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Keywords:

  • Alzheimer’s disease;
  • long-term potentiation;
  • N-methyl-d-aspartate;
  • okadaic acid;
  • phosphorylation;
  • synaptic transmission;
  • tau

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Kinases and phosphatases act antagonistically to maintain physiological phosphorylation/dephosphorylation at numerous intracellular sites critical for neuronal signalling. In this study, it was found that inhibition of serine/threonine phosphatases by exposure of hippocampal slices to okadaic acid (OA) or cantharidin (CA; 100 nmol/L) for 2 h resulted in reduced basal synaptic transmission and blocked the induction of synaptic plasticity in the form of long-term potentiation as determined by electrophysiological analysis. Fura-2 Ca2+ imaging revealed a bidirectional modulation of N-methyl-d-aspartate (NMDA) -mediated Ca2+ responses and reduced KCl-mediated Ca2+ responses in neonatal cultured hippocampal neurons after phosphatase inhibition. While OA inhibited NMDA-induced Ca2+ influx both acutely and after incubation, CA-enhanced receptor-mediated Ca2+ signalling at low concentrations (1 nmol/L) but reduced NMDA and KCl-mediated Ca2+ responses at higher concentrations (100 nmol/L). Changes in Ca2+ signalling were accompanied by increased phosphorylation of cytoskeletal proteins tau and neurofilament and the NMDA receptor subunit NR1 in selective treatments. Incubation with OA (100 nmol/L) also led to the disruption of the microtubule network. This study highlights novel signalling effects of prolonged inhibition of protein phosphatases and suggests reduced post-synaptic signalling as a major mechanism for basal synaptic transmission and long-term potentiation impairments.

Abbreviations used
aCSF

artificial cerebrospinal fluid

AD

Alzheimer diseases

CA

cantharidin

CA1

hippocampal subfield cornu ammonis 1

HBS

HEPES-buffered saline

IC50

inhibition concentration 50

LTP

long-term potentiation

MEM

minimal essential medium

NMDA

N-methyl-d-aspartate

NMDAR

NMDA receptor

NR1

NMDA receptor subunit 1

NR2A

NMDA receptor subunit 2A

NR2B

NMDA receptor subunit 2B

OA

okadaic acid

PI

propidium iodide

PKA

protein kinase A

PKC

protein kinase C

PP

protein phosphatase

ROI

region of interest

Ser/Thr

serine/threonine

VGCC

voltage-gated calcium channels

Kinases and phosphatases are well-established modulators of neuronal excitability. Through phosphorylation and dephosphorylation they regulate a host of cellular proteins including ion channels and receptor subunits (for review, see Manusy and Shenolikar 2006). Of the phosphatases, two main classes are distinguished: serine/threonine (Ser/Thr) phosphatases, investigated in the present study, and protein tyrosine phosphatases. Protein phosphatase 1 (PP1), 2A (PP2A), the Ca2+-dependent 2B (PP2B or calcineurin) and 2C (PP2C) are traditional catalytic subtypes of the Ser/Thr phosphatases. However, novel members of this group have been identified, i.e. PP4, PP5, PP6 and PP7 (Huang and Honkanen 1998; and see Cohen 1997 for review). With the exception of the retinally restricted PP7, these phosphatases are widely expressed in the central nervous system and share close homology with PP1 and PP2A. The diverse functions of Ser/Thr phosphatases are based on a variety of accessory subunits, substrate specificity and subcellular localisation (see Herzig and Neumann 2000 for review).

Tight regulation of substrate properties via phosphorylation/dephosphorylation is a general principle underlying the rapid integration of multiple signalling events and some forms of plasticity, particularly in neurons. Accordingly, the inhibition of kinases impairs or blocks the induction of multiple forms of synaptic plasticity including long-term potentiation (LTP) (Hussain and Carpenter 2005). However, acute inhibition of phosphatases including PP1 and PP2A only appears to have small modulatory effects on LTP induction (Jouvenceau et al. 2003). Kinases (Ninan and Arancio 2004), and Ser/Thr phosphatases (Sim et al. 1993) have been found to modulate receptors of the principal neurotransmitters glutamate (Blackstone et al. 1994 and Wang et al. 1994) and GABA (Kapur and Macdonald 1996), implementing regulation of basal synaptic transmission. A potential key target of phosphorylation and an essential protein for both LTP and learning is the N-methyl-d-aspartate receptor (NMDAR). Phosphorylation-induced potentiation of NMDA currents can occur at several residues of the NMDAR subunit 1 (NR1), 2A (NR2A) and 2B (NR2B) subunits (Leonard and Hell 1997) and is mediated by protein kinase A (PKA) (Raman et al. 1996) and protein kinase C (PKC) (Ben-Ari et al. 1992); dephosphorylation of the same residues is mediated by Ser/Thr phosphatases PP1 and/or PP2A (Leonard and Hell 1997). Similarly, there are various phosphorylation sites on voltage-gated ion channels such as voltage-gated calcium channels (VGCCs) and K+ channels (see Smart 1997; Herzig and Neumann 2000 for reviews) sensitive to PKC (Bartschat and Rhodes 1995) and PKA (Hell et al. 1995), resulting in increased activity and an overall enhanced excitability. Both PP1 and PP2A have been implicated in the dephosphorylation of VGCCs (Bartschat and Rhodes 1995).

In addition to the importance of phosphorylation in key mechanisms of neuronal excitability, dysregulation of phosphorylation in the long-term can be cytotoxic and has therefore been linked to a number of diseases. High concentrations of PP inhibitors, especially okadaic acid (OA), can activate apoptotic pathways (Ko et al. 2000; Yan et al. 1997) and markers of resulting neurotoxicity share common hallmarks with neurodegenerative diseases such as Alzheimer’s disease (AD) and tauopathies in general, including hyperphosphorylated tau protein, β-amlyoid expression (Arendt et al. 1995, 1998) and AD-like memory impairments (Riedel 1999). Use of OA and other phosphatase inhibitors reportedly increase neuronal excitability, resulting in tau hyperphosphorylation (Ekinci et al. 2003; Niewiadomska et al. 2006 for a recent review), reorganisation of the cytoskeleton (Merrick et al. 1997) and distinctive patterns of neurodegeneration (Kim et al. 1999).

In this study, we aimed to assess the functional consequences of long-term PP inhibition on excitability, plasticity and Ca2+signalling in hippocampal neurons. We report that in slices Ser/Thr phosphatase inhibition reduced basal synaptic transmission and blocked LTP induction. In hippocampal neuronal cultures, low levels of phosphatase inhibition (CA, 1 nmol/L) enhanced, whilst higher levels of inhibition (OA 1–100 nmol/L and CA 100 nmol/L) reduced Ca2+ influx through NMDARs or VGCCs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Inhibitors of phosphatases

Two phosphatase inhibitors were used in the present study: (i) OA (Sigma-Aldrich, Gillingham, UK), a marine sponge toxin first isolated from a genus of sponges Halichondria, reported to directly inhibit the catalytic subunits of multiple phosphatases; (ii) Cantharidin (CA; Sigma-Aldrich), a blister beetle toxin (Carrel and Eisner 1974), that inhibits the activity of PP1, PP2A but not PP2B or PP2C (Li and Casida 1992), but with lower potency relative to OA (Hastie and Cohen 1998; Borthwick et al. 2001). Both OA and CA were dissolved in dimethyl sulfoxide to produce a 100 μmol/L stock solution. For relative inhibition concentration 50 (IC50) values of both compounds (see Table 1).

Table 1.   Range of IC50 values of protein phosphatase inhibition
DrugPP1PP2APP2BPP2CPP4PP5PP6
  1. aBorthwick et al. 2001; bCohen 1997; cBialojan and Takai 1988; dHastie and Cohen 1998; eHonkanen 1993; fHerzig and Neumann 2000. All values expressed as nanomolar (nmol/L). NE, not effective; NT, not known to be tested; CA, cantharidin; OA, okadaic acid; PP, protein phosphatase; IC50, 50% inhibition concentration.

OA10b–270c0.2b–2c3600cNE0.2b2b–7a2b
CA1700e40f–160eNENE50d50aNT

Slice preparation and electrophysiology

As described previously (Algaidi et al. 2006) and in agreement with Home Office regulations, Lister-hooded rats (3 to 4-months old, females) were terminally anesthetized with halothane, decapitated, the brain removed, hippocampi dissected out and placed in ice-cold artificial cerebrospinal fluid [aCSF; (in mmol/L): 129.5 NaCl, 1.5 KCl, 1.3 MgSO4, 2.5 CaCl2, 1.5 KH2PO4, 25 NaHCO3 and 10 glucose (pH 7.4)]. Hippocampal slices were prepared (300–400 μm) using a McIlwain tissue chopper (Intracel, Herts, UK), transferred and stored in pre-warmed aCSF (37°C) for 1 h before a 2 h drug incubation with OA (100 nmol/L) or CA (100 nmol/L). After a washout period in aCSF of 0–1 or 2–3 h slices were transferred to a submerged recording chamber.

For field recordings of population spikes, a monopolar stimulating electrode (0.5 MΩ; World Precision Instruments, Stevenage, UK) was positioned in the Schaffer-collateral fibre pathway and a borosilicate glass aCSF filled AgCl recording electrode (3–4 MΩ) was inserted into the CA1 pyramidal cell body layer. Basal synaptic transmission was established by fibre stimulation (20 μs duration) increasing in 2.5 V increments from 7.5 V until the evoked response was saturated (input/output curves, two repeats averaged). If subsequent baseline recordings indicated unstable responses (>10% variability), slices were discarded. Baseline recordings and LTP induction was conducted at 50% maximal stimulation with responses recorded every 30 s (two repeats averaged to yield one value per minute). LTP was induced by a theta (5 Hz) -like tetanus [150 bursts of four stimuli (100 Hz), 200 ms inter-burst interval for 30 s]. Post-tetanus responses were recorded for a further 1 h or 30 min (depending on establishment of potentiation). Control LTP experiments were conducted at regular intervals to ascertain fully reproducible recording conditions.

For data analysis, input/output curves of population spike amplitudes were compared with controls using two-way analysis of variance (anova). For LTP, post-tetanus values were compared using a repeated measure anova. Post hoc time point analysis was used to determine successful potentiation of signals within groups (paired t-test between baseline and each min post-tetanus). All values are presented as % of baseline + SEM. p < 0.05 was taken as significant and p < 0.01 as highly significant.

Hippocampal cell culture

As described previously (e.g. Drysdale et al. 2006), neonatal rat pups were killed in agreement with Home Office regulations. Hippocampi were dissected out and placed in ice-cold HEPES-buffered saline (HBS; mmol/L: 130 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES and 25 d-glucose; pH adjusted to 7.4 with NaOH). Hippocampi were placed in protease solution [protease X and protease XIV (Sigma-Aldrich) dissolved in HBS] for 40 min after mechanical dissociation. Tissue was washed with HBS and a series of three trituration and centrifugation steps were conducted. The supernatant was removed and replaced with minimal essential medium (MEM) solution [MEM (Invitrogen, Paisley, UK) containing 2 μmol/L l-glutamine (Sigma-Aldrich) and 10% foetal bovine serum (Gibco-Invitrogen)]. The final cell suspension was plated on poly-l-lysine (Invitrogen) coated dishes for Ca2+ imaging and on coated glass coverslips for immunocytochemistry and tubule tracker staining. Cultures were incubated for 2–3 days before MEM was replaced with Neurobasal medium (Invitrogen), with 2 μmol/L l-glutamine, 0.25 μmol/L glutamate (Tocris, Bristol, UK) and 2% B-27 supplement (Gibco-Invitrogen). All cultures were used within 5–10 days in vitro. Drug exposure was performed in 1 mL of Neurobasal medium solution prior to fura-2 imaging or staining. OA and CA were administered at 100 nmol/L for 2 h and at 1 nmol/L for 24 h, but CA was also assessed at 100 nmol/L for 24 h.

Fura-2 Ca2+ imaging

Hippocampal cultures were loaded with the cell-permeable fluorescent calcium indicator fura-2AM (1 mmol/L stock solution in dimethylformamide, final concentration 10 μmol/L; Molecular Probes-Invitrogen) for 1 h in the dark. Cultures were perfused, via a gravity perfusion system, with low Mg2+ HBS (composition in mmol/L: 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.01 MgCl2, 10 HEPES and 25 d-glucose; pH adjusted to 7.4 with NaOH) at a rate of 15 mL/min. For incubated cultures, 2 min applications of 1, 10 and 100 μmol/L NMDA in the presence of 50 μmol/L glycine (Tocris) were conducted in low Mg2+ HBS followed by a 1 min application of 30 mmol/L KCl (VWR, Poole, UK).

For acute application of OA, 10 μmol/L NMDA and 15 mmol/L KCl were applied prior to and immediately after 20 min incubation with 100 nmol/L OA, intermediate concentrations were selected here to allow bidirectional modifications of Ca2+ responses. All solutions contained 0.5 μmol/L tetrodotoxin (Caltag-Invitrogen). Generally, [Ca2+]i levels were allowed to return to baseline prior to an additional application of drug-containing solution. Cells were imaged via a digital CCD camera (Hamamatsu, Shizuoka, Japan) with a 40× water immersion lens mounted on a microscope (Olympus BX51W1; Southall, UK). Fura-2 was excited via a Lambda DG-4 illumination system (Sutter Instruments, Novato, CA, USA) generating alternating excitation wavelengths of 380 and 340 nm; emission was captured at 510 nm and images were taken every 5 s. Region of interest (ROI; 1 per cell body) within a given field were pre-selected by means of a transmission image overlay, and neuronal cell bodies were selected based on visually assessed morphology and NMDA responses.

For data analysis, ratiometric values obtained from Openlab (V. 4.02, Improvision, Coventry, UK) were plotted against time and the peak rise in fluorescence for each ROI was determined, shown as arbitrary fluorescence units in sample traces. All values were transformed to fluorescence intensity as the percentage change from pre-drug baseline fluorescence (%ΔF/F). The percentage of cells which responded >10%ΔF/F towards a given drug application was calculated relative to the total neuronal ROIs to establish potentially drug-induced changes in responder rates and as an indicator of general viability. Within NMDA control replications, the SEM ranged from 4–5% for 1, 10 and 100 μmol/L NMDA therefore, any change >5% is thus considered significant.

Data analysis was preformed by means of a Kruskal–Wallis test. Comparisons between data pairs were conducted using a non-parametric Mann–Whitney U-test for incubated cells and a Wilcoxon matched pairs test for acute OA application; p < 0.05 was taken as significant and p < 0.01 as highly significant. N’s provided refer to the number of neurons responding to the particular drug challenge; each experiment was repeated at least three times, using different culture batches.

Immunocytochemistry and Tubulin tracker

Morphological and immunohistochemical analysis utilized primary antibodies for tau phosphorylated at serine 396 (rabbit polyclonal tau-PS396, 1 : 200; Santa Cruz, Santa Cruz, CA, USA), phosphorylated 200 and 160 kDa neurofilament (SMI34, 1 : 200; Abcam, Cambridge, UK) and phosphorylated NMDAR1 at serine 890 (1 : 100; Cell Signaling, Danvers, MA, USA).

Incubated dishes were washed in HBS prior to 10 min fixation with 4%p-formaldehyde in 0.1 mol/L phosphate-buffered saline. Epitopes were unmasked by microwave-assisted antigen retrieval (Munoz et al. 2004). Cells were then incubated in blocking solution (1.5% normal goat serum, 2% bovine serum albumin, 1% milk powder and 0.001% Triton in HBS) for 1 h at 20–22°C. Primary antibody incubation was conduced in HBS overnight at 4°C. Secondary antibody incubation [chicken anti-rabbit Alexa 488, goat anti-mouse Alexa 488 and goat anti-rabbit Alexa 598 (1 : 200; Invitrogen)] was for 1 h at 20–22°C in modified HBS (2% bovine serum albumin containing HBS). Stained images were visualized via a Zeiss microscope (Axioskop 2 plus), and images taken with a digital camera (Axocam) using Axiovision software (Zeiss, Hertfordshire, UK).

Labelling of microtubules was conducted on live hippocampal cultures using tubule tracker (Invitrogen) according to manufacture’s instruction. Labelled cultures were imaged as for Ca2+ imaging, except with a 60× immersion lens and a fluorescein (FITC) filter (excitation 494 nm and emission 518 nm). For all staining, treated and control dishes were run in parallel and images taken with constant exposure times, to allow for direct comparison. Secondary antibody controls were also always run in parallel to ascertain the absence of unspecific staining.

All stains were conducted in three batch replications, five randomly selected fields were imaged from each dish. Intensity staining was measured using the Improvision Volocity software (Version 4). Stained neuronal cell bodies were selected based on threshold intensities after background subtraction. Somatic fluorescence was quantified, with nuclear regions subtracted based on 4′,6-diamidino-2-phenylindole (DAPI)-stained regions for tau-PS396 and SMI34. For NMDAR1-Ser890, whole cell body regions were selected, as no nuclear artefact was obtained. Individual intensity values were averaged to give a mean fluorescent intensity per dish, and values were compared between treated and untreated controls. Intensity values were analysed by one-way anova and post hoc Dunnett multiple comparison test.

Neuronal viability

To assess putative viability changes, as indicated by gross morphological changes and notable reductions in percentage responders towards NMDA and KCl challenges after 100 nmol/L 2 h OA incubation, a comparison between treated dishes and controls was conducted using a Live/Dead kit (Sigma-Aldrich), according to manufactures guidelines. The double-stain utilizes Calcein-AM, which upon esterase processing in healthy cells produces Calcein, a green fluorescent molecule, and the cell death marker propidium iodide (PI), a red-fluorescing nuclear stain that only penetrates compromised membrane (Platt et al. 2007). Stained cultures were imaged as above, with five randomly selected fields taken per dish (three replications) and the number of viable versus dead cells counted.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Slice physiology after phosphatase inhibition

Electrophysiological stimulation and recording in the CA1 region investigated the effects of a 2 h PP inhibition on synaptic transmission and plasticity. To assess basal transmission, slices were stimulated over the range of 7.5–45 V and the resulting field population spikes recorded. In control slices (n = 12), responses rose toward incremental stimulation with a threshold of 7.5 V eliciting mean responses of 0.6 mV and 42.5 V eliciting a mean maximal response of 4.8 mV (Fig. 1a). For slices incubated with OA, more than 80% displayed altered waveforms consisting of a large reduction and widening of the population spike. Notably, a large number of slices (>50%) failed to produce responses >0.5 mV even after 5 h of wash (data not shown), suggesting irreversible damage to hippocampal cells. After phosphatase inhibition with OA (100 nmol/L for 2 h followed by drug-free recovery in aCSF) the input–output relationship was significantly reduced (Fig. 1a) independent of recovery time [0–1 h: F(1,320) = 104.5, p < 0.001; 2–3 h: (F(1,304) = 122.2, p < 0.001]. Both groups of OA-treated slices failed to reach similar levels of maximal response (Fig. 1a), with the maximal spike amplitude being reduced by about half.

image

Figure 1.  Protein phosphatase inhibition suppresses basal synaptic transmission and blocks the induction of long-term potentiation (LTP). (a) Input–output relationship for population spike amplitudes, recorded from hippocampal slices. Data are shown as mean + SEM. Slices were incubated in phosphatases inhibitor containing aCSF; okadaic acid (OA) 100 nmol/L (n = 9, 0–1 h recovery shown) and cantharidin (CA) 100 nmol/L (n = 7) for 2 h; controls (n = 12) were incubated in non-drug containing aCSF. Phosphatase inhibitor incubated slices showed reduced maximal responses compared with controls (p < 0.001) and altered waveforms. Sample traces of population spikes, recorded in response to 45 V stimulation are shown on the right. (b) LTP time courses of population spike amplitudes in percentage of baseline (mean + SEM). Control slices (n = 14) showed robust LTP, while slices incubated in 100 nmol/L OA for 2 h with either 0–1 h (n = 9) or 2–3 h (n = 9) recovery period prior to recording failed to produce LTP. No potentiation was seen in 0–1 h OA slices, whilst only short-term potentiation was established in the 2–3 h recovery group. (c) LTP time course for CA. CA (n = 8) treated slices were given a 0–1 h recovery prior to recording. No potentiation was observed with amplitudes being significantly reduced compared with control. Sample traces of population spikes (baseline and 30 min post-tetanus) are depicted in the right, stimulus artifact truncated.

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Two hours incubation of slices with 100 nmol/L CA produced a similar deficit to that of OA (Fig 1a). The input–output relationship was significantly reduced in slices 0–1 h after CA [F(1,288) = 54.8, p < 0.001] relative to controls. The latency of population spikes was also measured and no significant effect was observed between any treatment and controls (data not shown).

To probe the ability of these slices to undergo plastic changes, LTP was induced by application of theta burst stimulation. Relative to control slices with peak LTP amplitudes of 169% and persistent elevation of population spike amplitudes after tetanisation (all p-values < 0.5–0.001 compared with baseline), OA-treated slices failed to produce LTP (Fig. 1b). A peak short-term potentiation of 148 ± 15% readily declined back to baseline levels after 17 min (for the 2–3 h washout group). Paired time point analysis confirmed a significant potentiation only from 14 to 17 min post-tetanus (p-values < 0.05). No significant potentiation was established for the 1 h washout group. Repeated measures anova with drug and time as factors confirmed a significant LTP impairment in both the 0–1 h [F (30,630) 6.725, p < 0.0001 for interaction] and 2–3 h washout groups [F (30,630) = 4.288, p < 0.0001 for interaction]; the two OA groups did not differ from each other (p > 0.05).

Treatment of slices with CA (Fig. 1c; 100 nmol/L, 0–1 h wash) also disrupted LTP [F (1,600) = 12.17, p < 0.01]. Amplitudes were not significantly potentiated at any post-tetanus time point reaching a maximum of 118 ± 11% at 18 min (all p-value > 0.05).

Exposure to OA alters Ca2+ signalling in hippocampal neurons

As synaptic transmission and synaptic plasticity rely on pre- and post-synaptic functional Ca2+ signalling, we explored next whether prolonged exposure to inhibitors of phosphatases (OA and CA) may alter Ca2+ signalling via NMDARs and VGCC in cultured hippocampal neurons. Cultures were challenged with a 20 min application of OA (100 nmol/L), flanked by brief pulses of 10 μmol/L NMDA and 15 mmol/L KCl (Fig. 2a). While basal Ca2+ levels were not affected by OA treatment, analysis of Ca2+ responses pre- versus post-OA application revealed a significant reduction in the amplitude of the NMDA-evoked (p = 0.0004; n = 50, Fig. 2b) and KCl-evoked Ca2+ signals (p < 0.0001; n = 57, Fig. 2c) compared with pre-incubation responses. The number of cells that responded to the challenge was slightly reduced (10% less cells responding towards 15 mmol/L KCl). Typically, repeat applications of NMDA in controls varied by ≤5%, thus, the alteration of responders after OA is considered significant.

image

Figure 2.  Acute okadaic acid (OA) application reduces N-methyl-d-aspartate (NMDA) and KCl Ca2+ signalling. (a) Mean trace of 10 neuronal regions in arbitrary fluorescence units; responses were evoked with 10 μmol/L NMDA (2 min ) and 15 mmol/L KCl (1 min ), applied pre- and post-exposure to 100 nmol/L OA (20 min ). Maximal mean NMDA (b) and KCl (c) responses pre- and post-OA; data are shown as mean percentage delta F/F + SEM; ***p < 0.001. The percentage responder rate for all groups is given within each bar.

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Post-incubation responses to 10 μmol/L NMDA and to 15 mmol/L KCl were robustly reduced in comparison with untreated controls (20 min, low Mg2+ HBS; p < 0.001, n = 51) (p < 0.001; KCl values correct for small reduction in control responses, see.

Our results were somewhat surprising since previous reports had suggested enhanced excitability and signalling after phosphatase inhibition (Wang et al. 1994), especially since further experiments with acute applications of OA at varying concentrations (0.1 nmol/L–1 μmol/L) and for a range of incubation periods (100 nmol/L, 5–20 min) resulted in either unaltered or suppressed signalling, with no evidence for the previously reported enhanced excitability (data not shown).

To investigate consequences of prolonged alterations in phosphorylation status, a series of hippocampal cultures were incubated with PP inhibitors followed by imaging experiments (Figs 3 and 4). Naïve control experiments in sister cultures produced a concentration-dependent increase in the magnitude of NMDA-induced [Ca2+]i responses (p-value < 0.001, n = 70, see Figs 3c and 4d), alongside an increasing number of cells responding. The subsequent application of 30 mmol/L KCl produced a rise in [Ca2+]i of 155 ± 13%ΔF/F with 75% of cells responding, similar to the highest NMDA challenge.

image

Figure 3.  Prolonged serine/threonine phosphatase inhibition by okadaic acid (OA) reduces neuronal excitability towards N-methyl-d-aspartate (NMDA) and KCl. (a and b) Mean time courses of neuronal responses in arbitrary fluorescence units; evoked with 1, 10 and 100 μmol/L NMDA (2 min) and 30 mmol/L KCl (1 min) applications; responses from 100 nmol/L OA (2 h) incubated neurones (n = 12) and 1 nmol/L OA (24 h, n = 12) are shown as a black line in (a and b), respectively, control responses (n = 12) in grey. (c) Maximal means responses toward NMDA and KCl applications. Responses towards 10 μmol/L NMDA and 30 mmol/L KCl were reduced compared with controls in 100 nmol/L OA (2 h) incubated cells. After 24 h incubation 1 nmol/L OA produced a reduction of NMDA responses at 1, 10 and 100 μmol/L NMDA but not to 30 mmol/L KCl compared with controls. The percentage responder rates (R) displayed above the corresponding bars were reduced particularly after 100 nmol/L OA incubations for 2 h. Data are shown as mean percentage delta F/F + SEM; *p < 0.05, **p < 0.01 and ***p < 0.001.

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image

Figure 4.  Biphasic modulation of N-methyl-d-aspartate (NMDA) responses and reduced KCl signalling after serine/threonine phosphatase inhibition with cantharidin (CA). (a–c) Mean time courses of neuronal responses (in arbitrary fluorescence units); evoked with 1, 10 and 100 μmol/L NMDA and 30 mmol/L KCl applications. (a) CA applied for 2 h at 100 nmol/L (n = 14), (b) 1 nmol/L CA for 24 h (n = 19) and (c) 100 nmol/L CA for 24 h (n = 8), illustrated as black lines, with control traces (n = 12) shown in grey. (d) Summary of mean responses toward NMDA and KCl. The magnitude of the responses was unchanged in 100 nmol/L CA 2 h incubated cells for all applications. Responses evoked by 10 and 100 μmol/L NMDA were enhanced in 1 nmol/L CA (24 h) incubated cells, and 100 μmol/L NMDA and 30 mmol/L KCl-induced responses were reduced in 100 nm CA 24 h incubated cells. The percentage responders displayed above the corresponding bars show a small increase in 100 nmol/L CA 2 h and 1 nmol/L CA 24 h incubated cells. Data shown as mean percentage delta F/F + SEM; *p < 0.05 and ***p < 0.001.

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Relative to controls, cultures incubated for 2 h with 100 nmol/L OA (thus mirroring the protocol used for slice incubations) showed significantly reduced responses (Figs 3a and c) to 10 μmol/L NMDA (p = 0.0021, n = 23). A decrease in percentage responders was seen towards NMDA at all concentrations suggesting a general deterioration of cellular viability. Accordingly, Ca2+ responses and responder rates towards 30 mmol/L KCl were also reduced compared with controls (p < 0.0001, n = 24) (Figs 3a and c). As a substantial decrease in responder rates in OA-treated neurons was observed (maximum rate 43% cf. 75% in controls), viability was assessed using a live/dead stain. Despite the reduction in responsiveness, general viability was not found to be impaired, only 8 ± 5% of OA (2 h, 100 nmol/L) treated neurons stained for PI (dead stain), relative to 5 ± 2% PI-positive cells in controls (p > 0.05, control: n = 131; 2 h 100 nmol/L OA: n = 83), all remaining neurons stained positive for calcein (data not shown). Overall, there was a reduction in neuronal density in OA incubated cultures (∼−35%); however, the viability staining and imaging data indicate that remaining neurons were not necrotic, thus, functional changes were a genuine result of altered phosphorylation status and not due to loss of viability.

To study whether a similar pattern of altered signalling would be obtained after long-term inhibition of phosphatases (as it is assumed to occur in neurodegenerative disease), we next conducted 24 h incubations, but with 100-fold lower concentration of OA (1 nmol/L OA) to avoid unspecific effects due to toxicity and apoptosis. Interestingly, comparisons of OA-treated cells with controls still revealed significantly reduced Ca2+ responses for all concentrations of NMDA (p = 0.0002, p < 0.0001 and p = 0.0124 for 1, 10 and 100 μmol/L NMDA, n = 27; Figs 3b and c) beyond that seen for the shorter application. Although fewer cells responded to the NMDA challenges than controls, this reduction was not as dramatic as seen after the 2 h treatment. In contrast to the shorter incubations with 100 nmol/L OA, no significant reduction of the KCl-mediated [Ca2+]i rise was seen after 24 h OA treatment and no alteration in percentage responders compared with controls was observed (Fig. 3c), thus indicating that general viability was not affected with this protocol.

CA causes bidirectional changes of Ca2+ responses in hippocampal neurons

In the next series of experiments, we tested the somewhat weaker PP inhibitor CA (see Table 1). In contrast to OA incubations, cultures incubated with 100 nmol/L CA for 2 h (Fig. 4a) displayed no significant alteration in responses to either NMDA or KCl (n = 29). Nevertheless, a trend of increased NMDA responses was noted and an elevation in responder rates ≥10% towards 10 and 100 μmol/L NMDA was observed, suggesting increased excitability. Moreover, although there were no changes in KCl-mediated Ca2+ responses, more cells were activated by KCl post-CA treatment (Fig. 4d).

In line with the protocols used for OA, a second CA incubation regime was performed with 1 nmol/L CA for 24 h (Fig. 4b). After the longer treatment, Ca2+ responses (Fig. 4d) were reliably enhanced for 10 and 100 μmol/L NMDA, with the responder rate remaining unchanged relative to controls (p < 0.001 for both, n = 38). Sensitivity to KCl and responder rates remained unaltered (Fig. 4d) compared with controls. These data provide compelling evidence that long-term CA exposure selectively increased NMDAR-mediated Ca2+influx, and this mode of action apparently differs from OA, which decreased both types of Ca2+ responses.

Given that CA is a less potent phosphatase inhibitor than OA, and no evidence for neurotoxicity was seen here, we next explored whether a pattern comparable with OAs action may be achieved with higher CA concentrations. Increasing the concentration over the long exposure time (24 h) should achieve this, and if the mode of action and targeted enzymes are identical, should result in a similar reduction of Ca2+ signalling as found for OA (Fig. 3). Results for this group are summarized in Figs 4c and d. In line with our hypothesis, CA at 100 nmol/L, applied for 24 h, caused a significant reduction in NMDA-mediated [Ca2+]i responses, which was significant for both 10 and 100 μmol/L NMDA, as well as KCl and coincided with a small, but reliable reduction in responder rates [p < 0.03 and p = 0.001 for 10 and 100 μmol/L NMDA (n = 41) and p = 0.028 for 30 mmol/L KCl (n = 36)]. Interestingly, this reduction in Ca2+ responses towards 100 μmol/L NMDA did not significantly differ from the 1 nmol/L 24 h OA incubated cells (p > 0.05), supporting the assumption that similar levels of phosphatase inhibition had been reached.

Ser/Thr phosphatase inhibition induces NMDAR and cytoskeletal phosphorylation

Using a marker for the neuronal cytoskeleton protein tubulin, alterations in general cell morphology such as branching and structural rearrangements caused by Ser/Thr PP inhibition were visualized (Fig. 5a). Most notably, only the short-term incubation with 100 nmol/L OA for 2 h led to dramatic alterations in cell morphology such as a reduction of neuronal branches, in agreement with generalized signalling impairments observed. Transmission images indicated that some processes were still maintained (but did not label for tubulin, data not shown). Under all other treatments cellular processes and tubulin density appeared unaltered. In light of this apparent reorganisation of the microtubule system, the phosphorylation of microtubule-associated protein tau was also examined with a phosphorylation-specific antibody (Fig 5b). Analysis of cell body PS396 tau immunoreactivty revealed an overall treatment effect (F > 1), post hoc Dunnett multiple comparison further showed that only OA-treated cultures display increased tau phosphorylation in the somatic cytoplasm (Fig 5c; both p-value < 0.05). After 1 nmol/L 24 h OA treatment, neurons also displayed the presence of PS396-positive neurites. In contrast, cultures treated with the weaker Ser/Thr phosphatase inhibitor CA showed either no change in PS396 levels or even a slight (Dunnett: NS, Mann–Whitney U-paired comparison: p < 0.05) decrease in PS396 immunoreactivty after 1 nmol/L (24 h) CA treatment.

image

Figure 5.  Serine/threonine phosphatase inhibition induces neuronal cytoskeleton disruption in conditions were neuronal excitability is suppressed. Cultures incubated with either 100 or 1 nmol/L of okadaic acid (OA) or cantharidin (CA) for 2 or 24 h, respectively. (a) Microtubule labelling in live cells. Notably, after 2 h of 100 nmol/L OA incubations staining of neuronal processes was lost and cell bodies appeared swollen. All other groups showed an intact cytoskeleton. (b) Cultures labelled with PS396 for phosphorylated tau. Treatment of cultures with OA resulted in a significant rise in cytoplasmic PS396 immunoreactivity, while CA failed to alter immunoreactivty. (c) Semi-quantitative analysis of PS396 immunoreactivty; **p < 0.01 and ***p < 0.001 relative to intensity fluorescence values of control.

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Strong immunoreactivity for phosphorylated forms of neurofilament was seen in the somata of hippocampal cultures treated with any of the incubation protocols (Figs 6a and c). Analysis of cell bodies revealed a dependence on treatment (F > 1) and all treatments were significantly elevated compared with controls (Fig 6b; all p < 0.05). Additionally, neurofilament labelling was not only considerably stronger but also extended more into the processes. In contrast, while immunoreactivity for phosphorylated NMDAR1 showed a trend for enhancement in all treatment groups, this was found to be significant only in the 2 h 100 nmol/L OA group (Fig. 6c).

image

Figure 6.  Serine/threonine phosphatase inhibition increases phosphorylation of neurofilament and the N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). After incubations with either OA or cantharidin (CA) for 2 or 24 h, cultures were labelled with SMI34 (for phosphorylated neurofilament, NF) and for phosphorylated NMDA NR1 subunits (Ser890). (a) Controls show little basal level of phosphorylated neurofilament in selective processes and low levels of phosphorylated NMDA NR1, localised in neuronal cell bodies only. Phosphatase inhibition with either CA or OA increased phosphorylated neurofilament immunoreactivity, particular in proximal processes and cell bodies. There was a significant increase of somatic NMDAR1 phosphorylation in short-term OA-treated cells. Merged images (right column) consists of neurofilament, NMDAR1 staining and transmission image overlay. (b) Mean fluorescence of phosphorylated neurofilament immunoreactivty. (c) Mean somatic NR1 immunoreactivity; **p < 0.01 relative to intensity fluorescence values of control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The overall outcome of our experiments can be summarised as: (i) PP inhibitors reduce basal synaptic transmission and synaptic plasticity in hippocampal slices; (ii) PPs are able to regulate intracellular Ca2+ signalling pathways including Ca2+ influx through NMDARs and VGCCs; (iii) Long-term inhibition of Ser/Thr phosphatases can lead to alterations in receptor phosphorylation and cytoskeletal restructuring, which may result in subsequent neurodegeneration.

Inhibition of PPs and evidence for neurotoxicity

Both phosphatase inhibitors, OA and CA, resulted in reduced basal synaptic transmission and blocked the induction of LTP in hippocampal cells. Even slices incubated for 2 h in 100 nmol/L OA followed by prolonged washout periods (up to 5 h, data not shown) failed to show full recovery of evoked responses but were capable of producing short-term potentiation. Overall, this indicates some level of irreversible damage, presumably due to hyperphosphorylation.

Prolonged exposure to phosphatase inhibitors can be pro-apoptotic. Ser/Thr phosphatase inhibition with 500 nmol/L OA or Calyculin A (30 nmol/L) has been shown to act directly on apoptosis mediators within 4 h, which in turn initiates programmed cell death (Yan et al. 1997; Ko et al. 2000). Such studies suggest that a fine-balanced regulation of phosphorylation/dephosphorylation is critical to maintain normal neuronal physiology and cell survival, although a 2 h administration of phosphatases inhibitors is unlikely to cause immediate cell death or necrosis. It is thus possible that early stages of apoptosis have been attained here; yet, our cell death studies show that physiological changes were seen in the absence of frank cell death. Previous studies with apoptosis inducers support such early stage changes; for example, soluble β-amyloid oligomers cause characteristic changes in mitochondria function as early as 2 h post-incubation, yet cell death is not elevated until 8 h after insult (Deshpande et al. 2006). Further indication of neuronal dysfunction is provided by a reduction in responders in hippocampal cultures treated with PP inhibitors as well as the gross morphological changes seen in cultures following 2 h 100 nmol/L OA exposure.

Our results draw into question previous reports of enhanced excitability after phosphatase inhibition. OA incubation of forebrain synaptosomes caused increased basal release of glutamate (Sim et al. 1993) and enhanced excitatory synaptic responses in striatal slices incubated with 10 μmol/L OA for 10 min (Colwell and Levine 1995). Prior studies applied phosphatase inhibitors acutely in the range of 10–30 min, i.e. before consequences of secondary excitotoxicity or phosphorylation cascade effects may become obvious. In agreement with this hypothesis, during a 90-min incubation of rat neuromuscular junction with OA (0.1–1 μmol/L) or CA (0.1–100 μmol/L) a biphasic response was seen with initial increases, followed by a decrease in muscle twitch (Hong 2000). This study, as well as our Ca2+ imaging data (discussed below), suggest that bidirectional changes in neuronal excitability may occur due to prolonged phosphatase inhibition.

The involvement of phosphatases in a range of synaptic plasticity phenomena is also well established. Lisman (1989) suggested that the amount of Ca2+ influx determines the direction of plastic changes in hippocampus with kinase activity being increased during LTP, and phosphatase activity being increased during long-term depression. In the hippocampus, blockade or genetic knockdown of phosphatases during high-frequency tetanisation increased LTP in some (Brown et al. 2000; Malleret et al. 2001; Zeng et al. 2001), but not all cases (Wang and Stelzer 1994; Lu et al. 1996a,b ), whilst phosphatase inhibition resulted in the block or attenuation of long-term depression in hippocampus (reviewed in: Riedel 1999). However, the reduction of basal synaptic transmission and altered waveforms of extracellular field potentials seen here implicate disruptions of basic neuronal physiology. Thus, the failure to achieve LTP induction more likely results from impaired basal synaptic transmission, rather than directly reflecting the requirement of protein dephosphorylation during LTP induction. A reduction in the post-synaptic signalling by PP inhibition may limit Ca2+ entry leading to a failure to trigger the Ca2+-dependent mechanisms required for LTP induction (Wang and Kelly 1995). Such a reduction in post-synaptic depolarisation and resulting blockade of LTP induction has, for instance, been reported in aged rats in the CA1 (Hsu et al. 2002), and reduced NMDAR activation has been implicated (Potier et al. 2000). While it has to be considered that changes in excitability may affect population spike generation without necessarily affecting synaptic transmission per se, such actions seem unlikely in light of the observed consistent and pronounced depression in population spikes alongside changes in waveform.

Post-synaptic modulations caused by Ser/Thr phosphatase inhibition

Evidence from our neuronal culture experiments suggests modulation of Ca2+ signalling induced by phosphatase inhibition, which may have contributed to the altered synaptic transmission and the failure to induce LTP in treated hippocampal slices. Our Ca2+ imaging data show that CA induces a bidirectional modulation of NMDA responses, with low exposure levels inducing a potentiation, and high exposure causing attenuated responses. In contrast, treatment of neurons with OA only led to reduced NMDA responses with either acute or incubation protocols. Equally, only incubations with high concentrations of inhibitors (100 nmol/L OA 20 min, 2 h or CA 100 nmol/L 24 h) caused reductions in KCl-mediated signalling. Such a relationship suggests concentration- or time-dependent effects on either selective members of Ser/Thr phosphatases or their targets.

Cantharidin-mediated NMDA augmentation is supported by previous reports. Direct application of PP1 or PP2A to inside-out patched hippocampal neurons was previously found to decrease NMDA currents, whilst acute bath application of 125 μmol/L OA for ∼15 min to hippocampal neurons increased currents (Wang et al. 1994). However, intracellular application of Calyculin (100 nmol/L, 5 min), a similar but more potent PP inhibitor, resulted in potentiation of NMDA currents in oocytes expressing hypothalamic RNA (Nijholt et al. 2000) and striatal neurons (Blank et al. 1997). In contrast to these, we found that short-term application of OA (100 nmol/L, 20 min) attenuated NMDA responses. This may reflect variability in cultures underlying basal phosphorylation turnover or selective differences in targets and concentrations achieved (see Table 1), e.g. involvement of PP2B inhibition, which also can regulate NMDA signalling (Choe et al. 2005). Additionally, experimental parameters may determine the net overall effects, whilst the above study measured fast (microseconds) peak NMDA currents, our sample rate and analysis method determine steady-state responses to more prolonged NMDA applications and thus provide an index of sustained Ca2+ responses. Such apparently contradictory observations have previously been stated with regards to NMDA and PKC activity (reviewed in MacDonald et al. 2001). Similar to NMDA, KCl-mediated signalling or VGCC activity is reported to be increased by OA treatment; Ca2+ imaging of hippocampal synaptosomes found that a 30-min incubation with 100 nmol/L OA-enhanced Ca2+ entry towards 100 mmol/L KCl depolarisation (e.g. Bartschat and Rhodes 1995). Interestingly, at high levels of Ser/Thr phosphatase inhibitors a reduction in Ca2+ responses towards high KCl challenges were observed here.

The OA-induced suppression of neuronal excitability and signalling provides a possible explanation for impaired slice physiology. However, treatment of cultures with CA differed from slice data, as it produced an enhancement of NMDA signalling. Nevertheless, suppression of neuronal signalling was attained following longer CA incubations. It is likely that cultures are less susceptible to apoptosis induction due to the incubation conditions, conducted in antioxidant- and nutrient-rich culturing medium, while freshly prepared slices are only viable for a period of hours in the standard aCSF solution. Additionally, differences in basal phosphorylation levels between the two approaches related to the preparation, age, differences in receptor expression after culturing and/or selective subfields (heterogeneous hippocampal population vs. CA3–CA1 pyramidal neurons) may also have contributed to the observed difference in susceptibility.

Potential phosphorylation targets and a potential link to dementias

Whilst this is the first report on suppressed NMDA/VGCC signalling after Ser/Thr phosphatase inhibition in hippocampal neurons, evidence for such action is found within the literature on direct protein kinase stimulation, an expected consequence of phosphatase inhibition. Treatment with PP inhibitors, specifically with OA, resulted in an increase in total serine residue phosphorylation, PKA and PKC substrate phosphorylation as well general tyrosine residue phosphorylation (Hill et al. 2006). The inhibition of phosphatases also increased the activity of many other kinases, including Ca2+/calmodulin-dependant kinase II and PKA (Li et al. 2004). Thus, phosphorylation at a multitude of both Ser/Thr but also tyrosine residues can be expected to increase over the incubation duration.

Regulation of NMDARs is diverse, PKC and PKA phosphorylation sites have been shown to be present on NR1, NR2A and NR2B subunits, and phosphorylation is enhanced in the presence of OA implicating PP1 and/or PP2A in the regulation of these sites (Leonard and Hell 1997). Both PKA (Raman et al. 1996) and PKC (Ben-Ari et al. 1992) stimulation have been shown to enhance NMDAR activity, similar to incubations with 1 nmol/L CA for 24 h. Equally, VGCCs are regulated via numerous Ser/Thr kinases, PKA, PKC and Ca2+/calmodulin-dependant kinase II (Bartschat and Rhodes 1995; Hell et al. 1995; Grueter et al. 2006), which are implicated in phosphorylation and potentiation of channels. However, low-level phosphatase inhibition failed to enhance VGCC activity. Higher or prolonged levels of PP inhibition may activate alternative or additional phosphorylation cascades. PKC-dependent phosphorylation has indeed also been implicated in a reduction of both VGCC (Doerner and Alger 1992) and NMDAR (Markram and Segal 1992 and Jackson et al. 2006) activity. These results suggest that bidirectional effects of kinases and phosphatases are commonly observed and are related to experimental conditions. In this study, we show that NMDA NR1 subunit phosphorylation at the PKC-mediated NR1 phosphorylation site Ser890 (Sánchez-Pérez and Felipo 2005) increased significantly only following 2 h 100 nmol/L OA treatment. This pattern does not show a correlation with changes in NMDA signalling and thus suggests that phosphorylation at this site may only play a role in short-term alterations, while further phosphorylation sites or additional pathways may dominate after longer incubations. In contrast, increased phosphorylation of tau at Ser396 showed a correlation with impaired NMDA signalling at both time windows.

We also observed a striking reorganisation of the microtubule network in cultures treated similar to hippocampal slices (2 h 100 nmol/L OA). Such reorganisation may be caused by tau dissociation from microtubules as seen in AD brains (Alonso et al. 1994) and may have a causative role in the signalling deficits described above. A reduction of the neuronal branching in the hippocampus would correlate with impaired synaptic contacts and communication, alongside diminished synaptic transmission, NMDA and VGCC signalling.

In AD brains, alterations in the expression and activity of a number of proteins and phosphatases relevant to the above study are found, e.g. PP2A (Vogelsberg-Ragaglia et al. 2001; Sontag et al. 2004) as well as PP5 (Liu et al. 2005a) are reduced and the activity of endogenous inhibitors of PP2A are up-regulated (Tanimukai et al. 2005) compared with healthy brains (for review see Manusy et al. 2006). Such reduced activity of phosphatases likely contributes to the formation of neurofibrillary tangles, primarily composed of hyperphosphorylated tau (Braak and Braak 1995; see Iqbal et al. 2005 for review).

The dephosphorylation of tau by Ser/Thr PP is well documented and includes PP1 2A, B and C (Gong et al. 1994a,b; Wang et al. 1995) and more recently PP5 (Liu et al. 2005b). Pseudo-hyperphosphorylated tau results in the induction of apoptotic neuronal death (Fath et al. 2002) and thus may be involved in the cellular impairments seen in the present study. Additionally, AD brains show evidence of reduced NMDAR subunits NR1 and NR2B protein (Sze et al. 2001) and mRNA (Mishizen-Eberz et al. 2004). In line with many reports on transgenic animal models of neurodegenerative diseases (e.g. Oddo et al. 2003; Hartman et al. 2005; Jacobsen et al. 2006), we have found impairments of basal synaptic transmission and LTP. Similar deficits are seen in animal models where Ser/Thr phosphatase inhibitors are infused into the brain, also leading to impaired learning and memory (Sun et al. 2003), expression of hyperphosphorylated tau, β-amyloid deposits and neuronal apoptosis (Arendt et al. 1998). Thus, the alterations in neuronal function observed here mirror some of the neuronal defects observed in neurodegenerative dementias such as AD.

In conclusion, we show that bidirectional modulation of excitability occurs during long-term inhibition of PPs, ultimately resulting in suppression of signalling at high levels of inhibition. This suppression is coincident with increased phosphorylation of cytoskeletal proteins and a disruption of the microtubule network. Long-term changes in phosphorylation closer resemble the situation in neurodegenerative dementia, and we propose that reduced neuronal transmission may be an immediate result of such changes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors would like to thank the Alzheimer Research Trust, UK, for financial support.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Fig. S1 Repetitive applications of 10 mol/L NMDA and 15 mmol/L KCl flanking 20 min static bath incubations. (a) Mean trace of 10 neuronal regions of interest in ratiometric fluorescence units; responses were evoked with 10 μmol/L NMDA (2 min) and 15mmol/L KCl (1 min) pre- and post-20 min static bath incubations in low Mg2+ HBS. (b) Maximal mean NMDA responses pre- and post 20min static bath. (c) Respective mean responses towards KCl. Data are shown as mean percentage delta F/F + SEM; ***p < 0.0001. The percentage responder rate for all groups is given within each bar.

FilenameFormatSizeDescription
JNC4579FigS1.TIF82KSupporting info item

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