Cognitive and histological disturbances after chlorpyrifos exposure and chronic Aβ(1–42) infusions in Wistar rats

NeuroToxicology 32 (2011) 836–844

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NeuroToxicology

Cognitive and histological disturbances after chlorpyrifos exposure and chronic Ab(1–42) infusions in Wistar rats ˜ oz a, Francisco A. Nieto-Escamez a, Susana Aznar c, Marı´a T. Colomina b, Ana M. Ruiz-Mun Fernando Sanchez-Santed a,* a b c

Department Neuroscience and Health Sciences, Faculty of Psychology, University of Almeria, Crta. Sacramento S/N, 04120 Almeria, Spain Laboratory of Toxicology and Environmental Health, and Research Center in Behavioural Assessment (CRAMC), Rovira i Virgili University, San Lorenzo 21, 43201 Reus, Spain Center for Integrated Molecular Brain Imaging, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 March 2011 Accepted 27 May 2011 Available online 6 June 2011

Exposure to pesticides has been linked to an increased vulnerability to neurodegenerative diseases. In order to study whether the exposure to the organophosphate chlorpyrifos renders the brain prone to amyloid-beta peptide deposition and accelerates its neuropathological and behavioural effects, Wistar rats were injected a single subcutaneous dose of chlorpyrifos (250 mg/kg) and subsequently infused with Ab(1–42) peptide (i.c.v.) for 15 days. No effects of either treatment were noted in the classic water maze test. The animals infused with Ab peptide showed worse performance when the platform was both hidden and moved from trial to trial. Both groups showed worse performance when the platform was visible and moved from trial to trial. No amyloid deposition was observed in hippocampus or cerebral cortex after the infusion period, although microtubule-associated protein 1A (MAP1A) immunoreactivity was significantly reduced in hippocampus and prefrontal cortex, whereas chlorpyrifos exposure produced a significant reduction of microtubule-associated protein 2 (MAP2) in the prefrontal cortex. Therefore, behavioural deficits could be related to a loss of dendrite and spine processes in these brain regions. ß 2011 Elsevier Inc. All rights reserved.

Keywords: Beta-amyloid Chlorpyrifos Learning Memory Flexibility MAPs

1. Introduction Accumulation of Ab peptide aggregates in the brain has been hypothesized to play a central role in Alzheimer disease (AD) neuropathology, and it has been reported that extracellular and intraneuronal formation of Ab deposits are involved in AD pathogenesis. However, the precise mechanism of Ab neurotoxicity is not completely understood. One of the recently proposed targets of Ab is microtubuleassociated proteins (MAPs), leading to cytoskeletal disorganization and neuronal degeneration associated with the early stages of AD (Fifre et al., 2006; Gevorkian et al., 2008). Thus, a loss of MAP1, MAP2 and normal tau associated with cytoskeleton breakdown has been associated to AD (Alonso et al., 1997; Iqbal et al., 2009). This

* Corresponding author. Tel.: +34 950 214631; fax: +34 950 214627. ˜ oz), E-mail addresses: [email protected] (A.M. Ruiz-Mun [email protected] (F.A. Nieto-Escamez), [email protected] (S. Aznar), [email protected] (M.T. Colomina), [email protected] (F. Sanchez-Santed). 0161-813X/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2011.05.014

loss of synaptic contacts has therefore been considered a hallmark of AD. It is important to remark that over 95% of all AD cases are sporadic, with genetics playing a minor role in the etiology of this pathology. Therefore, it becomes crucial to investigate the environment as primary risk factor in AD. An important group of environmental toxics are organophosphate (OP) pesticides. Some epidemiological studies have reported a relationship between pesticides and dementia. Additionally, occupational exposure to pesticides has also been related to a higher risk of Alzheimer disease (Abdollahi et al., 2004; Baldi et al., 2003; Hayden ˜ ez et al., 2007). et al., 2010; Santiba´n One pesticide which has spurred renewed interest is chlorpyrifos (CPF). CPF is a broad spectrum OP insecticide utilized extensively throughout the world. It has been shown that exposure to CPF induces cognitive deficits and emotional alterations in humans and laboratory animals. In this line, data from our ˜ adas laboratory have shown cognitive impairments in rats (Can et al., 2005; Sa´nchez-Santed et al., 2004) and humans (Rolda´nTapia et al., 2005, 2006). Although OP pesticides have been classically described as inhibitors of acetylcholinesterase (AChE) activity, there are

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recent evidences supporting the hypothesis that these compounds do also act through non-cholinergic pathways, especially those related to cognitive processes. For instance, it has recently been described that CPF exposure produces a selective reduction in the availability and functional effects of MAP-2 proteins preceding neuronal injury ‘‘in vitro’’ (Prendergast et al., 2007), and inhibits kinesin-dependent microtubule motility (Gearhart et al., 2007). All these data permit us to hypothesize a possible interaction between exposure to CPF and an increased risk of developing AD. In this way, rats exposed to CPF would be more prone to the effects of intracerebral Ab infusions. Thereby, we designed a study in which multiple i.c.v. injections of Ab(1–42) were conducted on rats that previously received an acute dose of CPF (compared to their controls). This would allow us to analyze the interaction between both substances on learning and memory variables along with histological markers for AD. Spatial learning and memory were evaluated in the water maze, and histological analyses were performed to measure the presence of Ab deposits along with MAP levels in cerebral cortex and hippocampus.

˜ adas subjects who gradually recover activity after 12 weeks (Can et al., 2005).

2. Materials and methods

The peptide (amyloid b-protein (1–42) hydrochloride salt, H-6466, BACHEM, Switzerland) was diluted at a concentration of 1 mg/166 ml of ultrapure water. Sterile PBS was added to complete a final volume of 1 ml. The solution was then aggregated at 37 8C for 72 h. Ab(1–42) is more readily fibrillated than Ab(1–40), and its HCl form facilitates such aggregation (Takata et al., 2003). Aggregated Ab(1–42) and PBS were infused during 15 days in a daily dose of 3 mg/3 ml, using a precision syringe (Hamilton 10 ml) connected to a micro-perfusion pump (Harvard apparatus, USA). The speed of injection was 1 ml/min. All infusion material was sterilized with sodium dichloroisocianurate (Suavinexß) tablets for 30 min before use. From the beginning to the end of infusions (the day before the behavioural assessment finished) animals were administered 10 mg/ kg of antibiotic (Enrofloxacine (Baytrilß) 0.5%, oral solution, BAYER, Barcelona) diluted in the drink water as a preventive treatment.

2.1. Animals Subjects were 32 male Wistar rats (Charles River Laboratories, Spain), weighing between 300 g and 350 g at the start of the experiment. Rats were housed in groups of three and kept in a regulated environment (temperature at 23  1 8C) with a 12:12 h light:dark cycle (lights on at 08:00 a.m.). Nourishment of the animals was ad libitum during the whole experiment. Rats were allowed to habituate to their new housing conditions for 7 days after their arrival and then handled for 5 days. All testing was carried out in accordance with the Spanish Royal Decree 1201/2005 on the protection of experimental animals, and with the European Community Council Directives (86/609/EEC). 2.2. Surgery After habituation and handling periods, rats were implanted with an intracerebroventricular cannula following stereotaxic procedures. Rats were previously anesthetized with equithesine (0.3 ml/100 g i.p.), receiving an injection of atropine (0.5 mg/kg i.p.) to counterbalance the cardiorespiratory effects. The cannula was implanted into the right ventricle (coordinates 0.9 mm posterior to bregma, 1.3 mm lateral to the midline and 3.5 mm ventral from dura mater, according to the brain atlas of Paxinos and Watson, 1998) and fixed with dental cement and 4 steel screws attached to the skull surface. Animals were kept in individual cages for the rest of the experiment. 2.3. CPF intoxication After a post-operative period of 2 weeks, rats were randomly divided into two groups, and then received a subcutaneous injection of 250 mg/kg of CPF (PESTANAL SIGMA–ALDRICH LABORCHEMIKALIEN, Germany) or its vehicle (corn oil, 1 ml/ kg body weight). Body weight and temperature were recorded 24 h, 48 h and 7 days later as an intoxication control. The dose used is based on our previous studies showing behavioural and cognitive deficits both short and long term after 250 mg/kg s.c. administration (Sa´nchez-Santed et al., 2004; Cardona et al., 2006). Typically it induces a profound acute AChE inhibition on

2.4. Amyloid b(1–42) administration A 2 week period was again left for recovery from CPF intoxication. Then, every group of rats (CPF and vehicle) was subdivided into two further groups for receiving either i.c.v. Ab(1– 42) or phosphate-buffered saline (PBS) infusions. Thus, the resulting experimental groups were as follows: (1) Rats receiving a s.c. injection of 1 ml of corn oil/kg of animal weight and being administered i.c.v. 3 ml of PBS daily: ‘‘oil/PBS group’’. (2) Rats receiving a s.c. injection of 1 ml of corn oil/kg of animal weight and being administered i.c.v. 3 mg/3 ml of Ab(1–42) daily: ‘‘oil/amyloid group’’. (3) Rats receiving an injection of 250 mg of CPF/kg of animal weight and being administered i.c.v. 3 ml of PBS daily: ‘‘CPF/PBS group’’. (4) Rats receiving an injection of 250 mg of CPF/kg of animal weight and being administered i.c.v. 3 mg/3 ml of Ab(1–42) daily: ‘‘CPF/amyloid group’’.

2.5. Water maze task The sixth day of i.c.v. Ab(1–42) or PBS administration, the behavioural phase in the water maze began. Escape latencies, swimming speed and path length were recorded as measures for performance. The apparatus was a circular black plastic pool (height: 50 cm, diameter: 150 cm). The tank was filled with clear water kept at 22 8C. Twelve black platform holders were attached to the floor of the pool arranged in concentric circles with 12 possible positions at 30 cm from the maze wall. The pool was divided into four imaginary quadrants, with four starting positions marked on the outer wall of the maze (N: north; E: east; S: south; W: west). Swimming activity of each rat was monitored by a small video camera 150 cm above the centre of the pool and connected to a computer running video-tracking software (Ethovision 3.1, Noldus, The Netherlands). A black escape platform (height: 38.5 cm, diameter: 10 cm) was submerged 2 cm below the water surface. Another platform with a white top, protruding 2 cm above the water surface, was used during the visual task. For a total of 10 sessions (one per day) the animals were trained and tested using the following four procedures. 2.5.1. Classical task (days 1–5) The invisible platform remained unchanged (east quadrant) during the five sessions, but the starting points were changed

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for each trial and day (conditions were equal for each animal). Animals were allowed to search for the platform for a maximum period of 90 s. If the animal found the platform within this period of time, it was left on the platform for 15 s. Otherwise the subject was hand-guided to the platform and allowed to stay there for the same period of time. Subsequently, the rat was placed in a bucket for 30 s, until the next trial began. At the end of each session the animals were dried with a towel and placed in a clean cage. 2.5.2. Transfer test (day 6) On the 6th day, the platform was removed from the pool and each animal was released into the pool for a period of 60 s. The starting point was the opposite quadrant to the platform during the previous spatial training phase. Swimming activity and time spent in every quadrant and in an imaginary circle (40 cm in diameter) around the position previously occupied by the platform were recorded. 2.5.3. Moving hidden platform (days 7–8) An invisible platform was used again but its position was changed for each session and placed in the centre of the new quadrant (north, south or west). All the remaining procedures were kept identical to the initial five sessions. 2.5.4. Visually cued task (days 9–10) Instead of a black submerged platform, a platform with a white top, protruding 2 cm above the water surface was used. Each session consisted of four trials, but the platform position and release sites were changed for each trial. Otherwise, procedures were as described for the classical task. 2.6. Histology and immunocytochemistry One day after the last training session in the water maze rats were deeply anesthetized with equithesine, and perfused transcardially (PBS pH 7.4 followed by 4% phosphate buffered paraformaldehyde). After 48 h of post-fixation in paraformaldehyde, all the brains were crioprotected with sucrose 15% (24 h), and then sucrose 30% (next 24 h). Brains were subsequently frozen with 2-methylbutane and stored at 20 8C until their histological processing. Coronal sections (50-mm), were taken throughout the prefrontal cortex (PFC) and hippocampus (HP), and then stored in cryoprotectant solution at 4 8C before use. 2.6.1. Cresyl violet staining In order to check the absence of cell death in brains, as well as the correct position of the cannula in the lateral ventricle, a series of sections were Nissl-stained (0.1% cresyl violet solution) and examined under light microscope (Olympus BX50). 2.6.2. MAP2 and MAP1A immunostaining Sections were processed using a free-floating immunocytochemistry method for MAP2 and MAP1A. Sections were first rinsed several times in Tris-buffered saline solution (TBS) 0.01 M + 0.2% Triton X-100 at room temperature and then placed in 0.3% hydrogen peroxide in TBS for 5 min to inactivate endogenous peroxidase activity. Sections underwent several TBS washes and were then incubated in a blocking solution at room temperature for 1 h to prevent non-specific protein binding. The blocking solution included 0.2% Triton X-100, 0.1% bovine serum albumin and 2% goat serum in TBS. Subsequently, sections were incubated in primary antibody in the blocking solution at 4 8C overnight. The primary antibody was either anti-MAP2 (clone

HM-2, 1:500, Sigma, St. Louis, MO, USA), which binds all forms of MAP2, namely MAP2a, MAP2b and MAP2c, or anti-MAP1A (ab11264, 1:500, Abcam, Cambridge, UK). Following the 24-h primary antibody incubation, sections were rinsed several times in TBS + 0.2% Triton X-100 and then placed in secondary antibody (1:500 biotinylated horse anti-mouse IgG; Sigma) in the blocking solution for 1 h at room temperature. After this incubation, sections were rinsed several times in TBS and incubated at room temperature for 1 h with streptavidin (Dako, Denmark). MAP2 or MAP1A immunoreactivity was then visualized using 3-30 diaminobenzidine (Liquid DAB Substrate Chromogen System, Dako North America Inc., Carpinteria, USA), a standard intensification process. Specificity of antibody binding was verified with tissue sections incubated without anti-MAP2 or anti-MAP1A; these sections were present in each immunocytochemical run and did not contain evidence of distinct process staining. There was one staining batch for each zone (PFC and HP) with each primary antibody (MAP2 and MAP1A), i.e., four batches including tissue from all animals from all groups (3 slices/animal, 6 animals/group) to decrease the contribution of batch effects to the variability in immunocytochemistry staining. 2.6.3. Amyloid b-protein deposits detection Sections from four Ab(1–42) administered animals underwent a very similar process to that described for the MAP2 and MAP1A immunostaining but using 1% hydrogen peroxide in TBS to inactivate endogenous peroxidase activity, and this time the blocking solution included 0.3% Triton X-100, 1% bovine serum albumin and 5% goat serum in TBS. In this case, several dilutions of the primary antibody (Beta Amyloid, 1–16 (6E10) Monoclonal Antibody (SIGNET)) were used in order to determine which one would best show the plaque depositions. These dilutions were 1:1000, 1:2000, 1:4000, 1:8000 or 1:16000. Furthermore, brain sections from 11-month-old transgenic mice (TgAPPSWE/PS1DE9) were used as positive controls, as previous studies have shown that these mice have detectable Ab-accumulation in neocortex and hippocampus as early as 4 months of age (Borchelt et al., 1997; Jankowsky et al., 2004). A deviation from the protocol described above was the incubation for 60 min in avidin:biotin:peroxidase complex diluted in TBS + 0.2% Triton X-100, following the incubation in secondary antibody (biotinylated anti-mouse IgG made in horse; Sigma) (1:1000), and the 10-min rinse in Tris–HCl pH 7.6 before being developed with 3-30 diaminobenzidine. Ab deposition was examined under a light microscope (Olympus BX50). 2.6.4. MAP2 and MAP1A quantification The density of MAP2 or MAP1A immunoreactive dendritic processes in PFC and HP was measured by a stereological approach using a random rotation grid intersection method. Sections were coded so the experimenter was blind to the experimental groups. Systematic random sampling (by a VIS new Cast program) was used to obtain a 10% sample of the area of interest in each of the three sections per brain region. The selected area (whole PFC or CA1–CA3 regions of the HP) was delimitated using a 1.25 objective of a Zeiss Axioskop 2 plus microscope, and quantification was performed with the 40 objective. A fixed plane of focus was chosen per sample (5 mm below the slice surface), and intersections between the randomly rotated grid lines and MAP2 or MAP1A processes were counted. MAP2 and MAP1A immunoreactive process densities (Sv) were calculated using the formula: Sv = I/A, where I = total number of intersections summed across all samples per brain, and A = total delimitated volume area, estimated using the

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Cavalieri principle by simultaneously overlaying the samples with a point grid. 3. Results 3.1. Acute toxicity evaluation Effects of exposure on body weight were compared, showing a significant group effect. In the control rats, body weight increased gradually in the course of the experiment, while animals receiving 250 mg/kg of CPF showed a temporary decrease in body weight that continued for 7 days post exposure. Subsequently, the rats gained weight again but their body weight was significantly lower compared to that of the controls 1 month after intoxication (F5,21 = 26.333; p < 0.001). A decrease of body temperature was also observed 24 h and 48 h after intoxication in the group receiving CPF (F4,18 = 3.398; p < 0.05). Previous experiments from our group have shown a similar body weight reduction with 250 mg/kg of CPF, together with an acute ˜ adas et al., 2005; Cardona et al., AChE inhibition above 85% (Can 2006). An unexpected result was the observation of further signs of toxicity (tremor, diarrhea and muscle twitching) in four of the CPF administered animals, two of which finally died. These ˜ adas et al., symptoms are not common for the dose used (Can 2005; Cardona et al., 2006; Lo´pez-Crespo et al., 2007; Sa´nchezSanted et al., 2004) and could be related to the previous surgical intervention conducted to implant the guide cannulae. In fact it has been determined that the maximum tolerated dose for subcutaneous CPF for rats is 279 mg/kg (established in Sprague– Dawley strain) (Pope et al., 1991). The cresyl violet staining revealed that all cannulae were located in the correct ventricle. It also showed, however, cerebral deformations in four subjects, which were discarded for further behavioural and histological analysis. These deformations were like extra-dilations of the right ventricle, possibly due to an excessive i.c.v. pressure. In addition, this staining showed no evidence of cell death in any of the brains. 3.2. Behavioural evaluation 3.2.1. Classical task To evaluate acquisition in the water maze, a two-way ANOVA for repeated measures was performed with ‘‘PESTICIDE’’ with two levels (OIL or CPF) and ‘‘INFUSION’’ with two levels (PBS or Ab1–42) as the between subject factors, and ‘‘SESSION’’ (with five levels) and ‘‘TRIAL’’ (with four levels) as the within subject

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factors. For each of the four groups of animals the time spent to find the submerged platform during the classical task is depicted in Fig. 1A. The figure represents the mean scores per each 4trials session. As shown, the decrease in escape latencies during the five sessions is noticeable (SESSION: F4,14 = 38.015, p < 0.001). There is also a significant intra-session improvement (TRIAL: F3,15 = 21.200, p < 0.001). An unexpected finding was the absence of significant differences between groups. The path length analysis did not show any difference between groups either. 3.2.2. Transfer test The repeated ANOVA measures with ‘‘QUADRANT’’ as within subject factor (with four levels: the four imaginary quadrants into which the pool was divided) and ‘‘PESTICIDE’’ and ‘‘INFUSION’’ as the between subject factors showed a strong preference for animals from all groups for the quadrant in which the platform was originally located (F3,19 = 12.738, p < 0.001) (Fig. 1B). Animals also traveled a similar distance in the platform quadrant. No group differences were found for the time spent or for the traveled distance, in the imaginary circle (40 cm in diameter) surrounding the position occupied by the platform or for any of the other 3 imaginary circular zones into which the pool was divided. 3.2.3. Moving hidden platform test A two-way ANOVA for repeated measures was performed with ‘‘PESTICIDE’’ and ‘‘INFUSION’’ as between subject factors and ‘‘SESSION’’ (with two levels) and ‘‘TRIAL’’ (with four levels) as within subject factors. For all four groups, the mean latencies during the first trial and session of this phase, were lower than those obtained during the first sessions of the initial spatial training. A main effect for SESSION (F1,17 = 8.050, p < 0.011), and TRIAL (F3,15 = 18.588, p < 0.001) was obtained for escape latencies. Nevertheless, the interaction between the SESSION and INFUSION factors did become significant (F1,17 = 6.254, p < 0.050). Analyzing separately each SESSION revealed no significance for the first one, but in the second session there was a significant effect for the INFUSION factor (F1,17 = 11.501, p < 0.010). Thus, whereas the latencies of animals receiving i.c.v. infusions of PBS (both CPF and oil groups) improved in the second training day, the performance of the oil/amyloid group was very similar to the previous one, with the CPF/amyloid group spending even more time in the second session to find the submerged platform (Fig. 2). Path length data followed the same tendency, although the interaction between SESSION and INFUSION was only marginally significant (F1,17 = 6.254, p = 0.057). Separate analyses for each SESSION again

Fig. 1. Classical task and transfer test. Escape latencies to find the hidden platform during the classical task are comparable for the four groups (A). All groups showed a significant preference during the transfer test for swimming in the quadrant in which the platform was previously placed (B).

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for the TRIAL, PESTICIDE and INFUSION factors (F3,15 = 5.012, p < 0.010). Separate ANOVAs of every session showed significant differences for the first visually cued session, but not for the second one. These differences were observed for the TRIAL (F3,15 = 10.842, p < 0.001) and PESTICIDE (F1,17 = 14.417, p < 0.001) factors, and marginally significant for the interaction between PESTICIDE and INFUSION (F1,17 = 4.096, p = 0.059), and the interaction between TRIAL, PESTICIDE and INFUSION (F3,15 = 3.129, p = 0.057). Analysis of path length data also showed a significant effect of PESTICIDE (F1,17 = 8.905, p < 0.010) and the interaction between SESSION and PESTICIDE (F1,17 = 9.074, p < 0.010), although CPF groups exhibited a worse performance during both sessions. There was also a significant interaction for the TRIAL, PESTICIDE and INFUSION (F3,15 = 4.250, p < 0.050) factors and a marginally significant effect for INFUSION (F1,17 = 3.706, p = 0.076), which resulted significant during the second session (F1,17 = 5.262, p < 0.050). Again the CPF/amyloid group obtained the highest scores in the task (Fig. 3).

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Session Fig. 2. Moving hidden platform test. Escape latencies showed that performance of Ab(1–42) infused animals was impaired during the second session. *Statistically significant differences p < 0.050.

showed significantly higher scores for animals receiving Ab(1–42), only for the second session (F1,17 = 13.529, p < 0.010).

3.2.5. Swim speed ANOVAs were conducted with every task for the mean speed of animals. Differences between groups were found in the classical task for the interaction between SESSION and PESTICIDE (F4,14 = 3.392, p < 0.05). The interaction between PESTICIDE and INFUSION (F1,17 = 8.333, p < 0.01) resulted in lower speed of the CPF/amyloid group, only during the transfer test.

3.2.4. Visually cued task Groups receiving the pesticide, and specially CPF/amyloid group, took longer to locate and reach the visible platform. Results obtained during the two sessions of the visual task are shown in Fig. 3. ANOVA of escape latencies showed a significant effect for SESSION (F3,15 = 6.887, p = 0.010), TRIAL (F3,15 = 16.390, p < 0.001) and their interaction (F1,17 = 8.278, p < 0.010), as animals performed the task better in subsequent executions. The analysis also revealed group differences: there was a significant effect of PESTICIDE (F1,17 = 7.726, p < 0.010) and a marginal significance for the interaction between PESTICIDE and INFUSION (F1,17 = 3.559, p = 0.076). There was also a significant interaction for the TRIAL and PESTICIDE factors (F3,15 = 3.463, p < 0.050), and

3.3. Immunocytochemistry 3.3.1. MAP2 and MAP1A immunostaining in prefrontal cortex ANOVAs with PESTICIDE and INFUSION as the between subjects factors were performed for every kind of microtubule-associated protein revealing a significant effect for the INFUSION factor

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Fig. 3. Visually cued task. Escape latencies are grouped by sessions (A) or trials during the first session (B). Both groups of chlorpyrifos-exposed animals showed higher latencies to reach the visible platform during the first session of the visually cued task compared to their controls. Moreover, Ab(1–42) infusions potentiated the impairment produced by CPF exposure, as the performance of these animals was even worse than that of their controls. Path length data grouped by sessions (C) or trials (D) show the same tendency; but the CPF/amyloid group also obtained higher scores during the second session. *Statistically significant differences p < 0.050.

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Fig. 4. MAP1A and MAP2 densities in prefrontal cortex. Ab(1–42) infusions but not CPF exposure produced a significant decrease of MAP1A immunoreactivity in prefrontal cortex (A and B). However, CPF but not amyloid produced a significant decrease of MAP2 immunoreactivity in this area (C and D). *Statistically significant differences p < 0.050.

(F1,16 = 24.38, p < 0.001) on MAP1A immunostaining processes, which were lower in both groups receiving the Ab(1–42) infusions, regardless of the CPF treatment (Fig. 4A). On the other hand, the PESTICIDE factor produced a significant reduction in the number of MAP2 immunopositive processes (F1,16 = 10.94, p < 0.010) (Fig. 4B) but had no effects on MAP1A. There were no interactions between PESTICIDE and INFUSION, each acting on a specific MAP type.

3.3.2. MAP2 and MAP1A immunostaining in CA1 and CA3 regions of the hippocampus The INFUSION factor produced the same pattern as seen in PFC: a significant decrease in MAP1A-positive processes was observed in CA1–CA3 hippocampal regions as a result of the Ab(1–42) infusions (F1,16 = 22.17, p < 0.001) (Fig. 5A). However, the number of MAP2 positive processes remained unchanged for all treatments (Fig. 5B).

Fig. 5. MAP1A and MAP2 densities in CA1 and CA3 regions of the hippocampus. Ab(1–42) infusions but not CPF exposure produced a significant decrease of MAP1A immunoreactivity in CA1–CA3 areas (A and B). However, none of the experimental treatment produced changes for MAP2 immunoreactivity in these areas (C and D). *Statistically significant differences p < 0.050.

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4. Discussion Amyloid plaques are one of the two neuropathological hallmarks of AD, together with the appearance of intraneuronal aggregates. Very few animal species, however, spontaneously develop all the cognitive, behavioural and neuropathological symptoms of Alzheimer’s disease; rare plaques have been described in monkeys, but none of the hystopathological alterations typical of AD occur in aging rodents (Pepeu et al., 1986). Only transgenic mice over-expressing human AD-related genes clearly show Ab deposits, and literature is contradictory for Ab deposits in brains of rats treated with i.c.v. injections of amyloid (Nakagawa et al., 2004, 2005; Perez et al., 2010; Stephan et al., 2001). Thus, it is not surprising that no Ab deposits were found in our subject brains. We also want to stress that cresyl violet staining showed no signs of cell death in any of the infused brains. However, it is important to point out that it has been recently proposed that chronic CPF is able to increase Ab levels in transgenic Tg2576 mice (Salazar et al., 2011). This leads us to consider that CPF may also potentiate other effects caused by the amyloid peptide, such as behavioural deficits. In spite of not finding amyloid deposits or cell death signs, behavioural data show effects of both CPF and amyloid treatment, together with an interesting interaction between them. Literature offers contradictory data on Morris water maze performance after i.c.v. Ab infusions. In contrast to some reports (Nakamura et al., 2001; Peng et al., 2009), we have been unable to find any deficit during the reference memory phase of the experiment (classical task acquisition and transfer test), apart from swimming speed. On the other hand, although our animals were able to learn and remember the platform position, they showed deficits during the moving hidden platform and visual tasks, but with normalized swim speed. Path length data reinforced the effects observed on escape latencies, proving that these results are not related to motor problems. For all groups the mean latencies during the first trial and session of the moving hidden platform test were lower than those observed at the beginning of the classical task. This would indicate that animals were already adapted to the swimming pool environment and basic task requirements (finding a hidden platform). As the scores of animals receiving saline solution declined in the subsequent session, however, performance of Ab(1–42) treated groups during the second session was very similar to that in the first one. The visually cued task procedure is usually followed as a control task aimed to assess sensory or motor impairments in the water maze; however, spatial learning was normal for all rats, deficits began to appear during the moving hidden platform phase and were exacerbated when animals were forced to change from spatial to visual searching strategies (de Bruin et al., 1994). Transient but significant differences between CPF and vehicle treated animals were detected during the first session. Additionally, animals receiving both Ab(1–42) and CPF treatments displayed the worst performance. Our results can be interpreted as a difficulty in developing a new navigation plan, and impaired cognitive flexibility (de Bruin et al., 1994). Alternatively, a memory problem, not detected during the first phase, could account for this result. On the other hand, increased variability of path length after failing to locate the platform could also explain the longer latencies. Thus, an interaction between CPF exposure and Ab(1–42) infusion was observed at behavioural level, adding concerns about the detrimental effects of OP exposure in the course of AD, and also in line with recent epidemiological data (Hayden et al., 2010; ˜ ez et al., 2007). Santiba´n

Our data show a significant decrease in dendritic MAP1A immunoreactivity for Ab(1–42) injected groups both in prefrontal cortex and hippocampus, and a significant reduction in MAP2 immunoreactivity in prefrontal cortex in groups receiving CPF. MAP2 is mostly present in dendrites, being considered a dendritic marker (Dehmelt and Halpain, 2005; Penzes et al., 2009), and MAP1A dynamics are related to spine plasticity (Jaworski et al., 2009). This would indicate that the changes observed in MAP1A reflect changes in synaptic density without being accompanied by dendritic retraction. However, our data do show a decrease in MAP2 density in PFC after CPF exposure, in line with previous results using organotypic slice cultures of immature rat hippocampus (Prendergast et al., 2007), which described that CPF exposure produces a selective reduction in the availability and functional effects of ‘‘in vitro’’ MAP2 proteins. This reduction in MAP2 density levels in PFC could be also related to the observed effects on cognitive flexibility that occurred in our study when an extra-dimensional shift in stimulus conditions was introduced (from spatial to visually cued task), and was worsened by the interaction with the effect of Ab(1–42) administration. In fact, Zaja-Milatovic et al. (2009) have also shown a drastic reduction in dentritic lengths and spine density after acute exposure to DFP (diisopropylfluorophosphate). Thus, the behavioural interaction between both treatments found in the present report could be explained by an interaction between their effects on both dendritic and spine density. Moreover, synaptic adaptations after CPF exposure, such as postsynaptic receptor density reduction, induce changes in synaptic activity, and Ab(1–42) has recently been reported to induce a decrease in rapid axonal transport (Nyakas et al., 2010). This indicates that both CPF and Ab(1–42) can transiently induce a decrease in the activity affecting dendritic and synaptic patterns. On the one hand, polymerization, stabilization, and dynamic properties of microtubules are influenced by interactions with microtubule-associated proteins (Fink et al., 1996), and different animal models of AD have shown impairments of microtubuledependent transport and axonopathies suggesting that these phenomena may underlie cognitive decline observed in patients (Avila et al., 2002; Kins and Beyreuther, 2006; Praprotnik et al., 1996). It has been demonstrated that the cytoskeleton is an early cellular target for intracellular Ab aggregates, and that the disruption of the microtubule network is required for Abinduced neuronal cell death (Butler et al., 2007; Mudher and Lovestone, 2002; Sponne et al., 2003). Fifre et al. (2006) report that soluble Ab oligomers induce a time-dependent degradation of MAP1A, MAP1B and MAP2 involving a perturbation of Ca2+ homeostasis with subsequent calpain activation. Increase in calpain activity has been demonstrated in the early stages of AD development, even before synaptic loss, neuronal degeneration, and tau hyperphosphorylation and aggregation (Saito et al., 1993). On the other hand, the disruption of axonal transport has also been proposed as a possible explanation for the learning and memory deficits caused by OPs. Microtubules are an essential component of axonal transport and in vitro and in vivo studies have shown that OP agents react with tubulin and dismantle the structure of microtubules (Grigoryan and Lockridge, 2009; Grigoryan et al., 2009; Jiang et al., 2010). The deterioration in microtubule function is also reflected in the inhibition of both fast antero- and retrograde axonal transport in animals suffering from growth retardation, behavioural disturbances and muscle weakness following CPF administration (Terry et al., 2003). These data are of particular interest as they suggest that microtubules and their associated proteins such as MAP proteins may be novel substrates for organophosphates in the CNS.

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5. Conclusions This study identifies detriments in the performance of various water maze tasks that require flexibility competences, because of the effect of Ab(1–42) infusions (moving hidden platform and visual task) and the effect of CPF (visual task), which was potentiated by Ab(1–42) administration. These behavioural disturbances occurred in absence of amyloid deposits but with chorpyrifos affecting MAP2 levels, and amyloid affecting MAP1A density. Therefore, although other histopathological or neurochemical candidates could also be considered, we have found a very suggestive hypothesis on interaction between OP exposure and AD susceptibility that warrants further research. Finally, different behavioural procedures specifically intended to assess behavioural flexibility, attentional, and working memory processes should be used to evaluate the effect of Ab infusions, CPF exposure and their interaction. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This study has been funded by the Spanish Ministry of Education and Science (project ref. SEJ2006-15628-C02-01 and SEJ2006-15628-C02-02) and FEDER funds from EU. We want to acknowledge the excellent technical support provided by Mr. Luis Ruedas and Mr. Hans Jørgen Jensen. Dr. Nobel Perdu reviewed and edited the English grammar and orthography of the manuscript. References Abdollahi M, Rainba A, Shadnia S, Nikfar S, Rezaie A. Pesticide and oxidative stress: a review. Med Sci Monitor 2004;10:141–7. Alonso AD, Grundke-Iqbal I, Barra HS, Iqbal K. Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau. Proc Natl Acad Sci USA 1997;94:298–303. Avila J, Lim F, Moreno F, Belmonte C, Cuello AC. Tau function and dysfunction in neurons: its role in neurodegenerative disorders. Mol Neurobiol 2002;25:213–31. Baldi I, Lebailly P, Mohammed-Brahim B, Letenneur L, Dartigues JF, Brochard P. Neurodegenerative diseases and exposure to pesticides in the elderly. Am J Epidemiol 2003;157:409–14. Borchelt DR, Ratovitski T, van Lare J, Lee MK, Gonzales V, Jenkins NA, et al. Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron 1997;19:939–45. Butler D, Bendiske J, Michaelis ML, Karanian DA, Bahr BA. Microtubule-stabilizing agent prevents protein accumulation-induced loss of synaptic markers. Eur J Pharmacol 2007;562:20–7. Can˜adas F, Cardona D, Da´vila E, Sa´nchez-Santed F. Long-term neurotoxicity of chlorpyrifos: spatial learning impairment on repeated acquisition in a water maze. Toxicol Sci 2005;85:944–51. Cardona D, Lo´pez-Grancha M, Lo´pez-Crespo G, Nieto-Escamez F, Sa´nchez-Santed F, Flores P. Vulnerability of long-term neurotoxicity of chlorpyrifos: effect on schedule-induced polydipsia and a delay discounting task. Psychopharmacology (Berl) 2006;189:47–57. de Bruin JP, Sa´nchez-Santed F, Heinsbroek RP, Donker A, Postmes P. A behavioural analysis of rats with damage to the medial prefrontal cortex using the Morris water maze: evidence for behavioural flexibility, but not for impaired spatial navigation. Brain Res 1994;652:323–33. Dehmelt L, Halpain S. The MAP2/Tau family of microtubule-associated proteins. Genome Biol 2005;6:204. Fifre A, Sponne I, Koziel V, Kriem B, Yen Potin FT, Bihain BE, et al. Microtubuleassociated protein MAP1A, MAP1B, and MAP2 proteolysis during soluble amyloid beta-peptide-induced neuronal apoptosis. Synergistic involvement of calpain and caspase-3. J Biol Chem 2006;281:229–40. Fink JK, Jones SM, Esposito C, Wilkowski J. Human microtubule-associated protein 1a (MAP1A) gene: genomic organization, cDNA sequence, and developmental- and tissue-specific expression. Genomics 1996;35:577–85. Gearhart DA, Sickles DW, Buccafusco JJ, Prendergast MA, Terry AV Jr. Chlorpyrifos, chlorpyrifos-oxon, and diisopropylfluorophosphate inhibit kinesin-dependent microtubule motility. Toxicol Appl Pharmacol 2007;218:20–9. Gevorkian G, Gonzalez-Noriega A, Acero G, Ordon˜ez J, Michalak C, Munguia ME, et al. Amyloid-beta peptide binds to microtubule-associated protein 1B (MAP1B). Neurochem Int 2008;52:1030–6.

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