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Properties of the new fluorescent Na+ indicator CoroNa Green: Comparison with SBFI and confocal Na+ imaging
Abstract
Neuronal activity causes substantial Na+ transients in fine cellular processes such as dendrites and spines. The physiological consequences of such Na+ transients are still largely unknown. High-resolution Na+ imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) has been the primary method of choice. Recently, a new Na+ indicator dye, CoroNa Green (CoroNa), that has its absorbance maximum at 492 nm, has become available. In the present study, we have compared the properties of SBFI with those of CoroNa by performing Na+ measurements in neurons of hippocampal slices. We show that CoroNa is suitable for measurement of Na+ transients using non-confocal wide-field imaging with a CCD camera. However, substantial transmembrane dye leakage and lower Na+ sensitivity are clearly disadvantages when compared to SBFI. We also tested CoroNa for its suitability for high-resolution imaging of Na+ transients using a confocal laser scanning system. We demonstrate that CoroNa, in contrast to SBFI, can be employed for confocal imaging using a conventional argon laser and report the first Na+ measurements in dendrites using this dye. In conclusion, CoroNa may prove to be a valuable tool for confocal Na+ imaging in fine cellular processes.
... After obtaining at least two complete images (each at 20 frames) under baseline conditions, cells were exposed to salines containing (in mM): 10 HEPES, 26 K + -gluconate, 0-150 [Na + ], 0-150 [K + ] (NaCl + KCl = 150 mM); pH titrated to 7.3 with KOH. Cell plasma membranes were permeabilized for Na + by additionally using 3 µM gramicidin (Na + -ionophore; Sigma-Aldrich Chemie), 10 µM monensin (Na + /H + -exchanger; Sigma-Aldrich Chemie), and 1 mM ouabain (NKA inhibitor; Calbiochem, Merck KGaA; Rose and Ransom, 1996a;Meier et al., 2006). ...
... CoroNaGreen is a fluorescent dye suitable for dynamic, intensity-based imaging of changes in intracellular sodium concentration ([Na + ] i ) of brain cells (Meier et al., 2006). Recent work has also shown that CoroNaGreen exhibits changes in FL with changes in the sodium concentration ([Na + ]) in vitro (Naumann et al., 2018). ...
... In HEK cells, when τ AVG (obtained from ROIs positioned over nuclear and cytosolic regions) was plotted against [Na + ], the data followed a sigmoidal curve revealing an apparent K d of 79.9 mM. Earlier calibrations of CoroNaGreen's sensitivity for Na + performed in hippocampal neurons only covered the lower concentration range, still suggesting that CoroNaGreen's K d for Na + is >60 mM (Meier et al., 2006). The data provided by Rungta et al. (2015), performing lifetime measurements with CoroNaGreen in hippocampal neurons, is also in line with this observation. ...
Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na ⁺ ]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na ⁺ ]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na ⁺ ], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na ⁺ ] between 0 and 150 mM, exhibiting an apparent Kd of ∼80 mM. Based on these calibrations, a [Na ⁺ ] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na ⁺ ] of 13.0 mM, whereas [Na ⁺ ] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na ⁺ /K ⁺ -ATPase by removal of extracellular K ⁺ caused significant [Na ⁺ ] increases in all cellular subcompartments. Using an alternative approach for data analysis (“Ratio FLIM”) increased the temporal resolution and revealed a sequential response to K ⁺ removal, with cytosolic [Na ⁺ ] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na ⁺ ]. This approach thus represents a valuable tool for quantitative determination of [Na ⁺ ] and changes thereof in different subcellular compartments.
... For imaging of [Na + ] i in CA1 pyramidal neurons, the membrane-permeant form of SBFI (SBFI-AM; 200 µM, Teflabs, Austin, TX, USA) was pressure-injected into the stratum radiatum and stratum oriens as reported before (Meier et al. 2006;Langer et al. 2017). Alternatively, individual CA1 neurons were subjected to whole-cell patch-clamp and loaded with the membrane-impermeant salt of SBFI (Teflabs) through the patch pipette for at least 15 min before starting imaging experiments. ...
... SBFI fluorescence was calibrated in situ as described previously (Rose et al. 1999;Meier et al. 2006;Gerkau et al. 2018). Briefly, SBFI-AM-loaded organotypic tissue slices were first superfused with nominally Na + -free calibration-ACSF, containing ouabain (100 µM), an inhibitor of the Na + /K + -ATPase, as well as the ionophores gramicidin (3 µM) and monensin (10 µM) to equilibrate intra-and extracellular Na + . ...
... Afterwards, slices were exposed to calibration-ACSFs containing different [Na + ] and resulting changes in SBFI fluorescence in neuronal somata were recorded using multiphoton excitation. As reported before (Rose et al. 1999;Meier et al. 2006;Gerkau et al. 2018), increasing [Na + ] caused a decrease in fluorescence emission of SBFI and traces were inverted for illustration purposes after normalization to fluorescence levels at 0 mM (Fig. 1A). Normalized changes in SBFI fluorescence were plotted against [Na + ] and fit using Michaelis-Menten kinetics (Fig. 1B). ...
Key points
Employing quantitative Na⁺‐imaging and Förster resonance energy transfer‐based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity‐induced Na⁺ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus.
Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations.
Different paradigms of stimulation that induced global Na⁺ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1–0.6 mm in somata and dendrites, while Na⁺ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP].
Our data suggest that global Na⁺ transients require global cellular activation of the Na⁺/K⁺‐ATPase resulting in a consumption of ATP that transiently overrides its production.
For recovery from locally restricted Na⁺ influx, ATP production as well as fast intracellular diffusion of ATP and Na⁺ might prevent a local drop in [ATP].
Abstract
Excitatory neuronal activity results in the influx of Na⁺ through voltage‐ and ligand‐gated channels. Recovery from accompanying increases in intracellular Na⁺ concentrations ([Na⁺]i) is mainly mediated by the Na⁺/K⁺‐ATPase (NKA) and is one of the major energy‐consuming processes in the brain. Here, we analysed the relation between different patterns of activity‐induced [Na⁺]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na⁺ imaging with sodium‐binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na⁺]i oscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3–0.4 mm in both compartments. Global [Na⁺]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na⁺ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i. Our results suggest that ATP consumption by the NKA following global [Na⁺]i transients temporarily overrides its availability, causing a decrease in [ATP]i. Locally restricted Na⁺ transients, however, do not result in detectable changes in local [ATP]i, suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na⁺ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.
... In the presence of K + , the dissociation constant (K d ) is 11.3 mM making it suitable for the detection of small changes in Na + concentration. Upon binding to Na + , SBFI's quantum yield increases causing a narrowing of the excitation peak and a shift in the excitation maximum to shorter wavelengths resulting in a significant change in the ratio of fluorescence excited at 340 nm/380 nm [14]. This property enables ratio-metric fluorescence analysis for the determination of Na + concentrations independent of fluorophore concentration [15]. ...
... Since SBFI has a low excitation wavelength in the UV range, the utilisation of this fluorophore necessitates UV lasers or two-photon imaging. The cost and complexity of these methods has been a barrier to widespread usage of this fluorophore [14]. One further disadvantage of the SBFI dye is that it has low cell permeability despite the addition of dispersion agents such as Pluronic F127. ...
... Sodium Green is an alternate fluorophore that has an excitation/emission in the visible light spectrum (507 nm/532 nm) enabling the utilisation of the Argon 488 laser. It also has greater selectivity for Na + ions than SBFI (41-fold versus 18-fold), a K d value of 21 mM in the presence of K + , making it suitable for measurements of physiological changes in Na + concentration [14,17]. However, Sodium Green has been shown to interact with proteins, measured by the attenuation of fluorescence and a shift in the life time in FLIM analysis in the presence of 5% BSA. ...
Sodium ions (Na⁺) are key regulators of molecular events in many cellular processes, yet the dynamics of this ion remain poorly defined. Developing approaches to identify and characterise Na⁺ microenvironments will enable more detailed elucidation of the mechanisms of signal transduction. Here we report the application of Spectral Phasor analysis to the Na⁺ fluorophore, CoroNa Green, to identify and spatially map spectral emissions that report Na⁺ microenvironments. We use differentiating stem cells where Na⁺ fluxes were reported as an antecedent. Myoblast stem cells were induced to differentiate by serum starvation and then fixed at intervals between 0 and 40-minutes of differentiation prior to addition of CoroNa Green. The fluorescent intensity was insufficient to identify discrete Na⁺ microenvironments. However, using Spectral Phasor analysis we identified spectral shifts in CoroNa Green fluorescence which is related to the Na⁺ microenvironment. Further, spectral-heterogeneity appears to be contingent on the distance of Na⁺ from the nucleus in the early stages of differentiation. Spectral Phasor analysis of CoroNa Green in fixed stem cells demonstrates for the first time that CoroNa Green has unique spectral emissions depending on the nature of the Na⁺ environment in differentiating stem cells. Applying Spectral Phasor analysis to CoroNa Green in live stem cells is likely to further elucidate the role of Na⁺ microenvironments in the differentiation process.
... To achieve higher spatiotemporal resolutions within tissues, Na + fluorescent dyes could be used. CoroNa Green AM is a cell-permeant, greenfluorescent dye, with absorbance/emission at 492 nm/ 520 nm (Meier et al. 2006). It has been employed to study Na + uptake in Arabidopsis (Park et al. 2009) and wheat (Wu et al. 2018) roots. ...
... It has been employed to study Na + uptake in Arabidopsis (Park et al. 2009) and wheat (Wu et al. 2018) roots. The Na + -sensing fluorescent probe sodium-binding benzofuran isophthalate (SBFI) is another dye that allows visualization of Na uptake (Meier et al. 2006). Cell-permeable SBFI-AM has K d = 3.8 mM in the absence of K + and 11.3 mM for solutions with combined Na + and K + concentration of 135 mM (physiological ionic strength). ...
Understanding mechanisms of nutrient allocation in organisms requires precise knowledge of the spatiotemporal dynamics of small molecules in vivo. Genetically encoded sensors are powerful tools for studying nutrient distribution and dynamics, as they enable minimally-invasive monitoring of nutrient steady-state levels in situ. Numerous types of genetically encoded sensors for nutrients have been designed and applied in mammalian cells and fungi. However, to date, their application for visualizing changing nutrient levels in planta remain limited. Systematic sensor-based approaches could provide the quantitative, kinetic information on tissue-specific, cellular and subcellular distributions and dynamics of nutrients in situ that is needed for the development of theoretical nutrient flux models that form the basis for future crop engineering.
Here, we review various approaches that can be used to measure nutrients in planta with an overview over conventional techniques, as well as genetically encoded sensors currently available for nutrient monitoring, and discuss their strengths and limitations. We provide a list of currently available sensors and summarize approaches for their application at the level of cellular compartments and organelles. When used in combination with bioassays on intact organisms and precise, yet destructive analytical methods, the spatiotemporal resolution of sensors offers the prospect of a holistic understanding of nutrient flux in plants.
... In recent years, some studies have employed an electrolyte indicator to clarify real-time electrolyte behaviour in nerve fibres and cardiomyocytes [12,13]. In the reproductive field, some studies used Ca 2+ indicators [14] but none have reported the use of Na + and K + electrolyte indicators. ...
... Unlike time-lapse K + imaging, fluorescence intensity reached a plateau in time-lapse Na + imaging because CoroNa Green AM was more easily excreted from the cells than was ION Potassium Green-2 AM. It has been reported that CoroNa Green AM leaks out of cells more easily than other Na + indicators [13]. Although a multidrug resistance protein is reportedly involved in the extracellular emission of fluorescent cellular indicators [24,25], details regarding the two indicators used in this study are unclear. ...
Studies have shown that some electrolytes, including Na ⁺ and K ⁺ , play important roles in embryonic development. However, these studies evaluated these electrolytes by using inhibitors or knockout mice, with no mention on the changes in the intracellular electrolyte concentrations during embryogenesis. In this study, we used the electrolyte indicators CoroNa Green AM and ION Potassium Green-2 AM to directly visualise intracellular concentrations of Na ⁺ and K ⁺ , respectively, at each embryonic developmental stage in mouse embryos. We directly observed intracellular electrolyte concentrations at the morula, blastocyst, and hatching stages. Our results revealed dynamic changes in intracellular electrolyte concentrations; we found that the intracellular Na ⁺ concentration decreased, while K ⁺ concentration increased during blastocoel formation. The degree of change in intensity in response to ouabain, an inhibitor of Na ⁺ /K ⁺ ATPase, was considered to correspond to the degree of Na ⁺ /K ⁺ ATPase activity at each developmental stage. Additionally, after the blastocyst stage, trophectoderm cells in direct contact with the blastocoel showed higher K ⁺ concentrations than in direct contact with inner cell mass, indicating that Na ⁺ /K ⁺ ATPase activity differs depending on the location in the trophectoderm. This is the first study to use CoroNa Green AM and ION Potassium Green-2 AM in mouse embryos and visualise electrolytes during embryonic development. The changes in electrolyte concentration observed in this study were consistent with the activity of Na ⁺ /K ⁺ ATPase reported previously, and it was possible to image more detailed electrolyte behaviour in embryo cells. This method can be used to improve the understanding of cell physiology and is useful for future embryonic development studies.
... Although small chemical fluorescent indicators sensitive for Na + are available, these probes do not represent applicable detection methods. These fluorescent chemical Na + indicators bind Na + via a crown ether structure within a certain cavity [177][178][179][180][181]. However, most available probes suffer from poor specificity for Na + over K + , and their affinity for Na + appears to be dependent on [K + ] or is far from the physiologic intracellular Na + concentration ([Na + ]) [177,181,182]. ...
... However, most available probes suffer from poor specificity for Na + over K + , and their affinity for Na + appears to be dependent on [K + ] or is far from the physiologic intracellular Na + concentration ([Na + ]) [177,181,182]. On the other hand, several probes only allow an intensiometric read-out, making quantifications of absolute [Na + ] challenging [178][179][180][181][182]. Additionally, all of these chemical probes are difficult to target to subcellular structures such as the endoplasmic reticulum (ER), the nucleus or the Golgi apparatus, although mitochondria localized Na + indicators are available [182]. ...
Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K + , alkaline earth metals including Mg 2+ and Ca 2+ , and transition metals including Cu + /Cu 2+ and Zn 2+. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe 2+ /Fe 3+ , Mn 2+ and Na + .
... Ion imaging and electrophysiology. For intracellular sodium imaging, brain slices were dye-loaded by injection of SBFI-acetoxymethyl ester (SBFI-AM; Invitrogen) as reported previously (Meier et al., 2006). For calcium imaging, the chemical indicator dye Oregon Green BAPTA-1 acetoxymethyl ester (OGB-AM; Invitrogen) was used. ...
... Further data analysis was performed offline using OriginPro9 Software (Origin Lab). Changes in SBFI fluorescence ratio were expressed as changes in sodium concentration based on in situ calibrations as described in detail previously (Rose and Ransom, 1996;Meier et al., 2006;Langer and Rose, 2009). ...
Activity-related sodium transients induced by glutamate uptake represent a special form of astrocyte excitability. Astrocytes of the neocortex, as opposed to the hippocampus proper, also express ionotropic glutamate receptors, which might provide additional sodium influx. We compared glutamate-related sodium transients in astrocytes and neurons in slices of the neocortex and hippocampus of juvenile mice of both sexes, using widefield and multiphoton imaging. Stimulation of glutamatergic afferents or glutamate application induced sodium transients that were twice as large in neocortical as in hippocampal astrocytes, despite similar neuronal responses. Astrocyte sodium transients were reduced by ∼50% upon blocking NMDA receptors in the neocortex, but not hippocampus. Neocortical, but not hippocampal, astrocytes exhibited marked sodium increases in response to NMDA. These key differences in sodium signaling were also observed in neonates and in adults. NMDA application evoked local calcium transients in processes of neocortical astrocytes, which were dampened upon blocking sodium/calcium exchange (NCX) with KB-R7943 or SEA0400. Mathematical computation based on our data predict that NMDA-induced sodium increases drive the NCX into reverse mode, resulting in calcium influx. Together, our study reveals a considerable regional heterogeneity in astrocyte sodium transients, which persists throughout postnatal development. Neocortical astrocytes respond with much larger sodium elevations to glutamatergic activity than hippocampal astrocytes. Moreover, neocortical astrocytes experience NMDA-receptor-mediated sodium influx, which hippocampal astrocytes lack, and which drives calcium import through reverse NCX. This pathway thereby links sodium to calcium signaling and represents a new mechanism for the generation of local calcium influx in neocortical astrocytes.SIGNIFICANCE STATEMENT Astrocyte calcium signals play a central role in neuron-glia interaction. Moreover, activity-related sodium transients may represent a new form of astrocyte excitability. Here we show that activation of NMDA receptors results in prominent sodium transients in neocortical, but not hippocampal, astrocytes in the mouse brain. NMDA receptor activation is accompanied by local calcium signaling in processes of neocortical astrocytes, which is augmented by sodium-driven reversal of the sodium/calcium exchanger. Our data demonstrate a significant regional heterogeneity in the magnitude and mechanisms of astrocyte sodium transients. They also suggest a close interrelation between NMDA-receptor-mediated sodium influx and calcium signaling through the reversal of sodium/calcium exchanger, thereby establishing a new pathway for the generation of local calcium signaling in astrocyte processes.
... Therefore, knowledge on the absolute concentration and the spatiotemporal extend of intracellular Na + is essential for understanding the role of Na + dynamics in cell biology. The development of Na + -sensitive fluorescent indicator dyes (Minta & Tsien, 1989) opened the possibility to study cellular Na + dynamics with conventional fluorescence microscopy (Lasser-Ross & Ross, 1992;Regehr, 1997;Despa et al., 2000a,b;Mondragão et al., 2016;Langer et al., 2017;Miyazaki & Ross, 2017), confocal microscopy (Winslow et al., 2002;Meier et al., 2006) and two-photon microscopy (2PM) (Rose et al., 1999;Lahn et al., 2011;Kleinhans et al., 2014;Langer et al., 2017). Two aspects, however, complicate quantitative Na + imaging. ...
... The subsequent development of dyes excited by visible light allowed using standard confocal microscopes for Na + imaging (for review see Schreiner & Rose, 2012). These 'green dyes' [CoraNa Green (Martin et al., 2005), ANG-2 and its lower-affinity analogue ANG-1 (Lamy & Chatton, 2011)], and Sodium Green, however, showed little Na + -dependent shifts in excitation or emission wavelengths, prohibiting ratiometric measurements, only allowing semiquantitative ' F/F' measurements (Meier et al., 2006). The development of 2P microscopy (Denk et al., 1990) allowed using SBFI for Na + imaging deep in neuronal tissue (Rose et al., 1999;Rose & Konnerth, 2001;Rose, 2012;Kleinhans et al., 2014;Ona-Jodar et al., 2017). ...
Two‐photon microscopy (2PM) offers great potential in fluorescence imaging of intracellular Na⁺ dynamics of live cells. A severe drawback, however, is that quantitative ratioing of fluorescence intensities at different wavelengths [possible in one‐photon imaging with the classical Na⁺‐indicator dye sodium‐binding benzofuran isophtalate (SBFI)] is not practical in 2PM. We aimed at establishing 2PM‐based time‐correlated fluorescence lifetime measurements as an alternative method for quantifying Na⁺ dynamics. We compared the photophysical properties of the four Na⁺‐sensitive fluorescent indicator dyes SBFI, CoroNa Green, Sodium Green and Asante NaTRIUM Green‐2 (ANG‐2) in cuvette calibrations. All four dyes showed Na⁺‐dependent intensity changes, with ANG‐2 having the most favourable properties for 2PM. All dyes but SBFI showed significant changes in their fluorescence lifetime upon Na⁺ binding, again with ANG‐2 being the most promising dye. We found that, unfortunately, the fluorescence lifetime of ANG‐2 is not only affected by Na⁺ but also by protons, K⁺ and dye impurities, rendering a quantitative description of the individual lifetime components impractical. However, a simplified calibration procedure, based on a published approach for Ca²⁺ imaging, allowed relating lifetimes to Na⁺ concentration. Using ANG‐2 and the simplified calibration will allow quantitative two‐photon Na⁺ imaging with millimolar sensitivity.
Lay Description
Dynamic changes of ion concentrations, which play crucial roles in cellular physiology, can be monitored with appropriate fluorescent indicator dyes. For intracellular sodium ions (Na⁺), certain dyes even allow quantitative measurements with standard microscopic techniques. However, for two‐photon microscopy, which allows resolving cells deep in intact tissue, imaging solutions that are fully quantitative are lacking. For the four commercially available Na⁺ dyes ‘SBFI’, ‘CoroNa Green’, ‘Sodium Green’, and ‘Asante NaTRIUM Green‐2’ (ANG2) we analyzed whether their fluorescent lifetime (LT), i.e., the nanosecond decay of emission of photons after a pulsed excitation, could serve as a quantitative measure of intracellular Na⁺. Pulsed excitation in the femtosecond range is an inherent feature of two‐photon microscopy and, in combination with fast, single‐photon counting microscopes, allows for easy‐to‐implement LT microscopy. We found that Sodium Green and ANG2 showed strong Na⁺‐dependent changes in the fluorescence LT, while SBFI showed no, and CoroNa Green only small changes. ANG2, as the brightest dye, was further characterized regarding effects of protons and potassium ions (K⁺), both also present in cells at significant concentrations, on the fluorescence LT. We found that the LT of ANG2 is affected in a predictable manner by Na⁺, K⁺, and protons. However, our data reveal that the commercial dye must also contain impurities with unexpected Na⁺‐ and K⁺‐binding characteristics, rendering a quantitative description of the individual lifetime components impractical. We, therefore, adapted a simplified calibration procedure, based on a published approach for Ca²⁺ imaging, that allows relating the average lifetime to Na⁺ concentration. With this simplified calibration procedure, ANG2 is well suited for quantitative two‐photon Na⁺ imaging with millimolar sensitivity.
... For intracellular sodium imaging, sodium-binding benzofuran isophthalate-acetoxymethyl ester (SBFI-AM) (TEFLabs Inc., Austin, TX, USA), the membrane-permeant form of SBFI, was injected into the corpus callosum, following a procedure reported before [39]. Ratiometric wide-field sodium imaging was performed as described earlier in detail [4] using a variable scan digital imaging system (Nikon NIS-Elements v4.3, Nikon GmbH Europe, Düsseldorf, Germany) attached to an upright microscope (Nikon Eclipse FN-PT, Nikon GmbH Europe, Düsseldorf, Germany) equipped with 40×/N.A. 0.8 LUMPlanFI water immersion objective (Olympus Deutschland GmbH, Hamburg, Germany) and an orca FLASH V2 camera (Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany). ...
... The fluorescence ratio was calculated from the emission at single wavelengths (F 340 /F 380 ). SBFI fluorescence was calibrated in situ as described earlier [4,39]. ...
Recent work has established that glutamatergic synaptic activity induces transient sodium elevations in grey matter astrocytes by stimulating glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Glial sodium transients have diverse functional consequences but are largely unexplored in white matter. Here, we employed ratiometric imaging to analyse sodium signalling in macroglial cells of mouse corpus callosum. Electrical stimulation resulted in robust sodium transients in astrocytes, oligodendrocytes and NG2 glia, which were blocked by tetrodotoxin, demonstrating their dependence on axonal action potentials (APs). Action potential-induced sodium increases were strongly reduced by combined inhibition of ionotropic glutamate receptors and glutamate transporters, indicating that they are related to release of glutamate. While AMPA receptors were involved in sodium influx into all cell types, oligodendrocytes and NG2 glia showed an additional contribution of NMDA receptors. The transporter subtypes GLT-1 and GLAST were detected at the protein level and contributed to glutamate-induced glial sodium signals, indicating that both are functionally relevant for glutamate clearance in corpus callosum. In summary, our results demonstrate that white matter macroglial cells experience sodium influx through ionotropic glutamate receptors and glutamate uptake upon AP generation. Activity-induced glial sodium signalling may thus contribute to the communication between active axons and macroglial cells.
... To functionally confirm that tricaine inhibits VGSCs in sea urchin embryos, we investigated the effect of tricaine treatment on transmembrane voltage (Vmem) and sodium ion levels by using the fluorescent reporters DiSBAC and CoroNa-AM, respectively (Epps et al., 1994;Meier et al., 2006;Adams and Levin, 2012;Rodriguez-Sastre et al., 2019). Since VGSC activity is required for skeletal patterning from 14 to 17 hpf, we asked how tricaine treatment affects Vmem and sodium ion levels during this developmental window. ...
Defining pattern formation mechanisms during embryonic development is important for understanding the etiology of birth defects and to inform tissue engineering approaches. In this study, we used tricaine, a voltage-gated sodium channel (VGSC) inhibitor, to show that VGSC activity is required for normal skeletal patterning in Lytechinus variegatus sea urchin larvae. We demonstrate that tricaine-mediated patterning defects are rescued by an anesthetic-insensitive version of the VGSC LvScn5a. This channel is expressed in the posterior ventrolateral ectoderm where it spatially overlaps with Wnt5. We show that VGSC activity is required to spatially restrict Wnt5 expression to this ectodermal region that is adjacent and instructive to clusters of primary mesenchymal cells (PMCs) where secretion of the larval skeleton is initiated as triradiates. Tricaine-mediated Wnt5 spatial expansion correlates with the formation of ectopic PMC clusters and triradiates. These tricaine-mediated defects are rescued by Wnt5 knock down, indicating that the spatial expansion Wnt5 is responsible for the patterning defects induced by VGSC inhibition. These results demonstrate a novel connection between bioelectrical status and the spatial control of patterning cue expression during embryonic pattern formation.
Summary statement
Inhibition of voltage-gated sodium channels perturbs Wnt5-mediated patterning of the sea urchin larval skeleton.
... Estimation of neuronal stimulation-induced changes to intracellular [Na + ] and Na + /K + pump activity SBFI calibration curves in rodent hippocampal neurons (Baeza-Lehnert et al., 2019;Diarra et al., 2001;Gerkau et al., 2019;Meier et al., 2006;Rose et al., 1999) reported apparent Na + K d values of 18-42 mM Na + . However, most were performed with ouabain concentrations of only 50-100 μM, well below the millimolar concentrations needed to fully inhibit the rodent Na + /K + pump α1 isozyme under physiological conditions; the relatively high ouabain-resistance of rodent α1 pumps would also be strongly increased in calibration solutions that substitute external Na + with high concentrations of K + since external K + competes with binding of the inhibitor to the pump's externally accessible E2 conformation (Hansen and Skou, 1973;Kanai et al., 2021). ...
Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca2+ influx, but is not activated by feedforward Ca2+ signaling (Díaz-García, Meyer, et al., 2021). Since ATP-powered Na+ and Ca2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na+, while intracellular Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na+/K+ pump, which is strongly potentiated by Na+ influx through the Na+/Ca2+ exchanger during extrusion of Ca2+ following stimulation.
... Since sos and moca1 mutants accumulate excessive Na + , we speculate that halotropism is triggered by ionic stress of the root tips. We next analyzed Na + accumulation in root tips using the Na + -specific fluorescent indicator CoroNa Green ( Figure 1F) (Meier et al., 2006;Park et al., 2009). As expected, Na + accumulation was significantly higher in sos and moca1 mutants as compared with wild-type (WT) root tips (Figures 1F and 1G). ...
Plants have evolved signaling mechanisms that guide growth away from adverse environments that can cause yield losses. Root halotropism is a sodium-specific negative tropism that is crucial for surviving and thriving under high salinity. Although root halotropism was discovered some years ago, the underlying molecular and cellular mechanisms remain unknown. Here, we show that abscisic acid (ABA)-mediated root twisting determines halotropism in Arabidopsis. An ABA-activated SnRK2 protein kinase (SnRK2.6) phosphorylates the microtubule-associated protein SP2L at Ser406, which induces a change in the anisotropic cell expansion at the root transition zone and is required for root twisting during halotropism. Salt stress triggers SP2L-mediated cortical microtubule reorientation, which guides cellulose microfibril patterns. Our findings thus outline the molecular mechanism of root halotropism and indicate that anisotropic cell expansion through microtubule reorientation and microfibril deposition has a central role in mediating tropic responses.
... CoroNa-AM enters the cells and exhibits an increase in fluorescence emission intensity upon binding Na + ions. Previously, it has been shown to detect intracellular Na + in neurons within brain slices [84], and we used an established protocol for modulating extracellular Na + [85]. A completely defined Tyrode solution was used as a starting extracellular solution with physiological levels of ions (high extracellular Na + -140 mM in comparison to cytosol). ...
All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to injury and reinnervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve repair and reinnervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.
... To account for [Na + ] i -independent changes in the dye emission due to photobleaching and/or loss in the presence of the ionophore (Meier et al., 2006), CoroNa green emission was corrected for its time-dependent decay, determined in 12 adult myocytes in [Na + ]-free medium containing gramicidin D. The correction was based on the monoexponential time course of the emission decay, which declined by 20% during the last 30 min exposure to gramicidin D (Fig. 1A). A typical set of traces is shown in Fig. 1B. ...
Little is currently known about possible developmental changes in myocardial Na⁺ handling, which may have impact on cell excitability and Ca²⁺ content. Resting intracellular Na⁺ concentration ([Na⁺]i), measured in freshly isolated rat ventricular myocytes with CoroNa green, was not significantly different in neonates (3–5 days old) and adults, but electrical stimulation caused marked [Na⁺]i rise only in neonates. Inhibition of L‐type Ca²⁺ current by CdCl2 abolished not only systolic Ca²⁺ transients, but also activity‐dependent intracellular Na⁺ accumulation in immature cells. This indicates that the main Na⁺ influx pathway during activity is the Na⁺/Ca²⁺ exchanger, rather than voltage‐dependent Na⁺ current (INa), which was not affected by CdCl2. In immature myocytes, INa density was two‐fold greater, inactivation was faster, and the current peak occurred at less negative transmembrane potential (Em) than in adults. Na⁺ channel steady‐state activation and inactivation curves in neonates showed a rightward shift, which should increase channel availability at diastolic Em, but also require greater depolarization for excitation, which was observed experimentally and reproduced in computer simulations. Ventricular mRNA levels of Nav1.1, Nav1.4 and Nav1.5 pore‐forming isoforms were greater in neonate ventricles, while a decrease was seen for the β1 subunit. Both molecular and biophysical changes in the channel profile may contribute to the differences in INa density and voltage‐dependence, and also to the less negative threshold Em, in neonates compared to adults. The apparently lower excitability in immature ventricle may confer protection against the development of spontaneous activity in this tissue.
Key points
Previous studies showed that myocardial preparations from immature rats are less sensitive to electrical field stimulation than adult preparations.
Freshly isolated ventricular myocytes from neonatal rats showed lower excitability than adult cells, e.g. less negative threshold membrane potential and greater membrane depolarization required for action potential triggering.
In addition to differences in mRNA levels for Na⁺ channel isoforms and greater Na⁺ current (INa) density, Na⁺ channel voltage‐dependence was shifted to the right in immature myocytes, which seems to be sufficient to decrease excitability, according to computer simulations.
Only in neonatal myocytes did cyclic activity promote marked cytosolic Na⁺ accumulation, which was prevented by abolition of systolic Ca²⁺ transients by blockade of Ca²⁺ currents.
Developmental changes in INa may account for the difference in action potential initiation parameters, but not for cytosolic Na⁺ accumulation, which seems to be due mainly to Na⁺/Ca²⁺ exchanger‐mediated Na⁺ influx.
... Sodium Green has visible light excitation and brighter fluorescence that enable its usage in flow cytometry (Amorino and Fox, 1995). CoroNa Green has a peak excitation wavelength at 492 nm and a peak emission wavelength at 516 nm (Meier et al., 2006;Iamshanova et al., 2016). NaTRIUM Green 2 (ANG-2) ( Figure 7B) is slightly red-shifted with peak excitation and emission wavelengths at 517 and 542 nm, respectively, (Roder and Hille, 2014;Iamshanova et al., 2016). ...
Monatomic ions play critical biological roles including maintaining the cellular osmotic pressure, transmitting signals, and catalyzing redox reactions as cofactors in enzymes. The ability to visualize monatomic ion concentration, and dynamic changes in the concentration, is essential to understanding their many biological functions. A growing number of genetically encodable and synthetic indicators enable the visualization and detection of monatomic ions in biological systems. With this review, we aim to provide a survey of the current landscape of reported indicators. We hope this review will be a useful guide to researchers who are interested in using indicators for biological applications and to tool developers seeking opportunities to create new and improved indicators.
... To quantify changes of [Na + ] i , we incubated cells with the fluorescent cell-permeable Na + indicator, CoroNa Green AM, where high intensity of emission corresponds to an increase of [Na + ] i and decreased intensity corresponds to decreased [Na + ] i . CoroNa Green is excited between 440-514 nm with peak emission at 490 nm [25]. CIBN-CRY2 dimerization occurs between 405 and 500 nm [20]. ...
The activity of the epithelial Na+ Channel (ENaC) is strongly dependent on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binds two distinct cationic clusters within the N termini of β- and γ-ENaC subunits (βN1 and γN2). The affinities of these sites were previously determined using short synthetic peptides, yet their role in sensitizing ENaC to changes in PIP2 levels in the cellular system is not well established. We addressed this question by comparing the effects of PIP2 depletion and recovery on ENaC channel activity and intracellular Na+ levels [Na+]i. We tested effects on ENaC activity with mutations to the PIP2 binding sites using the optogenetic system CIBN/CRY2-OCRL to selectively deplete PIP2. We monitored changes of [Na+]i by measuring the fluorescent Na+ indicator, CoroNa Green AM, and changes in channel activity by performing patch clamp electrophysiology. Whole cell patch clamp measurements showed a complete lack of response to PIP2 depletion and recovery in ENaC with mutations to βN1 or γN2 or both sites, compared to wild type ENaC. Whereas mutant βN1 also had no change in CoroNa Green fluorescence in response to PIP2 depletion, γN2 did have reduced [Na+]i, which was explained by having shorter CoroNa Green uptake and half-life. These results suggest that CoroNa Green measurements should be interpreted with caution. Importantly, the electrophysiology results show that the βN1 and γN2 sites on ENaC are each necessary to permit maximal ENaC activity in the presence of PIP2.
... This dye can pass the cell membranes while being attached to Ca 2+ and permeate ion channels [144], where the fluorescence can be measured in comparison to background fluorescence (ΔF/F 0 ). This can similarly be performed for Na + activity using CoroNa Green AM, a sodium ion-indicating dye [145]. ...
Recently, the development of smart materials and the study of their properties has provided an innovative approach to the field of tissue engineering. Piezoelectrics, which are able to generate electric charge in response to mechanical stress or strain have been utilised in the stimulation of electrophysiologically responsive cells , including those found in bone, muscle, and the central and peripheral nervous systems. This area of study has experienced tremendous growth in the past decade in terms of both the array of piezoelectric materials and analytical methods by which they are evaluated in relation to specific tissue types. This review provides a critical and comprehensive overview of the most recent advances in this emerging field. Furthermore, it will extend the scope to examine the most recent developments in piezoelectric biomedical devices that extract energy from physiological processes to either power biomedical implants or act as biomedical sensors .
... Thus, calibration for potassium has been attempted with gramicidin [4,5], a combination of valinomycin and nigericin [6], valinomicin/ nigericin/ouabain [7,8], gramicidin/valinomycin for 3 min [9], bacterial toxins [10,11], by incubation in buffers without any added ionophores [12], and in a cell-free buffer [13]. Likewise, sodium probes have been calibrated with gramicidin [14,15], monensin [16], monensin/gramicidin [17], or gramicidin/monensin/ouabain [18,19]. It is unclear, which of these protocols are most efficient in achieving the desired result. ...
The response of fluorescent ion probes to ions is affected by intracellular environment. To properly calibrate them, intracellular concentration of the measured ion must be made equal to its extracellular concentration. In the first, computational, part of this work, we show, using the example of potassium, that the two requirements for ion equilibration are complete dissipation of membrane potential and high membrane permeability for both potassium and sodium. In the second part, we tested the ability of various ionophores to achieve potassium equilibration in Jurkat and U937 cells and found a combination of valinomycin, nigericin, gramicidin and ouabain to be the most effective. In the third part, we applied this protocol to two Asante Potassium Green probes, APG‐4 and APG‐2. APG‐4 shows good sensitivity to potassium but its fluorescence is sensitive to protein density; therefore, calibration buffer had to be supplemented with 50 mM sucrose to keep cell volume constant. With these precautions taken, the average potassium concentrations in U937 and Jurkat cells were measured as 132 mM and 118 mM, respectively. The other tested probe, APG‐2, is non‐selective for cations; this is, however, a potentially useful property because the sum [K ⁺ ] + [Na ⁺ ] determines the amount of intracellular water.
Support or Funding Information
The research was partly supported by the Kent State University Research Council (MM) and by the Russian Fedration grant 0124‐2018‐0003 (VY and AV)
This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
... SOS1) has been shown to function in the exclusion of sodium from the root meristematic region and protects cells of the elongation zone (Oh et al., 2009). To determine whether overexpression of OsSOS1 resulted in the regulation of sodium uptake at the roots, sodium uptake was also determined using CoroNa-Green, a membrane permeable fluorescent dye routinely used for visualization of intracellular sodium (Meier et al., 2006). Strong fluorescence at the root tip as well as elongation zone of WT plants indicated that sodium ions could penetrate into the cells. ...
In rice (Oryza sativa), Si nutrition is known to improve salinity tolerance; however, limited efforts have been made to elucidate the underlying mechanism. Salt-Overly Sensitive (SOS) pathway contributes to salinity tolerance in plants in a major way which works primarily through Na⁺ exclusion from the cytosol. SOS1, a vital component of SOS pathway is a Na⁺/H⁺ antiporter that maintains ion homeostasis. In this study, we evaluated the effect of overexpression of Oryza sativa SOS1 (OsSOS1) in tobacco (cv. Petit Havana) and rice (cv. IR64) for modulating its response towards salinity further exploring its correlation with Si nutrition. OsSOS1 transgenic tobacco plants showed enhanced tolerance to salinity as evident by its high chlorophyll content and maintaining favorable ion homeostasis under salinity stress. Similarly, transgenic rice overexpressing OsSOS1 also showed improved salinity stress tolerance as shown by higher seed germination percentage, seedling survival and low Na⁺ accumulation under salinity stress. At their mature stage, compared with the non-transgenic plants, the transgenic rice plants showed better growth and maintained better photosynthetic efficiency with reduced chlorophyll loss under stress. Also, roots of transgenic rice plants showed reduced accumulation of Na⁺ leading to reduced oxidative damage and cell death under salinity stress which ultimately resulted in improved agronomic traits such as higher number of panicles and fertile spikelets per panicle. Si nutrition was found to improve the growth of salinity stressed OsSOS1 rice by upregulating the expression of Si transporters (Lsi1 and Lsi2) that leads to more uptake and accumulation of Si in the rice shoots. Metabolite profiling showed better stress regulatory machinery in the transgenic rice, since they maintained higher abundance of most of the osmolytes and free amino acids.
... For Na + imaging, cells are dyeloaded, e.g., with the membrane-permeable form of SBFI (SBFIacetoxymethyl ester). After cleavage by endogenous esterases SBFI allows ratiometric imaging of [Na + ] i (Figure 5; Minta and Tsien, 1989;Meier et al., 2006). Na + load into a single cell can be achieved via direct electrical stimulation. ...
Astrocytes and oligodendrocytes are main players in the brain to ensure ion and neurotransmitter homeostasis, metabolic supply, and fast action potential propagation in axons. These functions are fostered by the formation of large syncytia in which mainly astrocytes and oligodendrocytes are directly coupled. Panglial networks constitute on connexin-based gap junctions in the membranes of neighboring cells that allow the passage of ions, metabolites, and currents. However, these networks are not uniform but exhibit a brain region-dependent heterogeneous connectivity influencing electrical communication and intercellular ion spread. Here, we describe different approaches to analyze gap junctional communication in acute tissue slices that can be implemented easily in most electrophysiology and imaging laboratories. These approaches include paired recordings, determination of syncytial isopotentiality, tracer coupling followed by analysis of network topography, and wide field imaging of ion sensitive dyes. These approaches are capable to reveal cellular heterogeneity causing electrical isolation of functional circuits, reduced ion-transfer between different cell types, and anisotropy of tracer coupling. With a selective or combinatory use of these methods, the results will shed light on cellular properties of glial cells and their contribution to neuronal function.
... In addition, cellular metal ions (Ca 2+ , Na + and K + ) in the WT, ∆fab1 and ∆fab1::fab1 strains were assessed by using specific fluorescent indicator dyes, including Ca 2+ -specific fluorescent probe Fura-2-AM (Fura-2 acetoxymethyl ester), Na + -specific fluorescent probe SBFI-AM (sodium-binding benzofuran isophthalate acetoxymethyl ester) and K + -specific fluorescent probe PBFI-AM (potassium-binding benzofuran isophthalate acetoxymethyl ester), according to previously described methods [36][37][38][39]. Upon binding metal ions, these specific fluorescence dyes can exhibit a fluorescence absorption shift from 380 to 340 nm of excitation. ...
Aspergillus flavus (A. flavus) is one of the most important model environmental fungi which can produce a potent toxin and carcinogen known as aflatoxin. Aflatoxin contamination causes massive agricultural economic loss and a critical human health issue each year. Although a functional vacuole has been highlighted for its fundamental importance in fungal virulence, the molecular mechanisms of the vacuole in regulating the virulence of A. flavus remain largely unknown. Here, we identified a novel vacuole-related protein in A. flavus, the ortholog of phosphatidylinositol-3-phosphate-5-kinase (Fab1) in Saccharomyces cerevisiae. This kinase was located at the vacuolar membrane, and loss of fab1 function was found to affect the growth, conidia and sclerotial development, cellular acidification and metal ion homeostasis, aflatoxin production and pathogenicity of A. flavus. Further functional analysis revealed that Fab1 was required to maintain the vacuole size and cell morphology. Additional quantitative proteomic analysis suggested that Fab1 was likely to play an important role in maintaining vacuolar/cellular homeostasis, with vacuolar dysregulation upon fab1 deletion leading to impaired aflatoxin synthesis in this fungus. Together, these results provide insight into the molecular mechanisms by which this pathogen produces aflatoxin and mediates its pathogenicity, and may facilitate dissection of the vacuole-mediated regulatory network in A. flavus.
... Apparent K d for ING-2 and IPG-2 were determined. ING-2 calibration was performed as described for other cell types (42,49), that is, ING-2-loaded platelets were diluted to 0.5 × 10 9 /L in buffers with 170 mM (Na + +K + ), 140 mM gluconate, 30 mM Cl − , 10 mM HEPES, adjusted to pH 7.0 with tetramethylammonium. Platelets were permeabilized with 0.5 μM gramicidin, incubated 3 min and fluorescence was acquired on cytometer. ...
The interaction of platelet agonists with their respective membrane receptors triggers intracellular signaling, among which cytosolic ion fluxes play an important role in activation processes. While the key contribution of intercellular free calcium is accepted, sodium and potassium roles in platelet activation have been less investigated in recent studies. Here, we implemented a novel flow‐cytometric method to monitor over time cytosolic free calcium, sodium, and potassium ion fluxes upon platelet activation and we demonstrate the feasibility of real‐time visualization of ion kinetics, in particular with a focus on sodium and potassium. Platelets were loaded with selective ion indicators, Fluo‐3 (Ca²⁺), ION NaTRIUM Green‐2 (Na⁺), and ION Potassium Green‐2 (K⁺). Fluorescence was monitored by flow cytometry. After measurement of a stable baseline, platelets were activated and ion indicator fluorescence was acquired over time, up to 10 min. Platelets were activated with either thromboxane analogue U46619, ADP, thrombin, TRAP6 (PAR‐1 agonist), AYPGKF (PAR‐4 agonist), convulxin (collagen receptor GPVI agonist), or combinations thereof. We evaluated preanalytical parameters (in particular dye loading time and concentration) to implement an accurate method. Subsequently, we characterized cytosolic calcium, sodium, and potassium kinetics in response to platelet agonists. We observed different patterns of agonist synergism. In conclusion, the present work highlights the use of cytosolic ion monitoring by flow cytometry to investigate characteristic calcium, sodium, and potassium mobilization patterns following platelet activation. This easy technique opens a new way to analyze signaling in different platelet subpopulations and it should prove useful for investigating platelet pathophysiology. © 2020 International Society for Advancement of Cytometry
... For this reason, membrane integrity, Na + and K + concentrations, and the ratio between Na + and K + are key parameters that differentiate sensitive and tolerant rice cultivars during salinity stress (Hoang et al., 2015). Under K + -starved conditions (normal watering) for 1 week, less Na + accumulated in roots of OsSIRH2-14-overexpressing plants were CoroNa-Green binds Na + ions only after it has been confined within cells (Meier, Kovalchuk, & Rose, 2006). Thus, fluorescence intensity with CoroNa-Green increases in response to increasing Na + accumulation (Nath et al., 2016). ...
Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programs. Here, we characterized the rice (Oryza sativa) RING H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na+ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared to wild‐type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na+ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.
... Thus, calibration for potassium has been attempted with gramicidin [4,5], a combination of valinomycin and nigericin [6], valinomicin/ nigericin/ouabain [7,8], gramicidin/valinomycin for 3 min [9], bacterial toxins [10,11], by incubation in buffers without any added ionophores [12], and in a cell-free buffer [13]. Likewise, sodium probes have been calibrated with gramicidin [14,15], monensin [16], monensin/gramicidin [17], or gramicidin/monensin/ouabain [18,19]. It is unclear, which of these protocols are most efficient in achieving the desired result. ...
... This disparity is in part due to the scarcity of suitable fluorescent indicator dyes with desirable photochemical properties. Na + indicator dyes such as Sodium Green, 3 CoroNa Green, 4 and Asante Natrium Green, 5 which have their absorption maximum around 488−492 nm, and more recently CoroNa Red, 6 with a longer emission wavelength, have proven useful in a variety of studies. 7 However, interactions of these dyes with cellular proteins and single wavelength emission can hinder reliable measurements. ...
Sodium flux plays a pivotal role in neurobiological processes including initiation of action potentials and regulation of neuronal cell excitability. However, unlike the wide range of fluorescent calcium indicators used extensively for cellular studies, the choice of sodium probes remains limited. We have previously demonstrated optode-based nanosensors (OBNs) for detecting sodium ions with advantageous modular properties such as tunable physiological sensing range, full reversibility, and superb selectivity against key physiological interfering ion potassium. (1) Motivated by bridging the gap between the great interest in sodium imaging of neuronal cell activity as an alternative to patch clamp and limited choices of optical sodium indicators, in this Letter we report the application of nanosensors capable of detecting intracellular sodium flux in isolated rat dorsal root ganglion neurons during electrical stimulation using transparent microelectrodes. Taking advantage of the ratiometric detection scheme offered by this fluorescent modular sensing platform, we performed dual color imaging of the sensor to monitor the intracellular sodium currents underlying trains of action potentials in real time. The combination of nanosensors and microelectrodes for monitoring neuronal sodium dynamics is a novel tool for investigating the regulatory role of sodium ions involved during neural activities.
... CoroNa Green-AM enters cells and, after hydrolysis (MW 564.54 after hydrolysis), depending on the Na + concentration, it produces a green fluorescence. 13 3T3-HER2 cells were seeded on culture dishes and incubated for 24 hours. Tra-IR700 (5 μg/mL) was added to the culture medium and the cells were incubated for 55 minutes at 37°C. ...
Near‐infrared photoimmunotherapy (NIR‐PIT) is a new cancer phototherapy using an antibody conjugated to the photosensitizer, IR700. When the conjugate binds to the plasma membrane and is exposed to NIR light, NIR‐PIT‐treated cells undergo swelling, and target‐selective necrotic/immunogenic cell death is induced. However, the cytotoxic mechanism of NIR‐PIT has not been elucidated. In order to understand the mechanism, it is important to elucidate how the damage to the plasma membrane induced by NIR light irradiation changes over time. Thus, in this study we have investigated the changes in plasma membrane permeability using ions and molecules of various sizes. Na⁺ flowed into cells immediately after NIR light irradiation, even when the function of transporters or channels were blocked. Subsequently, fluorescent molecules larger than Na⁺ entered the cells, but the damage was not large enough for dextran to pass through at early time points. To assess these phenomena quantitatively, membrane permeability was estimated using radiolabeled ions and molecules: ¹¹¹InCl3, ¹¹¹In‐DTPA, and ³H‐H2O, and comparable results were obtained. Although tiny plasma membrane perforations usually do not induce cell death, our results suggest that the tiny damage induced by NIR‐PIT was irreversibly extended with time. In conclusion, the tiny plasma membrane damage would be a trigger for the increase in plasma membrane permeability, cell swelling, and necrotic/immunogenic cell death in NIR‐PIT. Our findings provide new insight into the cytotoxic mechanism of NIR‐PIT.
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... Imaging of intracellular sodium and cell viability. For Na + imaging of choroid plexus bathed in aCSF-HEPES, the epithelial cells were loaded (~20 min) with the membrane-permeable form of SBFI (sodium-binding benzofuran isophthalate acetoxymethyl (AM) ester, 200 µM, Teflabs) To this end, the dye was pressureapplied (5 s) directly onto the cells on several positions 70 . Afterwards, the tissue was perfused with aCSF-HEPES for at least 20 min to allow for de-esterification of the dye before imaging experiments were commenced. ...
Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na+/K+/2Cl- cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.
... Five-day-old Arabidopsis seedlings treated with 100 mM NaCl with or without HA (860 mg L -1 ) for 14 h were stained with 5 μM CoroNa-Green AM (Invitrogen) for 3 h in the presence of pluronic acid (Sigma-Aldrich) at a final concentration of 0.02% in the dark (Leshem et al., 2006;Mazel et al., 2004;Meier et al., 2006). The stained roots were examined under a confocal microscope (Olympus FV1000) at excitation and emissions wavelengths of 488 nm and 516 nm, respectively. ...
Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.
The early neurodevelopmental contributions of ion pumps remain poorly characterized. Combining analysis of public human embryo single-cell transcriptomic datasets and an embryonic chicken model, we found a conserved differentiation sequence whereby spinal cord neurons switch on neuron-specific alpha3 subunit (ATP1A3) of Na ⁺ /K ⁺ ATPases. In the chicken model, ATP1A3 is distributed along axons and growth cones. Its knockdown alters axon pathfinding of dorsal interneurons (DIN) that wire spinocerebellar circuits. In mirror of reported electric field (EF)-driven cell migration, we found that DIN axons align in EFs, which was abolished by Na ⁺ /K ⁺ ATPase inhibitor Ouabain and ATP1A3 knockdown. We recorded an embryonic trans-neural-epithelial potential generating EF whose pharmacological and surgical manipulation mimicked ATP1A3 knock-down-induced altered DIN axon pathfinding. Using DINs transplantation paradigm, we found that ATP1A3 is required cell-autonomously for EF-mediated long-range guidance. Finally, dominant-negative ATP1A3 mutation causing an early ATP1A3 childhood disease disrupts this fundamental developmental process, revealing unexpected pathogenic mechanisms.
Citation: Everaerts, K.; Thapaliya, P.; Pape, N.; Durry, S.; Eitelmann, S.; Roussa, E.; Ullah, G.; Rose, C.R. Inward Operation of Sodium-Bicarbonate Cotransporter 1 Promotes Astrocytic Na + Loading and Loss of ATP in Mouse Neocortex during Brief Chemical Ischemia. Cells 2023, 12, 2675. https://doi. Abstract: Ischemic conditions cause an increase in the sodium concentration of astrocytes, driving the breakdown of ionic homeostasis and exacerbating cellular damage. Astrocytes express high levels of the electrogenic sodium-bicarbonate cotransporter1 (NBCe1), which couples intracellular Na + homeostasis to regulation of pH and operates close to its reversal potential under physiological conditions. Here, we analyzed its mode of operation during transient energy deprivation via imaging astrocytic pH, Na + , and ATP in organotypic slice cultures of the mouse neocortex, complemented with patch-clamp and ion-selective microelectrode recordings and computational modeling. We found that a 2 min period of metabolic failure resulted in a transient acidosis accompanied by a Na + increase in astrocytes. Inhibition of NBCe1 increased the acidosis while decreasing the Na + load. Similar results were obtained when comparing ion changes in wild-type and Nbce1-deficient mice. Mathematical modeling replicated these findings and further predicted that NBCe1 activation contributes to the loss of cellular ATP under ischemic conditions, a result confirmed experimentally using FRET-based imaging of ATP. Altogether, our data demonstrate that transient energy failure stimulates the inward operation of NBCe1 in astrocytes. This causes a significant amelioration of ischemia-induced astrocytic acidification, albeit at the expense of increased Na + influx and a decline in cellular ATP.
Defining pattern formation mechanisms during embryonic development is important for understanding the etiology of birth defects and to inform tissue engineering approaches. In this study, we used tricaine, a voltage-gated sodium channel (VGSC) inhibitor, to show that VGSC activity is required for normal skeletal patterning in Lytechinus variegatus sea urchin larvae. We demonstrate that tricaine-mediated patterning defects are rescued by an anesthetic-insensitive version of the VGSC LvScn5a. Expression of this channel is enriched in the ventrolateral ectoderm where it spatially overlaps with posterolaterally expressed Wnt5. We show that VGSC activity is required to spatially restrict Wnt5 expression to this ectodermal region that is adjacent and instructive to clusters of primary mesenchymal cells that initiate secretion of the larval skeleton as triradiates. Tricaine-mediated Wnt5 spatial expansion correlates with the formation of ectopic PMC clusters and triradiates. These defects are rescued by Wnt5 knock-down, indicating that the spatial expansion Wnt5 is responsible for the patterning defects induced by VGSC inhibition. These results demonstrate a novel connection between bioelectrical status and the spatial control of patterning cue expression during embryonic pattern formation.
Soil salinity negatively affects plant growth, productivity, and metabolism. Rice is known to have more sensitive phenotypes than other cereal crops such as wheat (Triticum aestivum), sorghum (Sorghum bicolor), and barley (Hordeum vulgare). We characterized the molecular function of rice (Oryza sativa) C3HC4 as a really interesting new gene (RING). O. sativa RING finger protein HC-2 (OsSIRHC-2) was highly expressed in 100 mM NaCl and was targeted to the cytosol. An in vitro ubiquitin assay demonstrated that OsSIRHC-2 possessed E3-ubiquitin ligase activity. Under salinity conditions, OsSIRHC-2-overexpressing plants exhibited higher chlorophyll, proline, and soluble sugar contents and lower H2O2 accumulation than wild-type plants, supporting transgenic plants with enhanced salinity tolerance phenotypes. OsSIRHC-2-overexpressing plants exhibited low Na+ accumulation and Na+/K+ ratios in their roots. Low expression of some Na+ transporter genes, especially OsHKT1;4 was consistent with the low Na+ accumulation in transgenic plants. These results suggest that OsSIRHC-2 may play a positive regulatory role in high salinity through the low absorption of Na+. Understanding the salt tolerance mechanisms of OsSIRHC-2 may provide a crucial strategy for plant adaptation to salinity.
A novel thiophene based pyrrolo [1, 2-a] quinoxaline (TAAPP) fluorescent sensor, suitable for the nanomolar detection of sodium ions was successfully developed and characterized by various spectroscopic techniques. The fluorescent sensor showed excellent selectivity for sodium ions over other biologically and environmentally important metal ions/biomolecules by a remarkable quenching of fluorescence. Stern-Volmer studies reveal that static quenching predominates over dynamic quenching. The detection limit was found to be 2.219 nM within the linear range of 3 to 30nM. DFT studies were performed to validate the results. The applicability of the probe can be extended to quantify sodium ions in real samples.
Aims:
Several studies reported that astrocytes support neuronal communication by the release of gliotransmitters, including ATP and glutamate. Astrocytes also play a fundamental role in buffering extracellular glutamate in the synaptic cleft, thus limiting the risk of excitotoxicity in neurons. We previously demonstrated that extracellular tau oligomers (ex-oTau), by specifically targeting astrocytes, affect glutamate-dependent synaptic transmission via a reduction in gliotransmitter release. The aim of this work was to determine if ex-oTau also impair the ability of astrocytes to uptake extracellular glutamate, thus further contributing to ex-oTau-dependent neuronal dysfunction.
Methods:
Primary cultures of astrocytes and organotypic brain slices were exposed to ex-oTau (200 nM) for 1 hour. Extracellular glutamate buffering by astrocytes was studied by: Na+ imaging; electrophysiological recordings; high-performance liquid chromatography; Western blot and immunofluorescence. Experimental paradigms avoiding ex-oTau internalization (i.e., heparin pre-treatment and amyloid precursor protein knockout astrocytes) were used to dissect intracellular vs. extracellular effects of oTau.
Results:
Ex-oTau uploading in astrocytes significantly affected glutamate-transporter-1 expression and function, thus impinging on glutamate buffering activity. Ex-oTau also reduced Na-K-ATPase activity because of pump mislocalization on the plasma membrane, with no significant changes in expression. This effect was independent of oTau internalization and it caused Na+ overload and membrane depolarization in ex-oTau-targeted astrocytes.
Conclusions:
Ex-oTau exerted a complex action on astrocytes, at both intracellular and extracellular levels. The net effect was dysregulated glutamate signalling in terms of both release and uptake that relied on reduced expression of glutamate-transporter-1, altered function and localization of NKA1A1, and NKA1A2. Consequently, Na+ gradients and all Na+ -dependent transports were affected.
Salinity stress is one of the most important abiotic stress factors affecting rice production worldwide. Using a forward genetics approach, a salt-insensitive TILLING line 1 (sitl1) rice mutant was isolated from a TILLING population. The sitl1 mutant exhibited reduced root growth and leaf chlorophyll content when grown under normal conditions, owing to a reduced ability to take up Mg²⁺; however, the defective phenotype could be restored by Mg²⁺ supplementation. Ionomic analysis revealed that the salinity insensitivity of sitl1 was the result of significantly reduced Mg²⁺ and Na⁺, and whole genome and RNA sequencing revealed that the sitl1 mutant caused a frameshift in the OsCZMT1 protein’s CorA-like ZntB cation transfer domain. Transient expression of an OsCZMT1-sGFP fusion protein revealed that OsCZMT1 was localized to the plasma membrane, and comprehensive expression analysis revealed that OsCZMT1 was mainly expressed in the roots and leaves of seedlings and highly upregulated in response to salinity stress. Complementation assays in yeast and CRISPR/Cas9-mediated knock-out mutants reveal that OsCZMT1 possesses both Mg²⁺ and Na⁺ transport activity. Taken together, these findings suggest that the identified OsCZMT1 frameshift mutation is responsible for the reduced Na⁺ and Mg²⁺ transport abilities, and that those reduced transport abilities confer salinity insensitivity.
Data availability statement
The study’s raw whole-genome sequencing and RNA-sequencing data have been accessioned in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under BioProject Accession Number PRJNA648465 (SRR12334077 – SRR12334084).
Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na+ indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na+ signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na+ influx, accompanied by considerable cell swelling. Both Na+ loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na+ transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na+ loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na+ transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na+ loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na+ changes on energy depletion.
Fluorescence-based sensors play a fundamental role in biological research. These sensors can be based on fluorescent proteins, fluorescent probes or they can be hybrid systems. The availability of a very large dataset of fluorescent molecules, both genetically encoded and synthetically produced, together with the structural insights on many sensing domains, allowed to rationally design a high variety of sensors, capable of monitoring both molecular and global changes in living cells or in in vitro systems. The advancements in the fluorescence-imaging field helped researchers to obtain a deeper understanding of how and where specific changes occur in a cell or in vitro by combining the readout of the fluorescent sensors with the spatial information provided by fluorescent microscopy techniques. In this review we give an overview of the state of the art in the field of fluorescent biosensors and fluorescence imaging techniques, and eventually guide the reader through the choice of the best combination of fluorescent tools and techniques to answer specific biological questions. We particularly focus on sensors for probing the bioenergetics and physicochemical status of the cell.
For about half a century, the pharmacology of electroneutral cation-chloride cotransporters has been dominated by a few drugs that are widely used in clinical medicine. Because these diuretic drugs are so good at what they do, there has been little incentive in expanding their pharmacology. The increasing realization that cation-chloride cotransporters are involved in many other key physiological processes and the knowledge that different tissues express homologous proteins with matching transport functions have rekindled interest in drug discovery. This review summarizes the methods available to assess the function of these transporters and describe the multiple efforts that have made to identify new compounds. We describe multiple screens targeting KCC2 function and one screen designed to find compounds that discriminate between NKCC1 and NKCC2. Two of the KCC2 screens identified new inhibitors that are 3-4 orders of magnitude more potent than furosemide. Additional screens identified compounds that purportedly increase cell surface expression of the cotransporter, as well as several FDA-approved drugs that increase KCC2 transcription and expression. The technical details of each screen biased them towards specific processes in the life cycle of the transporter, making these efforts independent and complementary. In addition, each drug discovery effort contributes to our understanding of the biology of the cotransporters.
Ants use venom for predation, defense, and communication; however, the molecular diversity, function, and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modeling, stability studies, and in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain cross-linked by two disulfide bonds in an antiparallel orientation. We chemically synthesized Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blowfly assays (LD50 = 3 nmol/g). Molecular modeling and circular dichroism studies revealed strong α-helical features, indicating its cytotoxic effects could derive from cell membrane pore formation or disruption. The native heterodimer was substantially more stable against proteolytic degradation (t1/2 = 13 h) than its homodimers or monomers (t1/2 < 20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterization of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability, a concept that is highly relevant for the design and development of peptide therapeutics, molecular probes, and bioinsecticides.
Domestication and cultivation of tree species, such as Pongamia pinnata is quite important because of its biofuel properties. Seedlings grown in modified hydroponic culture were morphologically similar to that of soil grown seedlings. Further, seedlings were allowed to grow without root limitation. Comparatively, our modified hydroponic growth system can be performed with minimal resources. Prior incubated root segments with CoroNa-Green AM dye retained maximum amount of dye when compared to CoroNa-Green AM dye incubated sections. Our modified protocol provides quantitative analysis of 2D and 3D imaging process at cellular and sub-cellular level. • Our protocol is customized to study individual plant behavior. • Additionally, it is customized for growing tap rooted trees species hydroponically. Changing the nutrient solution with regular intervals provides continuous supply of nutrients to the plants. • Prior incubation of root segments with Na+ probe (CoroNa-Green AM) provides better resolution in imaging process. Additionally, both 2D and 3D imaging provides a means to acquire and analyze entirety of the sample. Method name: Hydroponic cultivation of tree species Pongamia pinnata (L.) pierre and Na+-fluorescence measurements, Keywords: CoroNa-Green AM dye, Na+ fluorescence intensity, Plant growth, Propidium iodide, Salt stress, Z-stack
The quantitative analysis of tear analytes in point‐of‐care settings can enable early diagnosis of ocular diseases. Here, a fluorescent scleral lens sensor is developed to quantitatively measure physiological levels of pH, Na+, K+, Ca2+, Mg2+, and Zn2+ ions. Benzenedicarboxylic acid, a pH probe, displays a sensitivity of 0.12 pH units within pH 7.0–8.0. Crown ether derivatives exhibit selectivity to Na+ and K+ ions within detection ranges of 0–100 and 0–50 mmol L−1, and selectivities of 15.6 and 8.1 mmol L−1, respectively. A 1,2 bis(o‐aminophenoxy)ethane‐N,N,‐N',N'‐tetraacetic‐acid‐based probe allows Ca2+ ion sensing with 0.02–0.05 mmol L−1 sensitivity within 0.50–1.25 mmol L−1 detection range. 5‐Oxazolecarboxylic acid senses Mg2+ ions, exhibiting a sensitivity of 0.10–0.44 mmol L−1 within the range of 0.5–0.8 mmol L−1. The N‐(2‐methoxyphenyl)iminodiacetate Zn2+ ion sensor has a sensitivity of 1 µmol L−1 within the range of 10–20 µmol L−1. The fluorescent sensors are subsequently multiplexed in the concavities of an engraved scleral lens. A handheld ophthalmic readout device comprising light‐emitting diodes (LEDs) and bandpass filters is fabricated to excite as well as read the scleral sensor. A smartphone camera application and an user interface are developed to deliver quantitative measurements with data deconvolution. The ophthalmic system enables the assessment of dry eye severity stages and the differentiation of its subtypes. A fluorescent scleral lens sensor is developed to quantitatively analyze electrolyte concentrations in tears. The multiplexed sensors integrated with a smartphone camera readout system provide accurate results using a data deconvolution algorithm. The ophthalmic system allows the determination of severity stages and subtype differentiation of dry eye syndrome.
Plants exhibit an intriguing morphological and physiological plasticity that enables them to thrive in a wide range of environments. To understand the cell biological basis of this unparalleled competence, a number ofmethodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants.
Mitochondrial Ca²⁺ transient is the earliest discovered organellar Ca²⁺ signaling pathway. It consist of a Ca²⁺ influx, mediated by mitochondrial Ca²⁺ uniporter (MCU), and mitochondrial Ca²⁺ efflux mediated by a Na⁺/Ca²⁺ exchanger (NCLX). Mitochondrial Ca²⁺ signaling machinery plays a fundamental role in linking metabolic activity to cellular Ca²⁺ signaling, and in controlling local Ca²⁺ concertation in distinct cellular compartments. Impaired balance between mitochondrial Ca²⁺ influx and efflux leads to mitochondrial Ca²⁺ overload, an early and key event in ischemic or neurodegenerative syndromes. Molecular identification of NCLX and MCU happened only recently. Surprisingly, MCU knockout yielded a relatively mild phenotype while conditional knockout of NCLX led to a rapid fatal heart failure. Here we will focus on recent functional and molecular studies on NCLX structure and its mode of regulation. We will describe the unique crosstalk of this exchanger with Na⁺ and Ca²⁺ signaling pathways in the cell membrane and the endoplasmic reticulum, and with protein kinases that posttranslationally modulate NCLX activity. We will critically compare selectivity of pharmacological blockers versus molecular control of NCLX expression and activity. Finally we will discuss why this exchanger is essential for survival and can serve as an attractive therapeutic target.
The use of fluorescent chemical indicator dyes enables the dynamic and quantitative imaging of intracellular sodium concentrations and activity-related sodium transients in astrocytes.Here we describe different approaches for the loading of cellular networks or single astrocytes with sodium-sensitive indicators in brain tissue. Fluorescence signals can then be detected and analyzed with conventional camera-based, wide-field imaging or by employing high-resolution multi-photon microscopy. We furthermore explain strategies for the induction of local and global sodium transients in astrocytes. Finally, we illustrate how fluorescence signals derived from such imaging experiments can be converted into absolute changes of sodium concentration in astrocytes based on an in situ calibration procedure.
The rich composition of solutes and metabolites in sweat and its relative ease of collection upon excretion from skin pores make this class of biofluid an attractive candidate for point of care analysis. Wearable technologies that combine electrochemical sensors with conventional or emerging semiconductor device technologies offer valuable capabilities in sweat sensing, but they are limited to assays that support amperometric, potentiometric, and colorimetric analyses. Here, we present a complementary approach that exploits fluorometric sensing modalities integrated into a soft, skin-interfaced microfluidic system which, when paired with a simple smartphone-based imaging module, allows for in situ measurement of important biomarkers in sweat. A network array of microchannels and a collection of microreservoirs pre-filled with fluorescent probes that selectively react with target analytes in sweat (e.g. probes), enable quantitative, rapid analysis. Field studies on human subjects demonstrate the ability to measure the concentrations of chloride, sodium and zinc in sweat, with accuracy that matches that of conventional laboratory techniques. The results highlight the versatility of advanced fluorescent-based imaging modalities in body-worn sweat microfluidics platforms, and they suggest some practical potential for these ideas.
One hallmark of adult neurogenesis is its adaptability to environmental influences. Here, we uncovered the epithelial sodium channel (ENaC) as a key regulator of adult neurogenesis as its deletion in neural stem cells (NSCs) and their progeny in the murine subependymal zone (SEZ) strongly impairs their proliferation and neurogenic output in the olfactory bulb. Importantly, alteration of fluid flow promotes proliferation of SEZ cells in an ENaC-dependent manner, eliciting sodium and calcium signals that regulate proliferation via calcium-release-activated channels and phosphorylation of ERK. Flow-induced calcium signals are restricted to NSCs in contact with the ventricular fluid, thereby providing a highly specific mechanism to regulate NSC behavior at this special interface with the cerebrospinal fluid. Thus, ENaC plays a central role in regulating adult neurogenesis, and among multiple modes of ENaC function, flow-induced changes in sodium signals are critical for NSC biology.
Intracellular sodium concentration plays important roles in many cellular processes, but methods to analyze dynamic change of [Na⁺] in organelles of interest are still lacking. To address this problem, controlled localization of fluorescent Na⁺ probes is a key issue. In this work, we developed a protein-coupled Na⁺-sensitive fluorescent probe, HaloNa-1, which covalently labelled a HaloTag protein expressed at specific organelles in living cells. Fluorescence intensity of HaloNa-1 increased by approximately three-fold when complexed with Na⁺, and the dissociation constant was within physiological range. Notably, by conjugating a fluorescent protein to the HaloTag protein and using it as internal standard, one can reduce unwanted variation of probe fluorescence due to different cell volume, HaloTag expression level, etc. With this technique, we could observe elevation of intracellular [Na⁺] after ionophore stimulation with higher reliability than conventional sensors. We believe our probe is potentially useful to understand Na⁺ dynamics inside cells.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
New fluorescent Na⁺ indicators, SBFI (short for sodium-binding benzofuran isophthalate) and SBFP (short for sodium-binding benzofuran phthalate) (Minta, A., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 19449–19457), were tested in Jurkat tumor lymphocytes and in REF52 rat embryo fibroblasts. Both dyes could be introduced by direct microinjection. However, when cells were incubated with the tetra(acetoxymethyl) esters of the dyes, only SBFI gave intracellular loading that was reasonably responsive to [Na⁺]i. Because some compartmentation of the SBFI was visible and because the indicator properties are somewhat affected by cytoplasm, the relationship between intracellular free Na⁺ ([Na⁺]i and the 340/385 nm excitation ratio of the indicator was calibrated in situ using poreforming antibiotics to equilibrate cytosolic [Na⁺] ([Na⁺]i) with extracellular [Na⁺]. The excitation ratio was sufficiently sensitive to resolve small changes, ≤1 mM, in [Na⁺]i in single cells. Basal [Na⁺]i values in lymphocytes and serum-starved fibroblasts were 9.4 and 4.2 mM, respectively. As expected, large increases in [Na⁺]i were elicited by blocking the Na⁺ pump with ouabain or withdrawal of extracellular K⁺. Mitogens such as phytohemagglutinin acting on the lymphocytes, or serum or vasopressin in fibroblasts, caused [Na⁺]i to increase up to 2-fold. In fibroblasts, the rise in [Na⁺]i was due at least partly to a stimulation of Na⁺ influx, which was not wholly through the Na⁺/H⁺ exchanger. The mitogen-induced increases in [Na⁺]i and the rate of Na⁺ influx are consistent with earlier estimates based on measurements of total [Na⁺] or tracer fluxes. However, the absolute values for free [Na⁺]i are much lower than previous values for total intracellular Na⁺, suggesting that much of the latter is bound or sequestered.
Why are some humans considered more beautiful than others? Theory
suggests that sexually reproducing organisms should choose mates
displaying characters indicative of high genotypic or phenotypic
quality. Attraction to beautiful individuals may therefore be an
adaptation for choosing high-quality mates. Culturally invariant
standards of beauty in humans have been taken as evidence favouring such
an adaptationist explanation of attraction; however, if standards of
beauty are instead no more than artefacts of culture, they should vary
across cultures. Here we show that male preference for women with a low
waist-to-hip ratio (WHR) is not culturally universal, as had previously
been assumed.
We investigated the effects of oxygen (O2)/glucose deprivation on intracellular sodium concentration ([Na+]i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O2/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na+]i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (D-amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na+/Ca2+-exchanger, affected these responses to O2/ glucose. The present results demonstrate that, in cortical neurons, O2/glucose deprivation induces an early rise in [Na+]i which cannot be ascribed to the activity of voltage gated Na+-channels, glutamate receptors or of the Na+/Ca2+-exchanger.
The spatial and temporal dynamics of many electrophysiological and biochemical processes in nerve cells are in turn dependent on the concentration dynamics of the second messenger calcium. We have used microfluorimetry of the calcium indicator fura-2 (Grynkiewicz et al., 1985) to measure and characterize synaptically activated calcium changes in individual CA1 pyramidal cells contained within guinea pig hippocampal slices. One component of the calcium changes was largely produced by influx through voltage-dependent Ca2+ channels (VDCCs). It consisted of large transient accumulations in the proximal-apical and basal dendrites; the amplitude was smaller in the distal-apical dendrites and the soma. This spatial profile was insensitive to the method of cell activation: stimulation of inputs located at different positions on the dendritic tree as well as antidromic stimulation produced only slight modifications. This component was not blocked by the NMDA antagonist 5-amino-4-phosphonovalerate (AP5) (Collingridge et al., 1983), was greatly reduced by Cd2+, partially reduced by nifedipine, and was increased by Bay-K 8644, providing the evidence that it was largely produced by influx through VDCCs. Blocking postsynaptic Na+ channels with QX-314 greatly reduced the accumulation amplitude, and spatial differences between proximal-dendritic and distal-dendritic regions were less pronounced, suggesting that active sodium conductances contribute significantly to the spatial activation of calcium conductances. Residual spatial differences that persist in QX-314 experiments are consistent with the idea that VDCCs have decreased density on distal-apical dendrites. A second component of accumulation was induced by ionic currents through NMDA receptor channels. It was blocked by AP5, unaffected by QX-314, attenuated and slowed down by elevated calcium buffering, and spatially localized to regions receiving activated synaptic inputs. The magnitude of this component was strongly dependent on the frequency and amplitude of synaptic activation. At high frequency, it was generally very large, often saturating the fura-2 (> 2 microM). Measurements made with the indicator furaptra also showed large localized AP5-sensitive fluorescence changes. Our results suggest that in dendritic regions near activated input fibers calcium levels may reach 2-10 microM. In general, our measurements of calcium dynamics provide an experimental basis for evaluating the spatial distribution of calcium conductances, the spatial distribution of calcium-activated electrophysiological and biochemical processes, and the spatial uniformity of calcium buffering and removal systems in CA1 hippocampal pyramidal cells. The time course and amplitude of Ca2+ transients we measured suggest that activation of Ca(2+)-dependent conductances [e.g., IK(Ca)] will be markedly different for different cellular regions.(ABSTRACT TRUNCATED AT 400 WORDS)
(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.
Fluorescent indicators sensitive to cytosolic concentrations of free Na+ have been synthesized and characterized. They consist of a crown ether, 1,7-diaza-4,10,13-trioxacyclopentadecane, linked via its nitrogens to fluorophores bearing additional liganding centers. In the currently preferred dye, SBFI (short for sodium-binding benzofuran isophthalate), the fluorophores are benzofurans linked to isophthalate groups. Selectivities for Na+ over K+ of about 20 are observed, resulting in effective dissociation constants for Na+ of about 20 mM against a background of 120 mM K+. Increasing [Na+] increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging can be performed with the same wavelengths as used with the well known Ca2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K+ over Na+.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
The spatial distribution of Na(+)-dependent events in guinea pig Purkinje cells was studied with a combination of high-speed imaging and simultaneous intracellular recording. Individual Purkinje cells in sagittal cerebellar slices were loaded with either fura-2 or the Na+ indicator sodium binding benzofuran isophthalate (SBFI) with sharp electrodes or patch electrodes on the soma or dendrites. [Na+]i changes were detected in response to climbing fiber and parallel fiber stimulation. These changes were located both at the anatomically expected sites of synaptic contact in the dendrites and in the somatic region. The variation in time course of these [Na+]i changes in different locations implies that Na+ enters at the synapse and diffuses rapidly to locations of lower initial [Na+]i. The synaptically activated somatic [Na+]i changes probably reflect Na+ entry through voltage-sensitive Na+ channels because they were detected only when regenerative potentials were recorded in the soma. [Na+]i changes in response to antidromically or intrasomatically evoked Na+ action potentials also were confined to the cell body. These observations are in agreement with other evidence that Na+ spikes are generated in the somatic region of the Purkinje neuron and spread passively into the dendrites. Plateau potentials, evoked by depolarizing pulses to the soma or dendrites, caused [Na+]i changes only in the soma, indicating that the noninactivating Na+ channels contributing to this potential also were concentrated in this region. The climbing fiber-activated [Na+]i changes were blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that these changes were not due to direct stimulation of the Purkinje neuron or activation of metabotropic receptors. Direct depolarization of the soma or dendrites never caused dendritic [Na+]i increases, suggesting that the climbing fiber-activated [Na+]i changes in the dendrites are due to Na+ entry through ligand-gated channels. A climbing fiber-like regenerative potential could be recorded in the soma after anode break stimulation, parallel fiber activation, or depolarizing pulses to the soma. The [Na+]i changes evoked by all of these potentials were confined to the cell body region of the Purkinje cell. [Ca2+]i changes in the dendrites evoked by the anode break potential were small relative to climbing fiber-activated changes, suggesting that a Ca2+ spike was not evoked by this response. The anode break and directly responses were blocked by tetrodotoxin. These results suggest that the somatically recorded climbing fiber response is predominantly a Na(+)-dependent event, consisting of a few fast action potentials and a slower regenerative response activating the same channels as the Na+ plateau potential.
The influx of Na+ is fundamental to electrical signalling in the nervous system and is essential for such basic signals as action potentials and excitatory postsynaptic potentials. During periods of bursting or high levels of discharge activity, large increases in intracellular Na+ concentration ([Na+]i) are produced in neuronal soma and dendrites. However, the intracellular signalling function of raised postsynaptic [Na+]i is unknown. Here we show that [Na+]i regulates the function of NMDA (N-methyl-D-aspartate) receptors, a principal subtype of glutamate receptor. NMDA-receptor-mediated whole-cell currents and NMDA-receptor single-channel activity were increased by raising [Na+]i and channel activity decreased upon lowering [Na+]i; therefore, the activity of NMDA channels tracks changes in [Na+]i. We found that the sensitivity of the channel to Na+ was set by a Src kinase that is associated with the channel. Raising [Na+]i selectively increased synaptic responses mediated by NMDA receptors, but not by non-NMDA receptors. Thus, the change in postsynaptic [Na+]i that occurs during neuronal activity is a signal for controlling the gain of excitatory synaptic transmission. This mechanism may be important for NMDA-receptor-dependent plasticity and toxicity in the central nervous system.
Dendritic spines are assumed to be the smallest units of neuronal integration. Because of their miniature size, however, many of their functional properties are still unclear. New insights in spine physiology have been provided by two-photon laser-scanning microscopy which allows fluorescence imaging with high spatial resolution and minimal photodamage. For example, two-photon imaging has been employed successfully for the measurement of activity-induced calcium transients in individual spines. Here, we describe the first application of two-photon imaging to measure Na+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices. Whole-cell patch-clamped neurons were loaded with the Na(+)-indicator dye SBFI (sodium-binding benzofuran-isophthalate). In situ calibration of SBFI fluorescence with ionophores enabled the determination of the actual magnitude of the [Na+]i changes. We found that back-propagating action potentials (APs) evoked Na+ transients throughout the proximal part of the dendritic tree and adjacent spines. The action-potential-induced [Na+]i transients reached values of 4 mM for a train of 20 APs and monotonically decayed with a time constant of several seconds. These results represent the first demonstration of activity-induced Na+ accumulation in spines. Our results demonstrate that two-photon Na+ imaging represents a powerful tool for extending our knowledge on Na+ signaling in fine cellular subcompartments.
We have explored the consequences of a [Na(+)](i) load and oxidative stress in isolated nerve terminals. The Na(+) load was achieved by veratridine (5-40 microM), which allows Na(+) entry via a voltage-operated Na(+) channel, and oxidative stress was induced by hydrogen peroxide (0.1-0.5 mM). Remarkably, neither the [Na(+)](i) load nor exposure to H(2)O(2) had any major effect on [Ca(2+)](i), mitochondrial membrane potential (Deltapsim), or ATP level. However, the combination of an Na(+) load and oxidative stress caused ATP depletion, a collapse of Deltapsim, and a progressive deregulation of [Ca(2+)](i) and [Na(+)](i) homeostasis. The decrease in the ATP level was unrelated to an increase in [Ca(2+)](i) and paralleled the rise in [Na(+)](i). The loss of Deltapsim was prevented in the absence of Ca(2+) but unaltered in the presence of cyclosporin A. We conclude that the increased ATP consumption by the Na,K-ATPase that results from a modest [Na(+)](i) load places an additional demand on mitochondria metabolically compromised by an oxidative stress, which are unable to produce a sufficient amount of ATP to fuel the ATP-driven ion pumps. This results in a deregulation of [Na(+)](i) and [Ca(2+)](i), and as a result of the latter, collapse of Deltapsim. The vicious cycle generated in the combined presence of Na(+) load and oxidative stress could be an important factor in the neuronal injury produced by ischemia or excitotoxicity, in which the oxidative insult is superimposed on a disturbed Na(+) homeostasis.
An experimental difficulty in unraveling circuits in the mammalian nervous system is the identification of postsynaptic targets of a given neuron. Besides ultrastructural reconstructions, simultaneous recordings from pairs of cells in brain slices have been used to identify connected neurons. We describe in this paper a technique using calcium imaging that allows rapid identification of potential postsynaptic targets. This method consists of stimulating one neuron ("trigger") while imaging a population of cells to detect which other neurons ("followers") are activated by the trigger. By using bulk-loading of calcium indicators in slices of mouse visual cortex, we demonstrate that neurons that display somatic calcium transients time-locked to the spikes of a trigger neuron can be monosynaptically connected to it. This technique could be applied to reconstruct and assay circuits in the central nervous system.
Spines and dendrites of central neurons represent an important site of synaptic signaling and integration. Here we identify a new, synaptically mediated spine signal with unique properties. Using two-photon Na(+) imaging, we show that suprathreshold synaptic stimulation leads to transient increases in Na(+) concentration in postsynaptic spines and their adjacent dendrites. This local signal is restricted to a dendritic domain near the site of synaptic input. In presumed active spines within this domain, the Na(+) level increases by 30-40 mm even during short bursts of synaptic stimulation. During a long-term potentiation induction protocol (100 Hz, 1 sec), the Na(+) level in the active spines reaches peak amplitudes of approximately 100 mm. We find that the Na(+) transients are mainly mediated by Na(+) entry through NMDA receptor channels and are detected during the coincident occurrence of synaptic potentials and backpropagating action potentials. The large amplitudes of the Na(+) transients and their location on dendritic spines suggest that this signal is an important determinant of electrical and biochemical spine characteristics.
G protein-gated K+channels (GIRK, or Kir3) are activated by the direct binding of Gβγ or of cytosolic Na+. Na+ activation is fast, Gβγ-independent, and probably via a direct, low affinity (EC50, 30–40 mm) binding of Na+ to the channel. Here we demonstrate that an increase in intracellular Na+ concentration, [Na+]in, within the physiological range (5–20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking
the direct Na+ effect. It is inhibited by a Gβγ scavenger, hence it is Gβγ-dependent; but it does not require GTP. We hypothesized that
Na+ elevates the cellular concentration of free Gβγ by promoting the dissociation of the Gαβγ heterotrimer into free GαGDP and Gβγ. Direct biochemical measurements showed that Na+ causes a moderate decrease (∼2-fold) in the affinity of interaction between GαGDP and Gβγ. Furthermore, in accord with the predictions of our model, slow Na+ activation was enhanced by mild coexpression of Gαi3. Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gβγ-dependent regulation
of GIRK by Na+. We propose that Na+ may act as a regulatory factor, or even a second messenger, that regulates effectors via Gβγ.
Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes. Previous attempts to use membrane-permeant forms of fluorometric calcium indicators to load populations of neurons have yielded satisfactory results only in cell cultures or in slices of immature brain tissue. Here we introduce a versatile approach for loading membrane-permeant fluorescent indicator dyes in large populations of cells. We established a pressure ejection-based local dye delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including calcium green-1 acetoxymethyl (AM) ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. We applied this dye-loading protocol successfully in mouse brain tissue at any developmental stage from newborn to adult in vivo and in vitro. In vivo two-photon Ca2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex. Thus, our results demonstrate the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.
Dendritic spines are assumed to be the smallest units of neuronal integration. Because of their miniature size, however, many of their functional properties are still unclear. New insights in spine physiology have been provided by two-photon laser-scanning microscopy which allows fluorescence imaging with high spatial resolution and minimal photodamage. For example, two-photon imaging has been employed successfully for the measurement of activity-induced calcium transients in individual spines. Here, we describe the first application of two-photon imaging to measure Na+ transients in spines and dendrites of CA1 pyramidal neurons in hippocampal slices. Whole-cell patch-clamped neurons were loaded with the Na+-indicator dye SBFI (sodium-binding benzofuran-isophthalate). In situ calibration of SBFI fluorescence with ionophores enabled the determination of the actual magnitude of the [Na+]i changes. We found that back-propagating action potentials (APs) evoked Na+ transients throughout the proximal part of the dendritic tree and adjacent spines. The action-potential-induced [Na+]i transients reached values of 4 mM for a train of 20 APs and monotonically decayed with a time constant of several seconds. These results represent the first demonstration of activity-induced Na+ accumulation in spines. Our results demonstrate that two-photon Na+ imaging represents a powerful tool for extending our knowledge on Na+ signaling in fine cellular subcompartments.
The emission spectra calibration curves for a fluorescence indicator and the F-min, F-max, and K-d formula were shown to be related. Using the known calibrated fluorescence emitted by Sodium Green (Na-Green) and photo-multiplier-tube quantum efficiency, we calculated the detection signal over a range of sodium concentrations. The calculated calibration curves were compared for optical filters passing a narrow band, medium band or full spectrum. We found that a method based on the full emission spectrum was the most appropriate. Given a known resting concentration of intracellular sodium, calibrated readings can be converted to concentration values. This method is applicable to any fluorescence indicator when curves for emission spectra over a range of concentrations are available. We measured sodium concentration changes during trains of action potentials (APs) at a crayfish motor axon's presynaptic terminals injected with Na-Green. During low frequency AP trains, net sodium increases asymptotically with frequency. Average net Na-flux per AP decreases for increasing terminal size. The terminals of crayfish motor axon have surface area to volume ratio which is 7700 times larger than for squid. Thus, in comparison to squid, crayfish terminals exhibit a larger change in [Na+](i) during equivalent AP activity.
Neurons and glial cells maintain a steep inwardly directed electrochemical gradient for Na+ across their plasma membranes. This gradient is provided by Na +, K+-ATPase activity and energizes a multitude of transmembrane processes. Recent investigations have sug gested that sustained increases in Na+i, following inhibition of Na+, K+-ATPase, or Na+ influx via voltage- or transmitter-activated ion channels, are key events during a variety of pathological conditions. Breakdown of the Na+ gradient causes reverse operation of Na+/Ca2+- and Na+/H+-exchange, resulting in intracellular Ca2+ increase and acidification. In addition, reversal of Na +-dependent glutamate transport promotes glutamate export, which can result in toxic glutamate concentrations in the extracellular space. Further stud ies of the mechanisms of Na+i regulation in neurons and glial cells may provide more insights into events initiating cell damage in the nervous system. NEUROSCIENTIST 3:85-88, 1997
This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkinet al., 1952,J. Physiol.116, 424–448; Hodgkin and Huxley, 1952,J. Physiol.116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).
• Possible mechanisms responsible for the increases in intracellular calcium ([Ca2+]i) and sodium ([Na+]i) levels seen during metabolic inhibition were investigated by continuous [Ca2+]i and [Na+]i measurement in cultured rat cerebellar granule cells. An initial small mitochondrial Ca2+ release was seen, followed by a large influx of extracellular Ca2+. A large influx of extracellular Na+ was also seen. • The large [Ca2+]i increase was not due to opening of voltage-dependent or voltage-independent calcium channels, activation of NMDA/non-NMDA channels, activation of the Na+i-Ca2+o exchanger, or inability of plasmalemmal Ca2+-ATPase to extrude, or mitochondria to take up, calcium. • The large [Na+]i increase was not due to activation of the TTX-sensitive Na+ channel, the Na+i-Ca2+o exchanger, the Na+-H+ exchanger, or the Na+-K+-2Cl− cotransporter, or an inability of Na+-K+-ATPase to extrude the intracellular sodium. • Phospholipase A2 (PLA2) activation may be involved in the large influx, since both were completely inhibited by PLA2 inhibitors. Moreover, melittin (a PLA2 activator) or lysophosphatidylcholine or arachidonic acid (both PLA2 activation products) caused similar responses. Inhibition of PLA2 activity may help prevent the influx of these ions that may result in serious brain injury and oedema during hypoxia/ischaemia.
1The present study investigated the regulatory mechanism of the Na+,K+-ATPase and the level of internal Na+ and Ca2+ in response to persistent Na+ influx in acutely dissociated rat thalamic neurones.2Whole-cell patch-clamp recordings and Na+ imaging revealed a stable [Na+]i and low background pump activity. Exposure to veratridine (50 μm) for 1 h resulted in a progressive rise in [Na+]i (ΔFNa= 64 ± 22 %) and [Ca2+]i (ΔFCa= 44 ± 14%) over 3 h. Increases in [Na+]i and [Ca2+]i were also observed during neuronal exposure to the Na+ ionophore monensin (50 μm).3Subcellular confocal immunofluorescence quantification of α3 catalytic Na+-K+ pump subunits showed that a veratridine-induced rise in [Na+]i was accompanied by a significant increase in pump density in both membrane and cytoplasmic compartments, by 39 and 54%, respectively. Similar results were also obtained in experiments when neurones were treated with monensin.4A fluorescent 9-anthroylouabain binding assay detected a 60 and 110% increase in phosphorylated (active) pumps after veratridine and monensin exposure, respectively.5During the entire experiment, application of ouabain or veratridine alone induced little cell swelling and death, but pump inhibition in cells pre-loaded with Na+ led to rapid cell swelling and necrosis.6The above results indicate that a persistent influx of Na+ may trigger rapid enhancement of pump synthesis, membrane redistribution and functional activity. However, these compensatory mechanisms failed to prevent persistent Na+ accumulation.
This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant
nerve fibre (Hodgkinet al., 1952,J. Physiol.
116, 424–448; Hodgkin and Huxley, 1952,J. Physiol.
116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical
form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).
Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CAI pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.
Following our previous observations that anoxia induces a drop in extracellular Na+ in the brain slice and that removal of extracellular Na+ prevents the anoxia-induced morphological changes in dissociated hippocampal neurons, we hypothesized that intracellular Na+ increases during anoxia in isolated neurons. Using the fluorophore Sodium Green in freshly dissociated rat CA1 neurons, and SBFI in cultured cortical neurons, we found that 10 min of anoxia caused an increase in Nai+ in both types of cells, with a latency of about 2 min. In CA1 neurons, fluorescence increased by an average of 20.34% (n = 8). The mean baseline Nai+ level (determined using SBFI) was 25 ± 2.5 mM, whichincreased to about an average of 52 ± 3 mM after 3–4 min. These and our previous results strongly suggest that Na+-mediated events are involved in anoxia-induced nerve injury.
(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.
Transient changes in sodium concentration in response to electrical activity were detected in Purkinje cells by using the fluorescent indicator SBFI (Minta & Tsien (J. biol. Chem. 264, 19,449 (1989)). Fast sodium action potentials caused large increases in internal sodium concentration, [Na]i, in the soma and axon, and were generally undetectable in the dendrites. No changes were detected in the dendrites corresponding to calcium action potentials. The spatial distribution of these transients corresponds to that expected if the increase in [Na]i were the result of Na+ entry through voltage-dependent Na channels generating sodium spikes in the axon hillock and soma. The [Na]i transients rapidly recovered (tau less than 1 s) in the axon hillock, probably by Na+ diffusion into the soma. Climbing fibre activation produced distinct [Na]i transients in the dendrites in addition to somatic and axonal signals. As regenerative potentials did not produce transients in this region, these signals may be caused by Na+ entry through ligand-gated channels. These results confirm and extend the description of channel distribution and electrical signalling in Purkinje cells.
The dendrites of many types of neurons contain voltage-dependent Na+ and Ca2+ conductances that generate action potentials (see ref. 1 for review). The function of these spikes is not well understood, but the Ca2+ entry stimulated by spikes probably affects Ca(2+)-dependent processes in dendrites. These include synaptic plasticity, cytotoxicity and exocytosis. Several lines of evidence suggest that dendritic spikes occur within subregions of the dendrites. To study the mechanism that govern the spread of spikes in the dendrites of hippocampal pyramidal cells, we imaged Ca2+ entry with Fura-2 (ref. 9) and Na+ entry with a newly developed Na(+)-sensitive dye. Our results indicate that Ca2+ entry into dendrites is triggered by Na+ spikes that actively invade the dendrites. The restricted spatial distribution of Ca2+ entry seems to depend on the spread of Na+ spikes in the dendrites, rather than on a limited distribution of Ca2+ channels. In addition, we have observed an activity-dependent process that modulates the invasion of spikes into the dendrites and progressively restricts Ca2+ entry to more proximal dendritic regions.
1. The fluorescent Na+ indicator SBFI was incorporated into isolated ventricular myocytes using the acetoxymethyl (AM) ester. 2. The excitation spectrum was found to be shifted about 20 nm in the cell compared to in vitro. In the cell, an increase of [Na+] decreased fluorescence at 380 nm (F380) and had no effect at 340 nm (F340). The ratio (R = F340/F380) was used as a measure of [Na+]i. 3. In vivo calibration of SBFI for [Na+]i was obtained by equilibrating [Na+] across the plasma membrane with a divalent-free solution in the presence of gramicidin D. 4. Selective removal of the surface membrane with saponin or digitonin released only about 50% of the indicator. Following saponin treatment, cyanide or carbonylcyanide m-chlorphenylhydrazone (CCCP) increased the apparent [Na+] measured by the remaining (presumably mitochondrial) SBFI. It is suggested that mitochondrial [Na+] is normally less than cytoplasmic. 5. Attempts to examine the effects of metabolic inhibition on [Na+]i were hampered by changes of autofluorescence due to changes of [NADH]. It is shown that this effect can be corrected for using the isosbestic signal (excited at 340 nm). 6. Inhibition of both aerobic metabolism (with CN-) and glycolysis (glucose removal or iodoacetate) produced a gradual increase of [Na+]i. This began before the resting contracture developed and may (via Na(+)-Ca2+ exchange) account for some of the rise of diastolic [Ca2+]i seen in previous work. The rise of [Na+]i began at about the same time as the decrease of systolic contraction and therefore at a time when [ATP]i had begun to fall.
New fluorescent Na+ indicators, SBFI (short for sodium-binding benzofuran isophthalate) and SBFP (short for sodium-binding benzofuran phthalate) (Minta, A., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 19449-19457), were tested in Jurkat tumor lymphocytes and in REF52 rat embryo fibroblasts. Both dyes could be introduced by direct microinjection. However, when cells were incubated with the tetra(acetoxymethyl) esters of the dyes, only SBFI gave intracellular loading that was reasonably responsive to [Na+]i. Because some compartmentation of the SBFI was visible and because the indicator properties are somewhat affected by cytoplasm, the relationship between intracellular free Na+ [( Na+]i and the 340/385 nm excitation ratio of the indicator was calibrated in situ using poreforming antibiotics to equilibrate cytosolic [Na+] [( Na+]i) with extracellular [Na+]. The excitation ratio was sufficiently sensitive to resolve small changes, less than or equal to 1 mM, in [Na+]i in single cells. Basal [Na+]i values in lymphocytes and serum-starved fibroblasts were 9.4 and 4.2 mM, respectively. As expected, large increases in [Na+]i were elicited by blocking the Na+ pump with ouabain or withdrawal of extracellular K+. Mitogens such as phytohemagglutinin acting on the lymphocytes, or serum or vasopressin in fibroblasts, caused [Na+]i to increase up to 2-fold. In fibroblasts, the rise in [Na+]i was due at least partly to a stimulation of Na+ influx, which was not wholly through the Na+/H+ exchanger. The mitogen-induced increases in [Na+]i and the rate of Na+ influx are consistent with earlier estimates based on measurements of total [Na+] or tracer fluxes. However, the absolute values for free [Na+]i are much lower than previous values for total intracellular Na+, suggesting that much of the latter is bound or sequestered.
Cerebellar long-term depression (LTD) is a persistent attenuation of the parallel fiber-Purkinje neuron (PF-PN) synapse induced by conjunctive stimulation of PF and climbing fiber (CF) inputs. A similar phenomenon is seen in the voltage-clamped PN in tissue culture when iontophoretic quisqualate application and PN depolarization are substituted for PF and CF stimulation, respectively. In this model, LTD induction requires activation of both AMPA and metabotropic receptors, together with PN depolarization. We have sought to determine the role of the AMPA receptor in LTD induction. The AMPA receptor does not appear to exert its effect by directly gating Ca2+ influx. Replacement of external Na+ during quisqualate/depolarization conjunction with permeant ions caused a blockade of LTD induction, suggesting that Na+ influx through the AMPA-associated channel is necessary for this process. Similarly, pairing quisqualate pulses with depolarizing steps near ENa also failed to induce LTD. The present results indicate that postsynaptic Na+ influx is necessary for LTD induction. While a portion of the relevant Na+ influx is provided by voltage-gated channels, the AMPA-associated ion channel is most important in this regard.
Changes in cytosolic Na+ ([Na+]i) caused by a toxic glutamate (GLU) or NMDA treatment of cultured hippocampal neurons were monitored by using SBFI fluorescent probe and imaging microscopy. Both GLU and NMDA (50 or 100 microM in Mg(2+)-free solution, 15 min) induced a marked increase in [Na+]i (from 6-8 to 30-45 mM) which persisted after the termination of a treatment. The competitive NMDA antagonist, APV (100 microM) when applied in the post-NMDA period failed to decrease the elevated [Na+]i. The results obtained strongly suggest that the main reason for an impairment of Na+/Ca2+ exchange in the post-glutamate period (see Febs Letters 1993, 324, 271-273) is a reduction of the transmembrane Na+ gradient caused apparently by inhibition of Na(+)-K+ pump.
We measured changes in [Ca2+]i and [Na+]i in the dendrites of cerebellar Purkinje cells and hippocampal pyramidal cells using high speed imaging of the fluorescence changes of intracellularly injected fura-2 and SBFI. These transients were detected in synchrony with intracellular recordings of membrane potential. In this way rapid calcium or sodium transients could be associated with specific electrical events. Using this technique we could determine the spatial distribution and source of transients evoked either intrasomatically or synaptically and could relate them to electrically recorded sodium and calcium spikes.
1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI-AM (acetoxymethylester of sodium-binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the 'Na+' signal. 2. Baseline [Na+]i was 14.6 +/- 4.9 mM (mean +/- S.D.) in CO2/HCO3(-)-containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/- 0.32 mM min-1) followed by a slower phase (0.15 +/- 0.09 mM min-1). 3. Changing from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)-K(+)-2Cl- cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage-gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)-ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min-1, respectively. This indicated that Na+, K(+)-ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o-induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/- 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)-HCO3- cotransport, Na(+)-K(+)-2Cl- cotransport and Na+, K(+)-ATPase, and that both Na+, K(+)-ATPase and inward Na(+)-K(+)-2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.
Propagation of action potentials in axons and dendrites increases intracellular Na+ ([Na+]i) and Ca2+ concentrations ([Ca2+]i). While the importance of [Ca2+]i in synaptic transmission is well established, a possible functional role of [Na+]i is unclear. In cultured hippocampal cells, [Na+]i was increased by veratridine. We have then measured spontaneous excitatory postsynaptic currents (sEPSCs) and, by means of fluorescent dyes, changes in [Na+]i, in [Ca2+]i, and in the turnover of the vesicular pool of individual boutons. An elevation of [Na+]i and a concomitant rise in [Ca2+]i, led to a large increase in sEPSC frequency and in the turnover of the presynaptic vesicular pool. Extracellular Ca2+ was essential for these effects of elevated [Na+]i on synaptic transmission. They probably occur via Na+/Ca2+ exchange.
1. We studied regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal neurones using fluorescence ratio imaging of the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). 2. In standard CO2/HCO3(-)-buffered saline with 3 mM K+, neurones had a baseline [Na+]i of 8.9 +/- 3.8 mM (mean +/- S.D.). Spontaneous, transient [Na+]i increases of 5 mM were observed in neurones on 27% of the coverslips studied. These [Na+]i increases were often synchronized among nearby neurones and were blocked reversibly by 1 microM tetrodotoxin (TTX) or by saline containing 10 mM Mg2+, suggesting that they were caused by periodic bursting activity of synaptically coupled cells. Opening of voltage-gated Na+ channels by application of 50 microM veratridine caused a TTX-sensitive [Na+]i increase of 25 mM. 3. Removing extracellular Na+ caused an exponential decline in [Na+]i to values close to zero within 10 min. Inhibition of Na+,K(+)-ATPase by removal of extracellular K+ or ouabain application evoked a [Na+]i increase of 5 mM min-1. Baseline [Na+]i was similar in the presence or absence of CO2/HCO3-; switching from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline, however, increased [Na+]i transiently by 3 mM, indicating activation of Na(+)-dependent Cl(-)-HCO3- exchange. Inhibition of Na(+)-K(+)-2Cl- cotransport by bumetanide had no effect on [Na+]i. 4. Brief, small changes in extracellular K+ concentration ([K+]o) influenced neuronal [Na+]i only weakly. Virtually no change in [Na+]i was observed with elevation or reduction of [K+]o by 1 mM. Only 30% of cells reacted to 3 min [K+]o elevations of up to 5 mM. In contrast, long [K+]o alterations (> or = 10 min) to 6 mM or greater slowly changed steady-state [Na+]i in the majority of cells. 5. Our results indicate several differences between [Na+]i regulation in cultured hippocampal neurones and astrocytes. Baseline [Na+]i is lower in neurones compared with astrocytes and is mainly determined by Na+,K(+)-ATPase, whereas Na(+)-dependent Cl(-)-HCO3- exchange, Na(+)-HCO3- cotransport or Na(+)-K(+)-2Cl- cotransport do not play a significant role. In contrast to glial cells, [Na+]i of neurones changes only weakly with small alterations in bath [K+]o, suggesting that activity-induced [K+]o changes in the brain might not significantly influence neuronal Na+,K(+)-ATPase activity.
Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na+]i in neighboring astrocytes was very similar (12.0 +/- 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na+]i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and alpha-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na+ load induced by removal of extracellular K+, indicating that octanol's effects on baseline [Na+]i were not due to inhibition of Na+, K+-ATPase activity. Under control conditions, increasing [K+]o from 3 to 8 mM caused similar decreases in [Na+]i in groups of astrocytes, presumably by stimulating Na+, K+-ATPase. During octanol application, [K+]o-induced [Na+]i decreases were amplified in cells with increased baseline [Na+]i, and reduced in cells with decreased baseline [Na+]i. This suggests that baseline [Na+]i in astrocytes "sets" the responsiveness of Na+, K+-ATPase to increases in [K]o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na+]i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na+ ions to equalize baseline [Na+]i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion.
We investigated the effect of changes in membrane‐voltage on intracellular sodium concentration ([Na ⁺ ] i ) of dopamine‐sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole‐cell patch‐clamp techniques were combined with microfluorometric measurements of [Na ⁺ ] i using the Na ⁺ ‐sensitive probe, sodium‐binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously
active dopamine neurons (recorded in current‐clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na ⁺ ] i . In dopamine neurons voltage‐clamped at a holding potential of −60 mV elevations of [Na ⁺ ] i were induced by long‐lasting (45–60 s) voltage jumps to more negative membrane potentials (–90 to −120 mV) but not by corresponding voltage jumps to −30 mV. These hyperpolarization‐induced elevations of [Na ⁺ ] i were depressed during inhibition of I h , a hyperpolarization‐activated inward current, by Cs ⁺ . Hyperpolarization‐induced elevations in [Na ⁺ ] i might occur also in other cell types which express a powerful I h and might signal lack of postsynaptic activity.
We investigated the effects of oxygen (O2)/glucose deprivation on intracellular sodium concentration ([Na+]i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O2/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na+]i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (D-amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na+/Ca2+-exchanger, affected these responses to O2/ glucose. The present results demonstrate that, in cortical neurons, O2/glucose deprivation induces an early rise in [Na+]i which cannot be ascribed to the activity of voltage gated Na+-channels, glutamate receptors or of the Na+/Ca2+-exchanger.
1. Whole-cell recordings were made from CA1 pyramidal cells in mouse hippocampal slices with patch pipettes containing the sodium indicator dye SBFI (sodium binding benzofuran isophthalate). Using a high-speed imaging system, we investigated changes in intracellular sodium concentration, [Na+]i, in response to hyperpolarizing pulses applied to the soma. 2. In current-clamp recordings, we detected increases in [Na+]i during negative current injection. Hyperpolarization-induced [Na+]i elevation was more prominent in the middle apical dendrites than in the soma. 3. In the voltage-clamp mode, hyperpolarization induced rapid increases in [Na+]i at the apical dendrites that were significantly faster than those at the soma. The signals were not affected by bath application of 1 microM TTX, but were reduced by 5 mM CsCl. 4. Changes in membrane potential recorded from the apical dendrites in response to negative currents were significantly smaller than those recorded from the soma. In the presence of 5 mM CsCl, the I-V relationships measured at the soma and the dendrites became almost identical, indicating that CsCl-sensitive components are predominantly in the apical dendrites. 5. These results suggest that hyperpolarization-induced [Na+]i elevations reflect Na+ influx through the non-selective cation channel (Ih channel), and that this channel is distributed predominantly in the apical dendrites. The non-uniform Na+ influx may contribute to integrative functions of the dendrites.
Glutamate uptake is coupled to counter-transport of K+, and high external K+ concentrations can induce reversal of glutamate uptake in whole-cell patch-clamp and isolated membrane preparations. However, high external K+ causes little or no reversal of glutamate uptake in intact astrocytes, suggesting a regulatory mechanism not evident in membrane preparations. One mechanism by which intact cells could limit the effects of altered extracellular ion concentrations on glutamate transport is by compensatory changes in intracellular Na+ concentrations. This possibility was examined using astrocyte cultures treated in two ways to reduce the driving force for glutamate uptake: incubation in high K+ (with reciprocal reduction in Na+), and incubation with metabolic inhibitors to induce ATP depletion. ATP depletion produced a rise in intracellular Na+, a collapse of the membrane sodium gradient and a massive reversal of glutamate uptake. By contrast, incubation in high K+/low Na+ medium did not significantly alter the sodium gradient and did not induce glutamate uptake reversal. The sodium gradient was shown to be maintained under these conditions by compensatory reductions in intracellular Na+ that approximately matched the reductions in extracellular Na+. These findings suggest a mechanism by which astrocytes may limit reversal of glutamate uptake under high K+/low Na+ conditions, and further suggest a general mechanism by which Na(+)-dependent transport processes could be shielded from fluctuating extracellular ion concentrations.
This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (tau2 = 2.4 +/- 0.2 ns) to that of the short component (tau1 = 0.4 +/- 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.
Cerebellar Purkinje cells express both ionotropic glutamate receptors and metabotropic glutamate receptors. Brief tetanic stimulation of parallel fibers in rat and mouse cerebellar slices evokes a slow excitatory postsynaptic current in Purkinje cells that is mediated by the mGluR1 subtype of metabotropic glutamate receptors. The effector system underlying this mGluR1 EPSC has not yet been identified. In the present study, we recorded the mGluR1 EPSC using the whole-cell patch-clamp technique in combination with microfluorometric recordings of the intracellular sodium concentration ([Na+]i) by means of the fluorescent sodium indicator SBFI. The mGluR1 EPSC was induced by local parallel fibre stimulation in the presence of the ionotropic glutamate receptor antagonists NBQX and D-APV and the GABAA receptor antagonists bicuculline or picrotoxin. The mGluR1 EPSC was associated with an increase in [Na+]i that was restricted to a specific portion of the dendritic tree. The mGluR1 EPSC as well as the increase in [Na+]i were inhibited by the mGluR antagonist S-MCPG. In the presence of NBQX, D-APV, pictrotoxin and TTX, bath application of the selective mGluR agonist 3,5-DHPG induced an elevation in [Na+]i which extended over the whole dendritic field of the Purkinje cell. This finding demonstrates that the mGluR1-mediated postsynaptic current leads to a significant influx of sodium into the dendritic cytoplasm of Purkinje cells and thereby provides a novel intracellular signalling mechanism that might be involved in mGluR1-dependent synaptic plasticity at this synapse.
The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.
Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to measure intracellular free Na+ concentrations ([Na+](i)), the in situ calibration techniques described to date do not favor the straightforward determination of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440-3450, 1985) to convert the ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a "full" in situ calibration are fit by a three-parameter hyperbolic equation; the apparent dissociation constant (K(d)) of SBFI for Na+ can then be resolved by means of a three-parameter hyperbolic decay equation. We also developed and tested a "one-point" technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at the end of an experiment during exposure to gramicidin D and 10 mM Na+ is used as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+](i) using a modification of the standard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Although the K(d) of SBFI for Na+ was relatively insensitive to changes in pH in the range 6.8-7.8, acidification resulted in an apparent decrease, and alkalinization in an apparent increase, in [Na+](i) values. The magnitudes of the apparent changes in [Na+](i) varied with absolute [Na+](i), and a method was developed for correcting [Na+](i) values measured with SBFI for changes in intracellular pH.
This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkinet al., 1952,J. Physiol.116, 424–448; Hodgkin and Huxley, 1952,J. Physiol.116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical form (Section 2) and to whow that they will account for conduction and excitation in quantitative terms (Sections 3–6).
Glutamate is the primary excitatory amino acid neurotransmitter in the central nervous system and its activity is carefully modulated in the synaptic cleft by glutamate transporters. A number of glutamate transporters have been identified in the central nervous system and each has a unique physiologic property and distribution. Glutamate transporter dysfunction may either be an initiating event or part of a cascade leading to cellular dysfunction and ultimately cell death. Animal models of glutamate transporter dysfunction have revealed a significant role for these proteins in pathologic conditions such as neurodegenerative diseases, epilepsy, stroke, and central nervous system tumors. Recent work has focused on glutamate transporter biology in human diseases with an emphasis on how manipulation of these transporter proteins may lead to therapeutic interventions in neurologic disease.
We report a method for the concurrent measurement of intracellular [Na+] ([Na+]i) and pH (pHi) in cells co-loaded with SBFI, a Na+-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H+-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na+]i and pHi in rat hippocampal neurons. Neither the Na+ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na+]i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower pK
a in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na+]i and pHi observed in response either to intracellular acid loads imposed by the NH4+ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na+]i and pHi.