In order to evaluate the morphological changes induced by Aβ-AChE complexes in hippocampal neurons, the following immunofluorescence studies were performed. Hippocampal neurons were treated with 5 μM of Aβ preparations: Aβ fibrils (Aβf), Aβ oligomers (Aβo), Aβ-AChE mostly fibers (Aβ-AChEf) and Aβ-AChE mostly oligomers (Aβ-AChEo) (see additional file 1). We used an Aβ reverse sequence (5 μM) and AChE (5 nM), for 1 hr as controls. Neurons were stained for MAP-1B (Fig. 1A) or NF-200 (Table 1). Neurons treated with Aβo (Fig. 1Ac) were observed to have a higher loss of their neurite network compared to neurons treated with Aβf (fig 1Ae). Furthermore, while neurons treated with Aβ-AChEo (Fig. 1Ab) appeared to have a small, loss of their neuritic network, neurons treated with Aβ-AChEf (Fig. 1Ad) were observed to have more damage on their neurite network than neurons treated with Aβf (Fig. 1Ae). Complementary immunofluorescence studies with a NF-200 antibody showed that Aβ-AChEf significantly decreased the length of the neurites by 40%, in comparison with Aβf-treated neurons (see Table 1). Additionally, we evaluated the effect of different Aβ and Aβ-AChE preparations on synaptic proteins (Additional file 2). We performed an immunofluorescence assay for presynaptic protein synapsin-1 and postsynaptic protein PSD-95. In agreement with previous studies, Aβo treatment decreased the immunostaining for PSD-95 whereas Aβf had no effect 17 . Also, Aβ-AChEo treatment induced a critical decrease in PSD-95 immunofluorescence similar to Aβo preparations, whereas Aβ-AChEf had no effect (Additional file 2), suggesting that Aβ oligomers formed in the presence of AChE have a similar synaptotoxicity to those formed in its absence.

To determine whether Aβ-AChE is more toxic than Aβ, we performed cell viability studies using MTT, as an indicator of cell viability. Aβf alone decreased cell viability by 35 ± 7% when compared with control, while Aβ-AChEf under the same conditions and concentration reduced viability by 50 ± 6%, suggesting that the complexes are significantly more toxic than Aβ aggregates alone at equal concentrations (Fig. 1B).

An increase in intracellular calcium has been observed in Aβ treated neurons as well as during glutamate excitotoxicity 18 . Therefore, we characterized calcium homeostasis after exposure of hippocampal neurons to Aβ-AChEf. Neurons were loaded for 30 min with Fluo-3 AM and then cytosolic calcium levels were evaluated in cells exposed to 5 nM AChE (Fig. 1C, silver circles), 5 μM Aβf (Fig. 1C, open circles) and 5 μM Aβ-AChEf (Fig. 1C, closed circles). Neurons treated with Aβf and Aβ-AChEf showed a marked and consistent calcium increase, while treatment with AChE enzyme alone showed no effect on the mobilization of intracellular calcium. However, the final fluorescence levels reached after 1 h treatment with Aβ-AChEf were significantly higher than Aβf, indicating that Aβ-AChEf complexes induces a sustained higher increase in intracellular calcium, in comparison with Aβ-treated neurons (Fig. 1C, inset). Then, we evaluated whether the calcium increase observed by treatments trigger the apoptotic cascade after 1 h treatment. The caspase-3 activity measurements showed that Aβ-AChEf significant increase the caspase-3 activity compared with Aβf or Aβo. On the other hand, AChE treatment did not show any effect (Fig. 1D). These results indicate that Aβ-AChE complexes showed a greater modification of intracellular calcium homeostasis than Aβ alone triggering the activation of the apoptotic pathway.

Given the effect of the Aβ-AChE on intracellular calcium homeostasis, we examined whether the Aβ-AChEf alter the integrity of the plasma membrane. We treated hippocampal neurons with 5 μM Aβ-AChEf and then loaded with calcein-AM and Fura-red. After 1 h treatment the fluorescence of Fura-red in neurons treated with Aβ-AChEf (Fig. 2Ad) decreased respect to the control (Fig. 2Ac), whereas the calcein fluorescence was not affected respect to the control (Fig. 2Aa and 2b), suggesting that the Aβ-AChEf treatment did not alters the integrity of the plasma membrane. We also evaluated whether Aβ-AChEo preparation interacts with the neuronal membrane through surface biotinyl assay. Fig. 2B shows that Aβ-AChEo interacts with the cell membrane, suggesting that the effects observed are because the complex is acting at the neuronal membrane level. Then, we evaluated the effect of different concentrations of Aβ-AChEf and the source of calcium responsible for the intracellular calcium increase. We determined the behaviour of calcium levels in neurons loaded with Fluo-3 AM and then treated with increasing Aβ-AChEf concentrations. We observed that the increase in the cytoplasmic calcium levels correlated with increasing Aβ-AChEf concentrations (Fig. 2C). In order to determine the source of the intracellular calcium increase induced by the complex, we treated neurons with the complex (5 μM Aβ-AChEf) in the presence of the extracellular calcium chelator, EGTA (2 mM), or the intracellular calcium chelator, BAPTA-AM (30 μM). We observed that treatment with EGTA prevented the increase of intracellular calcium induced by the complex (Fig. 2D). Additionally in neurons pre-incubated with BAPTA-AM and treated with the complex, we observed a ~ 2.8-fold increase in the intracellular calcium (Fig. 2D). These results suggest that intracellular calcium deregulation induced by the complex is dependent on extracellular calcium and that massive entrance of the ion could release intracellular calcium reservoirs. In order to study the role of the intracellular calcium deregulation in the neurotoxic properties of Aβ-AChE, we performed MTT cell viability assays in the presence of 2 mM EGTA, as indicated in Fig. 2E. Five μM Aβ-AChEf induced ~ 55% cell death, while EGTA partially reduced neuronal death triggered by the Aβ-AChEf complex (~ 25% reduction of viability), suggesting that extracellular calcium has an important role, but is not the only player in the mechanism of toxicity induced by the complex.

To determine whether the cytosolic calcium dysregulation induced by Aβ-AChE complexes alter mitochondrial function; we analyzed the variations in mitochondrial membrane potential (ΔΨ_mit) of hippocampal neurons treated with the Aβ-AChEf complex, AChE and Aβf, respectively (Fig. 3). In vivo confocal images of hippocampal neurons exposed to Aβ-AChEf showed a severe loss of mitochondrial membrane potential using TMRM⁺, a mitochondrial potential indicator which detects alterations in mitochondrial membrane polarity (Fig. 3A) 19 20 . Photographs registered at 30 min showed a large decrease in the fluorescence of the mitochondrial potential indicator after addition of 5 μM Aβ-AChEf when compared to the beginning of the experiment (Fig. 3Aa, b). Quantification of the reduction in the mitochondria membrane potential shows a decrease of -1 normalized fluorescence unit when compared to start levels (Fig. 3A, c). Treatment with Aβf (5 μM) alone induced partial disturbances in ΔΨ_mit levels represented by a decrease of ~ -0.4 normalized fluorescence units (Fig. 3B, a) and AChE treatment (5 nM) exhibited no alteration of ΔΨ_mit levels (Fig. 3B, b). Fig. 3C shows the quantification of mitochondrial potential levels reached at the end of the experiment (1 h) in each condition. Analysis of three independent experiments showed that Aβ-AChEf induced a significant decrease in ΔΨ_mit that was 2-fold higher than the decrease induced by Aβf treatment, while AChE had no effect on ΔΨ_mit (Fig. 3C). To evaluate the reversibility of the effect produced by Aβ-AChEf treatment on calcium homeostasis and ΔΨ_mit levels, hippocampal neurons were loaded with Fluo-3 AM and TMRM⁺, followed by the addition of 5 μM Aβ-AChEf for 15 min, and then washed with fresh KRH-glucose buffer. In these studies, the Aβ-AChEf induced calcium increase was completely reversed to basal levels (Fig. 3D, closed circles), however, mitochondrial membrane potential loss was not recovered after washing out of Aβ-AChEf (Fig. 3D, open circles). These results suggest that Aβ-AChE complexes could be acting at the neuronal surface, triggering an entrance of calcium depending on the presence of the complex; however, this intracellular calcium deregulation produces an irreversible effect on the mitochondrial membrane potential that could be responsible for the higher toxicity levels presented by Aβ-AChE.

Treatment with Aβ-AChE complexes caused a calcium influx in hippocampal neurons and a severe reduction in mitochondrial membrane potential. In order to study the role of mitochondria in the calcium deregulation induced by Aβ-AChE, we treated hippocampal neurons with Aβ and Aβ-AChE for 1 h and stained with MitoTracker. In control neurons without treatment we observed that most of the active mitochondria were localized through neurites (Fig. 4Aa). In the case of neurons treated with Aβ-AChEf, the fluorescence of active mitochondria decreased proportional to increasing Aβ-AChEf concentrations (Fig. 4Ab, c). Aβf (Fig. 4Ad, e) reduced the fluorescence of active mitochondria more than Aβo (Fig. 4Af, g), however both showed lower effect than Aβ-AChEf (Fig. 4B). AChE treatment had no effect (Fig. 4Ah). The quantification of MitoTracker fluorescence intensity indicated that Aβf and Aβo significant decreased the mitochondrial potential respect to control neurons. However, Aβ-AChEf treatment produced around 50% decrease in the mitochondrial potential. Then, we evaluated the mitochondrial calcium uptake 20 21 . Hippocampal neurons were loaded with Rhod-2 and then treated with Aβf and Aβ-AChEf. Aβf treatment produced an increase in the calcium uptake whereas Aβ-AChEf induced an acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels (Fig. 4C). Interestingly, decrease in mitochondrial calcium uptake correlates with severe mitochondrial potential loss induced by Aβ-AChE complexes in neurons. These results suggest that Aβ-AChEf produces the release of mitochondrial calcium, according with our previous result with the intracellular calcium chelator BAPTA-AM (Fig. 2D).

Previous studies in our laboratory showed that lithium and Wnt ligands protect neurons from Aβ toxicity by activation of the Wnt signaling pathway 22 . This pathway is also implicated in the development of the nervous system, and recently it has been demonstrated to regulate synaptic differentiation and neurotransmission 23 24 . We performed calcium experiments using Fluo-3 AM in neurons treated with Aβ-AChEf alone (Fig. 5Aa) or co-incubated with lithium or neurons pre-treated with Wnt-7a ligand conditioned-medium for 24 h and then exposed to Aβ-AChEf (Fig. 5Ab). Lithium (10 mM) prevented the intracellular calcium influx induced by the complex (Fig. 5Ab, open squares). In addition, Wnt-7a ligand also prevented the cytoplasmic calcium increase induced by the complex in hippocampal neurons (Fig. 5Ab, open circles). The quantification of the changes in fluorescence levels at 30 min into the experiment showed a significant prevention in the calcium increase triggered by Aβ-AChEf by the presence of lithium (Fig. 5Ac) and Wnt-7a ligand (Fig. 5Ad). Additionally, we tested the role of the NMDA receptor in the intracellular calcium deregulation induced by the Aβ-AChE complexes. In these studies, we incubated hippocampal neurons with 5 μM Aβ-AChEf in the presence of NMDA receptor antagonist MK-801 and cytoplasmic calcium changes were monitored (Fig. 5B). The inhibition of NMDA receptor activity blocked cytoplasmic calcium influx produced by the complex, (Fig. 5Ba) and the quantitative analysis of three independent experiments at 30 min into the experiments, corroborating the complete inhibition of calcium influx induced by Aβ-AChEf in hippocampal neurons (Fig. 5Bb). Additionally, inhibition of NMDA receptors prevented cytosolic calcium increase induced by 5 μM Aβ treatment (data not shown). These results suggest that both the activation of Wnt pathway and the inhibition of NMDA receptor prevent the cytoplasmic calcium deregulation induced by Aβ-AChE complexes in hippocampal neurons.

It has been suggested that Aβ oligomers are responsible for the toxic effects of Aβ 25 26 27 . Additionally, Aβ oligomers induced an accelerated calcium influx, mitochondrial depolarization, decreased ATP levels, and cytochrome c release in cortical neurons 28 . Therefore, we decided to evaluate whether Aβ-AChE containing Aβ oligomers, show the same toxicity found in hippocampal neurons exposed to the complex containing Aβ fibrils. Aβ was aggregated, at 37°C, in the absence or presence of AChE enzyme and aliquots were taken at different times of Aβ aggregation and analyzed by electron microscopy. The formation of Aβ oligomers was faster in the presence of enzyme AChE (compare picture Fig. 6Ac with 6Ad). The microphotographs show oligomeric structures present at 1 h of AChE and Aβ incubation (Fig. 6Ad also, Additional file 1), where in the case of Aβ alone, oligomeric structures appear only after 2 h incubation (Fig. 6Ae). Clearly, there are more Aβ oligomers formed in the presence of AChE, which correspond to either protofibrils (Fig. 6Af arrow), or amylospheroid assemblies (Fig. 6Ah, arrow head). Previous studies indicated that high Aβ oligomer neurotoxicity is associated with an accelerated calcium influx and mitochondrial impairment 28 . In addition, Aβ oligomers prepared in the presence of AChE were subjected to non-denaturing electrophoresis gels and western blotting for Aβ (Fig. 6Ba). The western blot showed 3 bands corresponding to monomers, dimmers and trimmers of Aβ when aggregation was performed without the presence of AChE enzyme (Fig. 6Ba, line 1). When Aβ was incubated with AChE, we observed the 3 bands for monomers, dimmers and trimmers plus a high molecular weight band of 74 KDa (Fig. 6Ba, line 2). In order to see whether AChE-Aβo affect intracellular Ca²⁺, hippocampal neurons were loaded with Fluo-3 AM. The use of an oligomeric-rich preparation (Aβ-AChEo) induced a faster and prominent calcium increase (~ 0.6 normalized fluorescence units during the first 10 min of treatment) respect to neurons treated with normal Aβ-AChEf complexes (~ 0.2 normalized fluorescence units during the first 10 min of treatment), indicating that the oligomeric-rich preparation had a fast effect on calcium homeostasis (Fig. 6Bb). These results are in agreement with the evidence that indicates that Aβ oligomers induce a similar effect on cell viability and calcium homeostasis in a human neuroblastoma cell line 28 .

Synthetic Aβ_1-42 and Aβ_42-1 peptides corresponding to the human Aβ wild-type and reverse sequence, respectively and both were obtained from Genemed Synthesis, Inc. (San Francisco, CA). Chemicals, culture media and sera were obtained from Sigma (St. Louis, MO), Roche (Alameda, CA), Merck (Darmstadt, Germany), Gibco BRL (Paisley, UK). Fluo-3 AM, Calcein-AM, Fura Red AM, and Bapta AM from Molecular Probes (Eugene, OR). Rhod-2AM, and TMRM⁺ (Xanthylium,3,6-bis(dimethylamino)-9-(2-(methoxycarbonyl)phenyl)-, perchlorate) were a kind gift of Dr. Luis Felipe Barros (CECS, Valdivia, Chile).

Tetrameric G₄ AChE form (sedimentation coefficient, 10.7 S) was purified from bovine caudate nucleus, using acridine affinity chromatography as previously described 44 . Both specific activity (6,000 U/mg protein) and staining intensity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to verify purity.

Aβ aggregates were formed in a turbidity assay as previously described 45 . Briefly, For Aβf and Aβ-AChEf, the Aβ peptide stock solution was prepared by dissolving freeze-dried aliquots of Aβ in dimethyl sulfoxide (DMSO) at 15 mg/ml (3.5 mmol/L). An aliquot of this stock solution equivalent to 15 μg of Aβ peptide was added to aqueous buffer (725 μl total volume; 100 μM PSB, pH 7.4). For the aggregation assay in the presence of AChE, an identical aliquot of the stock solution was added to a buffer containing AChE (100 nM). The solutions were stirred continuously (1,350 rpm) at room temperature for 24 h and then left at 4°C for another 48 h, then an aliquot was taken and observed by electron microscopy. Aggregation was measured by turbidity at 405 nm against a buffer blank. Amyloid aggregates obtained were characterized by Thioflavin-T (Th-T) binding 45 .

Aβoligomers were formed using Aβ peptide in the presence of AChE. Briefly, a 50 μM Aβ stock solution was prepared in the presence of 50 nM AChE. The solution was kept at 37°C for 1 h and then used immediately. An aliquot of Aβo and Aβ-AChEo was separated by centrifugation at 14,000 rpm (15 min) for electron microscopy. The supernatant was saved for non-denaturing SDS-PAGE gels using SDS-free and β-mercaptoethanol-free loading buffer and then regular western blot.

Fresh aliquots of samples were diluted 1:3 in water and 5 βl placed on Parlodion/carbon-coated 300-mesh copper grids for 60s. Excess sample was removed and 5 μl of 2% aqueous uranyl acetate was placed onto the grid for 10s, followed by removal of excess staining solution with filter paper and air-drying. Observations were carried out using a Philips Tecnai 12 electron microscope operated at 80 kV. Photographs were taken at original magnifications of 49,000×. Copies from the negatives were made with a further 3× magnification of given areas and used for figure presentation.

Hippocampi from Sprague-Dawley rats at embryonic day 18 were dissected and primary rat hippocampal cultures were prepared as described previously 39 45 . Hippocampal cells were seeded in polylysine-coated wells and cultivated in Neurobasal medium supplemented with B27, on day 3 of culture; cells were treated with 2 μM 1-β-D-arabinofuranosylcytosine (AraC) for 24 h to reduce most of the glial cells present in the culture. Seven days later, cultured hippocampal neurons were used for various experiments. The average number of neurons in each experiment corresponded approximately to 98% of total cells present in the cultures.

Cell viability was measured by the modified 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay as described previously 45 .

Hippocampal neurons plated on polylysine-coated covers (25,000 cells/cover) were treated and then fixed with 4% paraformaldehyde/4% sucrose in PBS for 20 min, permeabilized with 0.2% Triton-X100 for 5 min, blocked with 0.2% gelatine and immunostained using an anti- Map-1B, neurofilament protein, synapsin-1 or PSD-95 antibody (1:500). Coverslips were mounted and then analyzed under a Zeiss confocal microscope.

The number and length of neurites were quantified using an Image-Pro plus software as described previously 38 .

Hippocampal neurons 15 DIV were treated with Aβ-AChEf, Aβo and Aβf for 1 h at 37°C, then the cells were homogenized with RIPA buffer and then lysates were centrifuged at 15,000 g for 20 min at 4°C, the supernatant were collected to determinate Caspase activity. Caspase-3, substrate chromogenic (Upstate Biotechnology Inc., Lake Placid, NY, USA) was prepared in 12.5 mM buffer Hepes; 31.25 w/v Sucrose; 0,3125 w/v CHAPS pH 7.4. The reaction was measured at 405 nm in a microtiter plate reader.

Hippocampal neurons 15 DIV were treated with Aβ-AChEo (1 or 5 μM) or AChE alone (1 or 5 nM) for 1 h and membrane surface proteins were biotinylated with sulfo-NHS-LC-biotin (Pierce) in a final concentration of 0.5 mg/mL for 45 min. After biotinylation step, the free biotin was quenched by incubation with 50 mM NH₄Cl for 10 min. Cells were lysed in ice-cold SA buffer (150 mM NaCl; 20 mM Tris pH 8.0; 5 mM EDTA; 1% Triton-X-100; 0.2% BSA and protease inhibitors). Nuclear and cellular debris was removed by centrifugation at 14,000 x g for 5 min at 4 °C and the biotinylated cell-surface proteins were then adsorbed to streptavidin agarose beads for 16 h at 4 °C. Beads were washed and bound proteins were analyzed by SDS-PAGE followed by immunoblotting against AChE. The values for biotinylated cargo proteins were normalized to total cargo proteins expressed in the cells.

Changes in mitochondrial membrane potential was determined by specific mitochondrial probe TMRM⁺ and detected in a confocal microscope 19 46 . Neurons were grown on poly-L-lysine-coated glass coverslips and cultured for 5 days. The cells were then loaded for 30 min with 30 nM TMRM⁺ in Krebs-Ringer-Hepes (136 mM NaCl; 20 mM Hepes; 4.7 mM KCl; 1.25 mM MgSO₄; 1.25 mM CaCl₂) (KRH)-glucose containing 0.02% pluronic acid, then washed, and allowed to equilibrate for 30 min. Coverslips were then mounted in a chamber on the stage of a confocal laser scanning microscope (LSM Pascal Zeiss model 510, Carl Zeiss Ltd.). The fluorescence changes determined by TMRM fluorescence indicated the mitochondria potential changes. Images were acquired using a 543-nm He-Ne laser to excite TMRM⁺ and the signals were collected at 570 nm 18 19 20 . Signal from control neurons and neurons treated with Aβ-AChE complexes were compared using identical settings for laser power, confocal thickness and detector sensitivity. The images were analyzed with Zeiss confocal software, the mean TMRM⁺ fluorescence signal was measured per live cell. Estimation of fluorescence intensity of TMRM⁺ was presented like the pseudoratio (ΔF/F_o) indicated by: ΔF/F_o = (F-F_base)/(F_base-B), where F is the measured fluorescence intensity of the indicator, F_base is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the cells. For mitochondrial membrane potential by MitoTracker fluorescence, hippocampal neurons 15 DIV were treated with Aβ-AChEf, Aβo and Aβf for 1 h at 37°C. Then, cells were stained with MitoTrackers-fx (Molecular Probes, Eugene, OR). In all treatments, individual coverslips were rinsed twice in PBS and loaded with 10-500 nM MitoTracker for 15 min at 37°C. Dyes were diluted in PBS from a 1 mM stock in anhydrous dimethyl sulfoxide. Coverslips were rinsed for 15 min in PBS at room temperature. Loading parameters were tested and optimized for assessing fluorescence intensity and localization in Confocal Zeis Microscope and the quantification was made using Image J Program.

Neurons grown on glass coverslip were loaded for 30 min (37°C) with the following fluorescent probes as their acetoxymethyl (AM) ester forms: 5 μM Fluo-3 AM and 10 μM Rhod-2 AM in Krebs-Ringer-Hepes (KRH)-glucose containing 0.02% pluronic acid. The fluorescence changes determined by Fluo-3 represent the cytoplasmic calcium [Ca²⁺]_cyt changes. Coverslips were washed three times with PBS and left in KRH-glucose for 10 min until cell fluorescence had reach plateau. Fluorescence was imaged with a confocal laser scanning microscope as described previously 19 . Images were acquired using a 488-nm Argon laser to excite Fluo-3 and Fura-Red fluorescence. The signals were collected at 505-530 nm (Fluo-3). Background was measured in parts of the field devoid of cells and found to be not significantly different from the signal recorded in cells depleted of dye with 100 μM digitonin. This value was subtracted from cell measurements. The fluorescence intensity variation was recorded from 15-20 neurons in average per experiment. Estimation of fluorescence intensity of Fluo-3 was presented like the pseudoratio (ΔF/F_o), as indicated before 18 19 20 .

Hippocampal neurons were loaded 30 min with Calcein-AM (Molecular Probes, Leiden, Netherlands) as an indicator of cell integrity 19 .

Hippocampal neurons were grown on 35-mm dishes and loaded with 5 ìM Rhod-2 AM in KRH-glucose buffer containing 0.02% pluronic acid. The fluorescence changes determined by Rhod-2 indicate calcium changes in the mitochondria 19 . To estimate Rhod-2 fluorescence pattern in live mitochondria, we used MitoTracker Green™ (MTG) to mark the mitochondria. Cells were washed 3 times and left in KRH-glucose buffer for 10 min until cell fluorescence equilibrated. Fluorescence was imaged with a confocal laser scanning microscope (Leica TCS SP1) using a 40× water immersion lens. Images were acquired using a 488-nm argon laser to excite MTG fluorescence and a 563-nm He-Ne laser to excite Rhod-2 fluorescence. The signals were collected at 505-530 nm (MTG) and at 590 nm (Rhod-2). Fluorescence background signal was subtracted from cell fluorescence measurements in every experiment. The fluorescence intensity variation was recorded from 5-10 cells on average per experiment. Estimation of fluorescence intensities were presented as the pseudoratio (ΔF/F_o), which was calculated using the formula ΔF/F_o = (F - F_base)/(F_base - B), where F is the measured fluorescence intensity of the indicator, F_base is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the cells 21 .

Results were expressed as mean ± standard error. Student's t test and the Mann-Whitney test were used for analyzing data for fluorescence measurements and image analysis. P < 0.05 was regarded as statistically significant.