete understanding of their role in remembering and forgetting. Humans continuously update memory, forgetting old, out-of-date information and encoding new, more relevant information. Updating processes can be studied with the list method of directed forgetting. METHODS: In the list method of directed forgetting task, subjects study two lists of items and, after study of list 1, are asked to either forget or remember the list for an upcoming memory test. Free testosterone level was quantified from saliva samples. Directed forgetting and saliva testosterone were evaluated in young men (aged between 18 and 28 years). RESULTS: Following a forget cue, recall of list-1 items was reduced and recall of list-2 items was enhanced. However, only recall of list-2 items was associated with free testosterone level. Following a forget cue, participants with low testosterone levels showed higher recall of list-2 items than participants with high testosterone levels. CONCLUSION: The selective association between testosterone level and list-2 recall is consistent with two-mechanism accounts of memory updating, where the forgetting effect is due to impaired retrieval and the enhancement effect to improved encoding. On the basis of this view, the present results indicate that low testosterone levels are associated with improved binding of the newly encoded memories to their context cue.

Prestimulus amplitudes modulate P1 latencies and evoked traveling alpha waves.

Traveling waves have been well documented in the ongoing, and more recently also in the evoked EEG. In the present study we investigate what kind of physiological process might be responsible for inducing an evoked traveling wave. We used a semantic judgment task which already proved useful to study evoked traveling alpha waves that coincide with the appearance of the P1 component. We found that the P1 latency of the leading electrode is significantly correlated with prestimulus amplitude size and that this event is associated with a transient change in alpha frequency. We assume that cortical background excitability, as reflected by an increase in prestimulus amplitude, is responsible for the observed change in alpha frequency and the initiation of an evoked traveling trajectory.

Mathematics anxiety reduces default mode network deactivation in response to numerical tasks.

Mathematics anxiety is negatively related to mathematics performance, thereby threatening the professional success. Preoccupation with the emotional content of the stimuli may consume working memory resources, which may be reflected in decreased deactivation of areas associated with the default mode network (DMN) activated during self-referential and emotional processing. The common problem is that math anxiety is usually associated with poor math performance, so that any group differences are difficult to interpret. Here we compared the BOLD-response of 18 participants with high (HMAs) and 18 participants with low mathematics anxiety (LMAs) matched for their mathematical performance to two numerical tasks (number comparison, number bisection). During both tasks, we found stronger deactivation within the DMN in LMAs compared to HMAs, while BOLD-response in task-related activation areas did not differ between HMAs and LMAs. The difference in DMN deactivation between the HMA and LMA group was more pronounced in stimuli with additional requirement on inhibitory functions, but did not differ between number magnitude processing and arithmetic fact retrieval.

Progesterone-associated increase in ERP amplitude correlates with an improvement in performance in a spatial attention paradigm.

Ovarian sex hormones modulate neuronal circuits not directly involved in reproductive functions. In the present study, we investigated whether endogenous fluctuations of estradiol and progesterone during the menstrual cycle are associated with early cortical processing stages in a cued spatial attention paradigm. EEG was monitored while young women responded to acoustically cued visual stimuli. Women with large mean amplitude of the event-related potential (ERP) (80-120 ms following visual stimuli) responded faster to visual stimuli. In luteal women, mean amplitude of the ERP as well as alpha amplitude, an indicator of attentional modulation, correlated positively with progesterone. Further, cerebral asymmetry in ERP amplitude in the alpha frequency band following target presentation was restricted to luteal women. Critically, early follicular women responded slower to right hemifield compared to left hemifield targets. In late follicular or luteal women, we did not detect a right hemifield disadvantage. Progesterone correlated negatively with RTs in luteal women. Therefore, whereas our behavioral data indicate a functional cerebral asymmetry in early follicular women, EEG recording reveal a physiological cerebral hemisphere asymmetry in the alpha frequency band in luteal women. We assume that a progesterone-associated enhancement in synchronization of synaptic activity in the alpha frequency band in luteal women improves early categorization of visual targets in a cued spatial attention paradigm.

50 years of hormonal contraception-time to find out, what it does to our brain.

Hormonal contraceptives are on the market for more than 50 years and used by 100 million women worldwide. However, while endogenous steroids have been convincingly associated with change in brain structure, function and cognitive performance, the effects of synthetic steroids contained in hormonal contraceptives on brain and cognition have barely been investigated. In this article we summarize the sparse findings, describing brain structural, functional and behavioral findings from the literature and suggest that synthetic steroids may contribute to masculinizing as well as feminizing effects on brain and behavior. We try to identify methodological challenges, explain, how results on endogenous steroids may transfer into research on hormonal contraceptives and point out factors that need to be controlled in the study of hormonal contraceptive dependent effects. We conclude that there is a strong need for more systematic studies, especially on brain structural, functional and cognitive changes due to hormonal contraceptive use. The hormonal contraceptive pill is the major tool for population control. Hence, such behavioral changes could cause a shift in society dynamics and should not stay unattended.

Resting state alpha frequency is associated with menstrual cycle phase, estradiol and use of oral contraceptives.

Ongoing intrinsic brain activity in resting, but awake humans is dominated by alpha oscillations. In human, individual alpha frequency (IAF) is associated with cognitive performance. Noticeable, performance in cognitive and emotional tasks in women is associated with menstrual cycle phase and sex hormone levels, respectively. In the present study, we correlated frequency of alpha oscillation in resting women with menstrual cycle phase, sex hormone level, or use of oral contraceptives. Electroencephalogram (EEG) was recorded from 57 women (aged 24.07 ± 3.67 years) having a natural menstrual cycle as well as from 57 women (aged 22.37 ± 2.20 years) using oral contraceptives while they sat in an armchair with eyes closed. Alpha frequency was related to the menstrual cycle phase. Luteal women showed highest and late follicular women showed lowest IAF or center frequency. Furthermore, IAF as well as center frequency correlated negatively with endogenous estradiol level, but did not reveal an association with endogenous progesterone. Women using oral contraceptives showed an alpha frequency similar to women in the early follicular phase. We suggest that endogenous estradiol modulate resting alpha frequency.

Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.

From the nucleus to the plasma membrane: translocation of the nuclear proteins histone H3 and lamin B1 in apoptotic microglia.

Nuclear autoantibodies have been found in patients with autoimmune diseases. One possible source for nuclear antigens are apoptotic cells. However, the mechanism of how apoptotic cells make nuclear factors accessible to the immune system is still elusive. In the present study, we investigated the redistribution of nuclear components after UV irradiation in the microglial cell line BV-2 and in primary mouse microglia at the ultrastructural level. We used transmission electron microscopy-coupled electron energy loss spectroscopy (EELS) to measure phosphorus as an indicator for nucleic acids and immunogold labeling to detect histone H3 and lamin B1 in apoptotic cells. EELS revealed elevated concentrations of phosphorus in nuclear and cytoplasmic condensed chromatin compared to the remaining cytoplasm. Furthermore, immunolabeling of lamin B1 and histone H3 was detected in apoptotic microglia not only in the nucleus, but also in the cytoplasm, and even at the plasma membrane. Confocal images of apoptotic microglia, which were not previously permeabilized, showed patches of histone H3 and lamin B1 labeling at the cell surface. The pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) prevented the occurrence of cytoplasmic condensed chromatin in apoptotic microglia. Our findings indicate that nuclear components leak from the nucleus into the cytoplasm in apoptotic microglia. At least histone H3 and lamin B1 reach the cell surface, this may promote autoreactive processes.

Chloride channel blockers suppress formation of engulfment pseudopodia in microglial cells.

BACKGROUND/AIMS: Phagocytosis depends on the formation of engulfment pseudopodia surrounding the target. We tested in microglia, monocyte-derived cells in the brain, whether a swelling-activated Cl(-)-current (I(Cl,swell)), required for global cell volume (CV) regulation, also contributes to local expansion and retraction of engulfment pseudopodia. METHODS: We used scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to visualize and quantify the uptake of polystyrene microbeads (MBs) by microglial cells. Flow cytometry was used for cell volume measurments and I(Cl,swell) was measured by whole-cell patch clamp. RESULTS: We found that exposure of microglial BV-2 cells to MBs in Cl(-)-free extracellular solution attenuated MB uptake and that the Cl(-)-channel blockers DIOA, flufenamic acid, NPPB and DCPIB suppressed the uptake of MBs in BV-2 cells and in primary microglial cells. Microglial cells exposed to MBs in the presence of Cl(-) channel blockers failed to extend engulfment pseudopodia. We observed that cells containing at least three MBs revealed an about twofold increase in current density of I(Cl, swell) compared to cells without MB. Osmotic challenges to stimulate global CV regulation before exposure to MBs modulated phagocytosis. Pre-conditioning of cells in hypo- or hypertonic medium for 12-16 hours caused a decrease in MB uptake. CONCLUSION: These findings indicate that I(Cl,swell) contributes to formation of engulfment pseudopodia and participates in engulfment and particle uptake in microglial cells.

Perturbation of intracellular K(+) homeostasis with valinomycin promotes cell death by mitochondrial swelling and autophagic processes.

Perturbation of cellular K(+) homeostasis is a common motif in apoptosis but it is unknown whether a decrease in intracellular K(+) alone is sufficient to replicate apoptotic hallmarks. We investigated, which mode of cell death is induced by decreasing the intracellular K(+) concentration using valinomycin, a highly K(+)-selective ionophore. Valinomycin treatment induced mitochondrial swelling and minor nuclear changes in cell lines (BV-2, C6, HEK 293), and in primary mouse microglia and astrocytes. In the microglial cell line BV-2, we identified and quantified three phenotypes in valinomycin-exposed cells. The first and most prevalent phenotype (62 ± 2%) was characterized by swollen mitochondria and no chromatin condensation, and the second (25 ± 3%) by swollen mitochondria and slight chromatin condensation. Only the third phenotype (11 ± 4%) fulfilled criteria of apoptosis by having normal-sized mitochondria and strongly condensed chromatin. Valinomycin-induced swelling of mitochondria was not altered by the adenine nucleotide translocase inhibitor bongkrekic acid (BA), the pan caspase inhibitor Z-VAD-FMK, changing extracellular K(+) or Cl(-) concentrations, or the membrane-permeable Ca(2+) chelator BAPTA-AM. Only co-exposure of cells to valinomycin and the Ca(2+) ionophore ionomycin in high K(+) Cl(-)-free extracellular solution suppressed mitochondrial swelling. Ionomycin alone caused shrinkage of mitochondria. Additionally, valinomycin promoted autophagic processes, which were further enhanced by preincubation with BA or with Z-VAD-FMK. Valinomycin-dependent chromatin condensation was inhibited by BA, Z-VAD-FMK, BAPTA-AM, and ionomycin. Our findings demonstrate that mitochondrial swelling and autophagy are common features of valinomycin-exposed cells. Accordingly, valinomycin promotes an autophagic cell death mode, but not apoptosis.

Beta-adrenergic stimulation suppresses phagocytosis via Epac activation in murine microglial cells.

Endogenous noradrenaline presumably prohibits neuroinflammation by stimulation of ß-adrenergic receptor-dependent suppression of the production of inflammatory mediators. Using the microglial cell line, BV-2, as well as primary murine microglial cells, we show here that the ß-adrenergic agonist, isoproterenol, suppresses uptake of hydrophobic polystyrene microspheres. The number of cells showing a specific number of engulfed microspheres followed a Poisson distribution. Isoproterenol decreased the number of engulfed particles per cell and the number of cells showing at least one incorporated particle. Elevation of intracellular cAMP by activation of adenylyl cyclase activity with forskolin, suppression of phosphodiesterase activity with 3-isobutyl-1-methylxanthine (IBMX), or application of the membrane-permeable cAMP analog, 8-bromo-cAMP, suppressed particle uptake. The protein kinase A inhibitor, H-89, did not prevent isoproterenol-dependent suppression of particle engulfment. However, activation of exchange protein activated by cAMP (Epac), specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, with the Epac1-specific cAMP analog, 8-pCPT-2'-O-Me-cAMP, mimicked the suppressive effect of isoproterenol on particle uptake. Our results suggest that ß-adrenergic receptor stimulation suppresses particle uptake in microglia by cAMP-dependent activation of Epac.

Predictors of performance in a real-life statistics examination depend on the individual cortisol profile.

In standardized stress conditions, cortisol responders and non-responders differ in their memory performance. To evaluate this association in a real-life situation, we investigated whether the individual cortisol response profile predicts the performance in an examination in statistics in 112 healthy participants. In addition, basic arithmetic abilities as well as mathematics anxiety were evaluated. We collected a baseline saliva sample before the examination day as well as pre- and post-examination samples. Participants were attributed to one of four cortisol profiles depending on their cortisol change in anticipation of the examination. 52% of participants showed an increase (A-profile) and 43% a decrease (D-profile) in cortisol before the examination. Cortisol changes did not predict performance in the examination. However, only in A-profile individuals, mathematics anxiety and arithmetic abilities predicted statistics performance. We conclude that in real-life situations, cortisol facilitates the influence of mathematics anxiety and mathematical abilities on examination performance.