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.
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.
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.
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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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.
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).
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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