A novel optical intracellular imaging approach for potassium dynamics in astrocytes.

Astrocytes fulfill a central role in regulating K+ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na+ ions and one proton and the efflux of one K+ ion. Thus, intracellular K+ concentration ([K+]i) is potentially influenced both by extracellular K+ concentration ([K+]o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K+ ion movements on [K+]i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K+]i using the recently developed K+ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K+]i at 133±1 mM. We first investigated the dependency of [K+]i levels on [K+]o. We found that [K+]i followed [K+]o changes nearly proportionally in the range 3-10 mM, which is consistent with previously reported microelectrode measurements of intracellular K+ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K+]i that depended on the glutamate concentration with an apparent EC50 of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K+]i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K+ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K+]i regulation with excellent spatial resolution

Control of Glutamate Transport by Extracellular Potassium: Basis for a Negative Feedback on Synaptic Transmission.

Glutamate and K+, both released during neuronal firing, need to be tightly regulated to ensure accurate synaptic transmission. Extracellular glutamate and K+ ([K+]o) are rapidly taken up by glutamate transporters and K+-transporters or channels, respectively. Glutamate transport includes the exchange of one glutamate, 3 Na+, and one proton, in exchange for one K+. This K+ efflux allows the glutamate binding site to reorient in the outwardly facing position and start a new transport cycle. Here, we demonstrate the sensitivity of the transport process to [K+]o changes. Increasing [K+]o over the physiological range had an immediate and reversible inhibitory action on glutamate transporters. This K+-dependent transporter inhibition was revealed using microspectrofluorimetry in primary astrocytes, and whole-cell patch-clamp in acute brain slices and HEK293 cells expressing glutamate transporters. Previous studies demonstrated that pharmacological inhibition of glutamate transporters decreases neuronal transmission via extrasynaptic glutamate spillover and subsequent activation of metabotropic glutamate receptors (mGluRs). Here, we demonstrate that increasing [K+]o also causes a decrease in neuronal mEPSC frequency, which is prevented by group II mGluR inhibition. These findings highlight a novel, previously unreported physiological negative feedback mechanism in which [K+]o elevations inhibit glutamate transporters, unveiling a new mechanism for activity-dependent modulation of synaptic activity

Extracellular Potassium and Glutamate Interact To Modulate Mitochondria in Astrocytes.

Astrocytes clear glutamate and potassium, both of which are released into the extracellular space during neuronal activity. These processes are intimately linked with energy metabolism. Whereas astrocyte glutamate uptake causes cytosolic and mitochondrial acidification, extracellular potassium induces bicarbonate-dependent cellular alkalinization. This study aimed at quantifying the combined impact of glutamate and extracellular potassium on mitochondrial parameters of primary cultured astrocytes. Glutamate in 3 mM potassium caused a stronger acidification of mitochondria compared to cytosol. 15 mM potassium caused alkalinization that was stronger in the cytosol than in mitochondria. While the combined application of 15 mM potassium and glutamate led to a marked cytosolic alkalinization, pH only marginally increased in mitochondria. Thus, potassium and glutamate effects cannot be arithmetically summed, which also applies to their effects on mitochondrial potential and respiration. The data implies that, because of the nonlinear interaction between the effects of potassium and glutamate, astrocytic energy metabolism will be differentially regulated

Modeling the Subsurface Structure of Sunspots

While sunspots are easily observed at the solar surface, determining their subsurface structure is not trivial. There are two main hypotheses for the subsurface structure of sunspots: the monolithic model and the cluster model. Local helioseismology is the only means by which we can investigate subphotospheric structure. However, as current linear inversion techniques do not yet allow helioseismology to probe the internal structure with sufficient confidence to distinguish between the monolith and cluster models, the development of physically realistic sunspot models are a priority for helioseismologists. This is because they are not only important indicators of the variety of physical effects that may influence helioseismic inferences in active regions, but they also enable detailed assessments of the validity of helioseismic interpretations through numerical forward modeling. In this paper, we provide a critical review of the existing sunspot models and an overview of numerical methods employed to model wave propagation through model sunspots. We then carry out an helioseismic analysis of the sunspot in Active Region 9787 and address the serious inconsistencies uncovered by \citeauthor{gizonetal2009}~(\citeyear{gizonetal2009,gizonetal2009a}). We find that this sunspot is most probably associated with a shallow, positive wave-speed perturbation (unlike the traditional two-layer model) and that travel-time measurements are consistent with a horizontal outflow in the surrounding moat.Comment: 73 pages, 19 figures, accepted by Solar Physic

An Observational Overview of Solar Flares

We present an overview of solar flares and associated phenomena, drawing upon a wide range of observational data primarily from the RHESSI era. Following an introductory discussion and overview of the status of observational capabilities, the article is split into topical sections which deal with different areas of flare phenomena (footpoints and ribbons, coronal sources, relationship to coronal mass ejections) and their interconnections. We also discuss flare soft X-ray spectroscopy and the energetics of the process. The emphasis is to describe the observations from multiple points of view, while bearing in mind the models that link them to each other and to theory. The present theoretical and observational understanding of solar flares is far from complete, so we conclude with a brief discussion of models, and a list of missing but important observations.Comment: This is an article for a monograph on the physics of solar flares, inspired by RHESSI observations. The individual articles are to appear in Space Science Reviews (2011

Imaging extracellular potassium dynamics in brain tissue using a potassium-sensitive nanosensor.

Neuronal activity results in the release of [Formula: see text] into the extracellular space (ECS). Classically, measurements of extracellular [Formula: see text] ([Formula: see text]) are carried out using [Formula: see text]-sensitive microelectrodes, which provide a single point measurement with undefined spatial resolution. An imaging approach would enable the spatiotemporal mapping of [Formula: see text]. Here, we report on the design and characterization of a fluorescence imaging-based [Formula: see text]-sensitive nanosensor for the ECS based on dendrimer nanotechnology. Spectral characterization, sensitivity, and selectivity of the nanosensor were assessed by spectrofluorimetry, as well as in both wide-field and two-photon microscopy settings, demonstrating the nanosensor efficacy over the physiologically relevant ion concentration range. Spatial and temporal kinetics of the nanosensor responses were assessed using a localized iontophoretic [Formula: see text] application on a two-photon imaging setup. Using acute mouse brain slices, we demonstrate that the nanosensor is retained in the ECS for extended periods of time. In addition, we present a ratiometric version of the nanosensor, validate its sensitivity in brain tissue in response to elicited neuronal activity and correlate the responses to the extracellular field potential. Together, this study demonstrates the efficacy of the [Formula: see text]-sensitive nanosensor approach and validates the possibility of creating multimodal nanosensors