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Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterised by the formation of insoluble amyloidogenic plaques and neurofibrillary tangles. Beta amyloid (Aβ) peptide is one of the main constituents in Aβ plaques, and is thought to be a primary causative agent in AD. Neurons are likely to be exposed to chronic, sublethal doses of Aβ over an extended time during the pathogenesis of AD, however most studies published to date using in vitro models have focussed on acute studies. To experimentally model the progressive pathogenesis of AD, we exposed primary cortical neurons daily to 1 μM of Aβ1-40 over 7 days and compared their survival with age-similar untreated cells. We also investigated whether chronic Aβ exposure affects neuronal susceptibility to the subsequent acute excitotoxicity induced by 10 μM glutamate and assessed how Ca2+ and K+ homeostasis were affected by either treatment.
We show that continuous exposure to 1 μM Aβ1-40 for seven days decreased survival of cultured cortical neurons by 20%. This decrease in survival correlated with increased K+ efflux from the cells. One day treatment with 1 μM Aβ followed by glutamate led to a substantially higher K+ efflux than in the age-similar untreated control. This difference further increased with the duration of the treatment. K+ efflux also remained higher in Aβ treated cells 20 min after glutamate application leading to 2.8-fold higher total K+ effluxed from the cells compared to controls. Ca2+ uptake was significantly higher only after prolonged Aβ treatment with 2.5-fold increase in total Ca2+ uptake over 20 min post glutamate application after six days of Aβ treatment or longer (P < 0.05).
Our data suggest that long term exposure to Aβ is detrimental because it reduces the ability of cortical neurons to maintain K+ and Ca2+ homeostasis in response to glutamate challenge, a response that might underlie the early symptoms of AD. The observed inability to maintain K+ homeostasis might furthermore be useful in future studies as an early indicator of pathological changes in response to Aβ.
Alzheimer's disease (AD) is the most common form of dementia within the ageing population and accounts for between 50% and 60% of dementia cases . Sufferers of AD experience progressive loss of memory and cognitive abilities that eventually lead to dementia and death. The pathological hallmarks of the disease include extracellular β-amyloid (Aβ) plaques, intracellular neurofibrillary tangles (NFTs) and dystrophic neurites (DNs) . The Aβ peptide is one of the main constituents in Aβ plaques, and is thought to be a primary causative agent in AD, significantly contributing towards AD pathogenesis.
AD is a progressive disease which develops over many years, even decades. Therefore the pathogenesis of AD does not entail a sudden insult of Aβ which causes widespread neuronal death within the brain. Instead, there appears to be a gradual progression of the disease which involves the accumulation of soluble Aβ within the brain due to the chronic imbalance between production and clearance of Aβ. This gradual accumulation of Aβ over extended periods of time leads to the formation of the insoluble Aβ aggregates which form the characteristic plaques, but it also modifies neuronal function. It is difficult to correlate early stages of AD pathogenesis with the accumulation of Aβ, as patients remain asymptomatic. Studies using transgenic mice which express human mutant Aβ precursor peptide (APP, Tg2576 mice) demonstrated that the increase of Aβ1-40 and Aβ1-42 over several months was accompanied by deficits in normal learning and memory [3,4]. When the Aβ load was minimal, mice demonstrated normal spatial learning and memory, indicating that the gradual build up of Aβ correlated with the physiological changes associated with AD. Studies have demonstrated that many molecular forms of Aβ are neurotoxic causing neuronal cell death in vitro and neuronal loss in vivo. For example, synthetic Aβ peptides were toxic to hippocampal and cortical neurons in culture [5,6].
Neurons are likely to be exposed to sublethal doses of Aβ over an extended time during the pathogenesis of AD. However studies to date have not developed an experimental model of this chronic exposure. There have been numerous in vitro models used to examine Aβ neurotoxicity using various forms and concentration of the peptide, over the short term. For instance Deshpande and colleagues examined the toxicity of a single dose of 5 μM fibrillar Aβ1-42 and Aβ-derived diffusible ligands on human cortical neuron cultures for up to 24 hours . At the same time studies examining the chronic exposure of neurons to Aβ at sublethal doses would provide valuable information relative to the physiological processes which occur in the AD brain.
The accumulation of toxic Aβ peptide aggregates in AD brain is thought to trigger the extensive synaptic loss and neurodegeneration linked to cognitive decline, an idea that underlies the 'amyloid hypothesis' of AD etiology in both the familial and sporadic forms of the disease . Recent reports strongly suggest that in the initial stages of AD, glutamate receptors are dysregulated by Aβ accumulation resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits . High concentrations of glutamate have been documented to cause neuronal degeneration in various in vivo and in vitro models [9,10]. Therefore, it would be of interest whether chronic neuron exposure to Aβ would sensitise them to excitotoxicity caused by increased glutamate concentration.
Excessive glutamatergic stimulation is associated with an increase in intracellular calcium ([Ca2+]i). Although Ca2+ is necessary for a number of physiological processes, excessive amounts may lead to neuronal dysfunction and cell death. Neuronal increases in [Ca2+]i can activate a number of enzymes, such as phospholipases, proteases, endonucleases and nitric oxide synthase (NOS) that are associated with neuronal cell death . Increases in cytosolic Ca2+ levels were observed in AD mouse models strongly supporting the theory that Ca2+ dysregulation is involved in AD [12-14]. Recent data also indicate that rodent and human Aβ modulate K+ currents . Voltage-dependent K+ (Kv) channels act as potent modulators of diverse excitatory events that are linked to glutamatergic neurotransmission  including modulation of Ca2+ uptake. Changes in ion fluxes are one of the earliest events in neural responses to Aβ treatment that precede neuron survival. Therefore, delineation between neuronal ability to maintain Ca2+ and K+ homeostasis during prolonged Aβ treatment and their viability might provide inside into mechanisms underlying processes of neurodegeneration.
To experimentally model the progressive pathogenesis of AD, primary cortical neurons were exposed to sublethal doses of Aβ1-40 over an extended period of time of 7 days. In this study we compared survival of cortical neurons treated for different periods of time with Aβ1-40 and correlated them to the neuron's ability to maintain homeostasis of Ca2+ and K+ ions under the same conditions. Finally, we investigated whether prolonged exposure to relatively low Aβ1-40 (1 μM) increased neuronal susceptibility to subsequent acute excitotoxicity induced by the neurotransmitter glutamate.
We first examined whether chronic exposure of mature neurons to low levels of Aβ caused cell death. Cortical neurons were maintained for 6 DIV, by which time they had formed a dense meshwork of neuritic processes. They were then treated with soluble monomeric Aβ1-40 (1 μM) daily for up to a further seven days. Daily determination of neuronal viability by an Alamar Blue assay revealed that Aβ1-40 treatment did not reduce cell viability for cells treated for up to six days (Figure (Figure1).1). However, by seventh day of the treatment neuronal viability decreased by 20% (P < 0.05).
Inability to maintain Ca2+ homeostasis has been strongly implicated in AD [12-14]. Therefore, we investigated whether ion homeostasis would be affected by Aβ. We first examined whether acute exposure of mature neurons to low levels of a soluble Aβ1-40 affect changes in K+ and Ca2+ fluxes. We therefore performed simultaneous recordings of K+ and Ca2+ fluxes from cortical neurons using Microelectrode Ion Flux Estimation (MIFE) after an acute application of 1 μMAβ1-40 to primary 14 DIV neurons (Additional file 1, Figure S1). MIFE enables sensitive, real time measurement of ion fluxes across cell membranes in a variety of cell models and is non-invasive [17,18]. No changes in net K+ and Ca2+ fluxes were observed within 15 min post treatment with 1 μM Aβ1-40 that served as a control for our further experiments. We also found no changes in net K+ and Ca2+ fluxes in response to vehicle (Additional file 2, Figure S2) further confirming our findings.
Under physiological conditions neurons are continuously exposed to low doses of Aβ. To simulate those conditions we treated neurons with 1 μM Aβ over a prolonged period of time measuring ion fluxes of interest. Therefore, we investigated whether chronic treatment with soluble Aβ1-40 can affect the ability of cortical neurons to maintain Ca2+ homeostasis (Figure (Figure2).2). We also tested whether K+ homeostasis would be affected by Aβ treatments since fluxes of this ion are pivotal to neuronal activity. No difference was observed in the level of Ca2+ fluxes between untreated cells, and cells treated with Aβ1-40 for up to eight days (Figure (Figure2A).2A). At the same time significant differences were observed in K+ fluxes (Figure (Figure2B).2B). While the K+ flux was similar in control cultures and those treated with Aβ1-40 for one and three days, treatment with a soluble monomeric Aβ1-40 (1 μM) daily for a period of six days or longer led to a significantly higher K+ efflux from the neural cells. Specifically, six and eight day Aβ treatment resulted in four- and three-fold increase in K+ efflux from the cells, respectively (P < 0.02)suggesting that chronic treatment with Aβ1-40 leads to a reduced capacity of neurons to maintain K+ homeostasis.
We next investigated whether neurons exposed to prolonged Aβ1-40 treatment had increased susceptibility to subsequent excitotoxicity induced by 200 μM glutamate. While glutamate treatment for 24 hours killed approximately 20% of neurons in the absence of any Aβ1-40 treatment (Figure (Figure3A),3A), we found that seven days of pre-treatment with Aβ1-40 did not further elevate neuronal susceptibility to glutamate-induced neurotoxicity (Figure (Figure3B).3B). We then examined K+ and Ca2+ fluxes in neurons which were treated with 1 μM Aβ1-40 daily for a period from one to eight days and then challenged with 10 μM of glutamate. Using MIFE, net fluxes of K+ and Ca2+ were monitored continuously throughout the experiment starting before the glutamate challenge and up to 30 min afterwards with data acquired every 6 sec. Typical examples of kinetics of net K+ and Ca2+ fluxes in response to challenge with glutamate are shown in Figures 4A-B and 5A-B, respectively.
Acute treatment of cortical neurons with 10 μM glutamate led to a dramatic K+ efflux from neurons that returned to pre-stress conditions within 20 min after the challenge (Figures 4A, B). Notably, the magnitude of K+ efflux was higher in cells treated with Aβ for six days or longer (peak values in the graphs). We therefore compared the magnitudes of the peak K+ efflux from neurons treated with Aβ with age-similar controls and found that K+ efflux was substantially (1.6-fold) higher even after one day of treatment with soluble Aβ1-40 (Figure (Figure6A).6A). This difference was more pronounced with increase of duration of the treatment with Aβ leading to more than 3.5-fold increase in the peak K+ efflux after six and eight days of treatment with soluble monomeric Aβ1-40 (1 μM), (Figure (Figure6A6A).
We also assessed capacity of the cortical neurons to return K+ flux to pre-stress levels after glutamate challenge. For this we compared steady-state values of K+ fluxes 20 min after glutamate challenge in Aβ treated and age-similar un-treated control neurons (Figure (Figure6B).6B). While the level of K+ flux had returned to basal levels in non Aβ treated cells, this was not the case when neurons had received continual treatment with Aβ, suggesting that Aβ1-40 treated neurons have reduced capacity to maintain K+ homeostasis in response to glutamate stimulation. We also calculated total K+ efflux over 20 min after acute glutamate application shown as shaded area in Figure Figure4B.4B. Total amount of K+ efflluxed from the cells over 20 min post glutamate application was substantially higher after treatment with low concentration of Aβ for six days or longer. For example, it was 2.8-fold higher in cells pre-treated with Aβ for six days than in age-similar controls (22228.1 ± 2800.13 vs 8033.46 ± 3799.76 nmol m-2, respectively, P < 0.05) further suggesting dysregulation of K+ homeostasis in Aβ treated cells.
We similarly used MIFE to examine Ca2+ fluxes following glutamate challenge to control and Aβ treated cortical neurons. Neurons which had been exposed to glutamate challenge (10 μM) rapidly took up Ca2+ followed by a return to pre-stress levels (Figure 5A, B). In neurons which had been exposed to Aβ for one and three days, glutamate induced an influx of Ca2+ that was not statistically different from age-similar control cells (Figure (Figure7A).7A). However, cortical neurons that received soluble Aβ1-40 for six consecutive days or longer had a significant impairment in their ability to maintain Ca2+ homeostasis, resulting in significantly greater Ca2+ uptake following excitotoxic stimulation with glutamate (Figure (Figure7A).7A). The magnitude of the peak Ca2+ influx was substantially higher in Aβ-treated cells than in the age-similar control cells (Figure (Figure7A).7A). At the same time steady-state values of Ca2+ fluxes measured 20 min after the glutamate challenge were not statistically different from the relevant values of age-similar control cells even after six and eight days of Aβ treatment, despite an apparent trend towards an increase in Ca2+ flux values (Figure (Figure7B).7B). However, a calculation of total Ca2+ uptake over 20 min of treatment with glutamate, showed that the amount of Ca2+ taken up by Aβ treated cells was 2.5-fold higher than in age-similar un-treated control cells after six days of treatment with Aβ (7964.47 ± 672.84 vs 3156.41 ± 1615.93 nmol m-2, respectively, P < 0.05) further confirming Ca2+ dysregulation in cortical neurons after prolonged exposure to a soluble Aβ.
The accumulation of Aβ protein in the brain is considered to be a key factor that causes AD. Most cultured cell models have examined the acute toxicity of Aβ, typically following 24 h treatment  which may not reflect the situation in vivo. In this study we examined the effect of daily exposure to moderate levels of soluble, monomeric Aβ1-40 (1 μM) for up to seven days and demonstrated, for the first time, that chronic exposure of primary cortical neurons to levels of Aβ which have no evident effect on viability over the short term, are indeed neurotoxic. A decrease in cell survival was only observed after seven days of exposure to 1 μM Aβ, with a 20% decrease in cell viability. This finding indicates that Aβ1-40 accumulation over time can induce neurodegenerative changes to neurons and thus contribute to the slowly progressing pathogenesis of AD.
Using MIFE, we observed an increase in K+ efflux concurrent with the decline in neuronal viability. While acute treatment with 1 μM Aβ did not affect net fluxes of K+ nor Ca2+ (Additional file 1: Figure S1) confirming earlier findings regarding intracellular Ca2+ changes , here we demonstrate that prolonged treatments with low concentrations of Aβ1-40 led to dysregulations of K+ but not Ca2+ homeostasis. This is in agreement with an earlier demonstration that exposure of neurons to Aβ peptide can enhance voltage gated K+ currents [20-22]. For example, preincubation of cells with recombinant human or rat Aβ1-40 was shown to significantly increase K+ channel current density and levels for Kv4.2 . Studies on brain slices at different stages of AD demonstrated that voltage gated K+ currents were already overexpressed in early stages of AD, and in advanced AD stages Kv3.4 was present at high levels in neurodegenerative structures . Other studies also demonstrated effects of Aβ on activity of Na+/K+-ATPase. For instance, it was shown that exposure of cultured neurons to Aβ1-40 caused selective reduction in Na+/K+-ATPase activity which preceded loss of Ca2+ homeostasis and cell degeneration . This was further confirmed in studies using APP and PS1 deficient mice . Together, enhancements of voltage gated K+ channels and decrease in Na+/K+-ATPase activity would increase overall K+ efflux, similar to Aβ effects observed in our experiments. The decrease in intracellular K+ concentration is also consistent with a decrease in cell viability since this is required for apoptosome formation and activation of caspases and endonucleases during apoptosis [26,27].
A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate may underlie chronic neurodegenerative disorders such as AD. Therefore, we investigated whether prolonged exposure to 1 μM Aβ1-40 increased neuronal susceptibility to subsequent acute excitotoxicity induced by glutamate. Surprisingly, no further decrease in neuronal viability was observed when cortical neurons treated with Aβ for seven days were exposed to glutamate (Figure 3A, B). Likewise, an earlier study has shown that low concentrations of Aβ peptide induced early and prolonged activation of proapoptoic markers in neurons without resulting in subsequent cell death in culture  most likely due to additional protective mechanisms in place at glutamate concentrations used. However, despite the absence of an overt change in neuronal viability following the combined treatments, we demonstrated that exposure to Aβ perturbed the ability of neurons to maintain K+ homeostasis following glutamate treatment. Treatments with 1 μM Aβ for one day were sufficient to lead to glutamate induced K+ dyshomeostasis in cortical neurons (Figures 6A, B). Indeed, peak K+ efflux after acute exposure to 10 μM glutamate was substantially higher in Aβ treated cells than in age-similar controls. This difference increased with time of Aβ exposure from 1.6 to 4.8-fold for one and six days of the treatment, respectively. Neurons were also unable to return K+ to pre-stress level as shown for steady-state K+ fluxes measured 20 minutes after the treatment with glutamate (Figure (Figure6B).6B). Another consequence of chronic pretreatment with Aβ was a higher level of K+ efflux even before challenge with glutamate (shown in Figure Figure22 for initial K+ values). Overall, this led to a 2.9-fold higher amount of K+ efflux from cells treated with Aβ1-40 for six days or longer after glutamate application than from age-similar control cells over 20 min of glutamate exposure. Taken together, our data suggest that chronic Aβ treatment impairs the ability of cortical neurons to maintain K+ homeostasis.
Ca2+ homeostasis undergoes subtle dysregulation during physiological ageing making neurons more vulnerable to additional stress which, in turn, can lead to neuronal degeneration. Recent evidence strongly supports the theory that dysregulation of intracellular Ca2+ homeostasis underlies the development of AD [12-14]. Therefore, we investigated whether prolonged exposure to continuous Aβ1-40 treatment followed by glutamate challenge would affect Ca2+ homeostasis in cortical neurons. Acute treatment with 10 μM glutamate after chronic pre-treatment with 1 μM Aβ1-40 in our experiments led to net Ca2+ uptake from external environment (Figure (Figure5B).5B). An increase in glutamate concentration leads to activation of ionotropic and metabotropic receptors [29,30] and Ca2+ influx through both glutamate-dependent pathways and voltage-dependent Ca2+ channels [31-33]. While the observed Ca2+ uptake in response to acute glutamate treatment was expected, in the current study we showed that the magnitude of Ca2+ uptake was substantially increased in cells treated with 1 μM Aβ1-40 for six consecutive days or longer compared to un-treated control cells (Figure (Figure7A).7A). Calculated total Ca2+ taken up by Aβ treated cells over 20 min post glutamate application was 2.5-fold higher than in age-similar untreated control cells (P < 0.05) suggesting dysregulation of Ca2+ homeostasis in cortical neurons after prolonged exposure to soluble Aβ. Overall, our data showed that prolonged Aβ accumulation renders cortical neurons more vulnerable to excitotoxicity leading to their inability to maintain Ca2+ homeostasis when glutamate concentration increases.
In this study we developed an in vitro model to test the effects of prolonged Aβ treatment. We demonstrated for the first time that continuous exposure to low levels of Ab1-40 induced neurodegeneration of cultured cortical neurons, with decreased viability evident after seven days of treatment. This decrease in viability correlated with increased neuronal K+ efflux. We also demonstrated that long term exposure to Aβ1-40 leads to imbalance in K+ and Ca2+ homeostasis and contributes to glutamate-induced vulnerability and cell death at later time point. This suggests that chronic accumulation of low levels of Aβ1-40 may contribute to the slowly progressing pathogenesis of AD. The results also suggest that the inability to maintain ion homeostasis is an early indicator of cell vulnerability that precedes cell death and might be used as a sensitive tool to cell susceptibility to the excytotoxic treatment.
Cortical tissue was removed from embryonic day 18 Hooded Wistar rat embryos and incubated in sterile 10 mM HEPES buffer (37°C; Sigma). The cortical tissue was trypsinised (0.25%; Sigma) and washed with fresh HEPES buffer. The cell pellet was gently triturated and filtered through 60 μm gauze. Cells were then plated onto glass coverslips (254 mm2) pre-coated overnight with poly-L-lysine (Sigma) at the density of 1 × 105 cells/well and culture medium added to the wells. The culture medium consisted of Neurobasal™ medium (Gibco), supplemented with 10% FCS (Gibco), 2% (f/c) B-27 supplement (Gibco), 5 mM (f/c) L-glutamine (Sigma), and 10 mg/ml gentamycin (Sigma). Cultures were maintained at 37°C in humidified air containing 5% CO2. The culture medium was replaced with serum-free culture medium after 24 hours, followed by a full media change three times a week.
1 μM Aβ1-40 was applied to 6 DIV cortical neuron cultures daily for 7 days. Fresh media was applied a week. A biological reducing agent, 300 μM ascorbate (the physiological concentration within the brain), was freshly applied with each media change for both control and the treatment. At days 2, 3, 4, 6, and 7 the neuronal viability was measured using an Alamar Blue assay, which measures cellular metabolic activity . The degree of cellular metabolic reduction of Alamar Blue was determined by fluorescence (excitation 535 nm, emission 595 nm), and expressed as the percentage of the signal obtained from the vehicle-treated culture. Day zero was the first day of treatment
To examine whether Aβ treatment increased neuron susceptibility to excitotoxicity, 6 DIV cultures treated daily with 1 μM of soluble Aβ1-40 for seven days were exposed to 200 μM glutamate. Glutamate was prepared as we have reported previously . Briefly, D-glutamic acid was dissolved in appropriate medium (Neurobasal medium for viability assay and aCSF for MIFE experiments). Neuronal viability was measured using the Alamar Blue assay after 24 hours of glutamate treatment.
The technique of Microelectrode ion flux estimation (MIFE) has been reviewed recently  and the complete experimental procedure including ion-selective microelectrode fabrication and cell preparation and immobilisation are given elsewhere [17,18]. Cortical neurons for the MIFE measurements were grown for 6 days at 1 × 105 cells/well on coverslips coated with poly-L-lysine as described above. By 6 DIV a dense monolayer of neurons had developed. Cells were washed in artificial CSF (aCSF) by dipping a coverslip with cells into a beaker with pre-warmed to 37°C aCMF. The coverslip then was placed into a measuring chamber with 2 mL of aCMF at 37°C, and left for adaptation for a further 40 min at room temperature (RT, 23°C) prior to an experiment. The composition of the aCSF was: 150 mM NaCl, 0.5 mM KCl, 0.5 mM CaCl2, 1.5 mM MgCl2, 1.25 mM NaH2PO4, 2 mM NaHCO3, 25 mM glucose, pH 7.2. Measurements were taken after one, three, six, and eight days post treatment with Aβ1-40. Control experiments used age similar cells that were not pre-treated with Aβ1-40. Additional controls such as addition of vehicle (aCMF) and Aβ1-40 were also used. For this co-focused K+ and Ca2+ microelectrodes were positioned ~5 μm above the cells and moved by a computer-driven hydraulic micromanipulator by 50 μm away from the cells and back to the primary position with 0.05 Hz frequency. Data was acquired at a rate of 10 samples/sec and averaged later over 6 second intervals. The ion fluxes were recorded for 5 min prior to the addition of 10 μM of glutamate, and recordings continued for a further 25 min. To achieve the required concentration of glutamate in the measuring chamber, an equal volume of the bath solution with a double concentration of glutamate was added resulting in the final concentrations of glutamate (10 μM). Net ion fluxes (nmol m-2s-1) were calculated using planar geometry of diffusion equations.
For each experiment unless otherwise stated, a minimum of four wells from at least three separate cultures (derived from different animals), were used for quantification. Statistical analysis was completed using SPSS 16.0 (SPSS). When data was unequally distributed, data was transformed so that the residuals were approximately normally distributed. Statistical significance was calculated using One-Way ANOVA with Tukey's Post Hoc Test and Student's t-test.
Aβ: beta amyloid; AD: Alzheimer's disease; APP: β-amyloid precursor peptide; DIV: days in vitro; MIFE: microelectrode ion flux measuring technique.
The authors declare that they have no competing interests.
LS carried out MIFE experiments, analysed and interpreted data, and drafted the manuscript. CH carried out the survival experiments and participated in data analyses. AKW participated in the design of the study, participated in its coordination and contributed to the preparation of the manuscript. RSC conceived the study, participated in overall direction of the study and preparation of the manuscript. All authors have read and approved the final manuscript.
Figure S1. Effects of acute application of Aβ1-40 on net K+ and Ca2+ fluxes. We tested whether acute application of Aβ1-40 to 14 DIV cortical neurons affected magnitudes of K+ and Ca2+ fluxes. Net fluxes of K+ (A) and Ca2+ (B) were recorded for 5 min (-5 to 0 min) followed by acute application of 1 μM Aβ1-40 (0 to 10 min) and 40 μM Aβ1-40 (final concentration, 10 to 25 min) to the bath. Neither concentration caused changes in net ion fluxes measured during the time course tested suggesting that prolonged treatment with Aβ1-40 is required to trigger disturbances in ion homeostasis. Error bars are SEM (n = 4).
Figure S2. Effects of experimental solution application to the measuring chamber on ion fluxes. An additional control was made to test a possible effect of solution disturbances in the measuring chamber on ion fluxes. Neurons at 14 DIV were used. Net K+ (A) and Ca2+ (B) fluxes were recorded continuously for 5 min (-5 to 0 min) and after vehicle (aCSF) application to the bath (0 to 10 min) with data acquired at a rate of 10 samples/sec and averaged over every 6 sec. Vehicle was applied at zero time as indicated by an arrow. No changes in K+ and Ca2+ fluxes were observed thus validating the approach used. Error bars are SEM (n = 4).
This work was supported by the National Health and Medical Research Council of Australia (#490025, 605528) to AKW and RSC, a Jack and Ethel Goldin Foundation Award (Alzheimer's Australia) to AKW and RSC, a Rising Stars Award from the University of Tasmania to RSC, and a research grant from the Australian Brain Foundation to LS. RSC holds a Research Fellowship from the Australian Research Council.