Lead (Pb+2) impairs long-term memory and blocks learning-induced increases in hippocampal protein kinase C activity

Toxicology and Applied Pharmacology 200 (2004) 27 – 39 www.elsevier.com/locate/ytaap

Lead (Pb+2) impairs long-term memory and blocks learning-induced increases in hippocampal protein kinase C activity Adrinel Va´zquez and Sandra Pen˜a de Ortiz * Department of Biology, University of Puerto Rico, San Juan 00931-3360, Puerto Rico Received 22 July 2003; accepted 18 March 2004 Available online 30 April 2004

Abstract The long-term storage of information in the brain known as long-term memory (LTM) depends on a variety of intracellular signaling cascades utilizing calcium (Ca2+) and cyclic adenosine monophosphate as second messengers. In particular, Ca+2/phospholipid-dependent protein kinase C (PKC) activity has been proposed to be necessary for the transition from short-term memory to LTM. Because the neurobehavioral toxicity of lead (Pb+2) has been associated to its interference with normal Ca+2 signaling in neurons, we studied its effects on spatial learning and memory using a hippocampal-dependent discrimination task. Adult rats received microinfusions of either Na+ or Pb+2 acetate in the CA1 hippocampal subregion before each one of four training sessions. A retention test was given 7 days later to examine LTM. Results suggest that intrahippocampal Pb+2 did not affect learning of the task, but significantly impaired retention. The effects of Pb+2 selectively impaired reference memory measured in the retention test, but had no effect on the general performance because it did not affect the latency to complete the task during the test. Finally, we examined the effects of Pb+2 on the induction of hippocampal Ca+2/phospholipiddependent PKC activity during acquisition training. The results showed that Pb+2 interfered with the learning-induced activation of Ca+2/ phospholipid-dependent PKC on day 3 of acquisition. Overall, our results indicate that Pb+2 causes cognitive impairments in adult rats and that such effects might be subserved by interference with Ca+2-related signaling mechanisms required for normal LTM. D 2004 Elsevier Inc. All rights reserved. Keywords: Lead neurotoxicity; Long-term memory; Protein kinase C; Holeboard task; Spatial learning; Heavy metals; Hippocampus

Introduction The central nervous system is one of the most important targets of Pb+2-mediated toxicity. The neurobehavioral effects of chronic and acute exposure to Pb+2 during development are well known (Bellinger et al., 1984; Booze et al., 1983; Brown, 1975; Carpenter et al., 2002; Ferguson and Bowman, 1990; Finkelstein et al., 1998; Jett et al., 1997; Minder et al., 1994; Moreira et al., 2001; Needleman et al., 1996; Nevin, 2000; Petit and Alfano, 1979; Rosen, 1995; Rummo et al., 1979). Developmental low-level chronic exposure to Pb+2 has been related to behavioral impairments such as hyperactivity (Ferguson and Bowman, 1990; Severo Rodrigues et al., 1993), attention deficits (Bellinger et al., 1984; Minder et al., 1994), and aggressive * Corresponding author. Department of Biology, University of Puerto Rico, PO Box 23360, JGD Building 108, Granada Avenue, San Juan 00931-3360, Puerto Rico. Fax: +1-787-764-3875. E-mail address: [email protected] (S. Pen˜a de Ortiz). 0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.03.011

or antisocial behavior (Dietrich et al., 2001; Needleman et al., 1996; Nevin, 2000). In addition, data obtained from epidemiological studies implicate a causal link between low-level chronic exposure to Pb2+ and deficiencies in intelligence quotients in children (Needleman et al., 1990; Schwartz, 1994; Wasserman et al., 2003). Accordingly, studies using rodents have shown that Pb2+ exposure in young animals interferes with spatial learning (Jett et al., 1997; Winneke et al., 1988). The effects of acute high-level exposure to Pb+2 in adult humans have also been recognized by the Agency for Toxic Substances and Disease Registry (ATSDR) and others (ATSDR, 1999; Staudinger and Roth, 1998). Cross-sectional epidemiological studies (Hogstedt et al., 1983; Williamson and Teo, 1986) suggest that exposure to Pb2+ in adults may result in poorer performance in specific cognitive functions at Pb+2 blood levels as low as 1.19 Amol/l (24.7 Ag/dl) and 1.31 Amol/l (27.1 Ag/dl). Only a few studies (Jett et al., 1996; Severo Rodrigues et al., 1993) have examined the effects of Pb2+ exposure during

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adulthood in animal models, and thus, little is known about the behavioral correlates between human and nonhuman exposure to Pb2+ on the matured central nervous system. A number of reports suggest that at least part of the neurobehavioral toxicity of Pb+2 is due to its interference with normal Ca+2 signaling in neurons (Bressler and Goldstein, 1991; Ferguson et al., 2000; Goldstein, 1993; Westerink et al., 2002, Zhang et al., 2002). In particular, it has been suggested that Pb+2 can mimic or block (depending on its concentration) the activation of Ca+2/phospholipid-dependent protein kinase C (PKC) (Hwang et al., 2001; Markovac and Goldstein, 1988a, 1988b; Murakami et al., 1993; Sun et al., 1999), which has also been closely associated with learning and memory processes (Abeliovich et al., 1993a, 1993b; Douma et al., 1998; Sweatt, 1999; Va´zquez et al., 2000; Young et al., 2002). The studies presented here examined the effects of repeated intrahippocampal administration of Pb+2 on the acquisition and long-term retention of the holeboard spatial discrimination task in adult rats. This task is dependent on the hippocampus and on the establishment of associations between extra maze cues and food-baited holes on the floor of the maze (Oades, 1981, 1982; Oades and Isaacson, 1978; Pen˜a de Ortiz et al., 2000). Most studies have chosen systemic methods for exposing rats to Pb2+ through food or water (Jett et al., 1997; Moreira et al., 2001; Nihei et al., 2000; Petit and Alfano, 1979; Zaiser and Miletic, 1997). This exposure modality allows for more accurate modeling of environmental exposure to Pb+2. In particular, this mode of exposure is especially relevant for the study of the effects of Pb+2 in the developing central nervous system. In contrast, the present study was designed to understand the effects of subchronic Pb+2 on the mature brain, with special attention to the process of memory consolidation in the hippocampus. Learning and memory of spatial or contextual information depends on the hippocampus, a structure that is important in the initial processing and long-term storage of information in the brain (Milner, 1970; Squire and Zola, 1996; Wong, 1997). We thus utilized direct intrahippocampal injections of Pb+2 to study the specific behavioral and cellular effects of Pb+2 on memory consolidation within this particular brain structure. Other studies have used this mode of exposure to Pb+2 and other toxico-pharmacological agents to address their specific effects on the hippocampus (Jett et al., 1996; Ohno et al., 1992, 1994; Walker and Gold, 1994). We also examined the consequences of such type of exposure to Pb+2 on Ca+2/phospholipid-dependent PKC activity, previously shown by us to be activated within the hippocampus during acquisition of the holeboard task (Va´zquez et al., 2000). Our results show that Pb+2 impairs the storage, rather than the acquisition, of information in the brain of adult rats by a mechanism that possibly involves blocking the learning-induced activation of Ca+2-dependent enzymes such as PKC.

Materials and methods Subjects. Male hooded Long Evans rats (Harlan Sprague – Dawley, Indianapolis, IN) weighing 270– 310 g were used in our studies. On arrival, rats were taken to the behavioral testing room and placed in home cages in pairs. Food and water were available at all times except when the rats entered the food restriction protocol, in which they were maintained at 85% of free feeding weight. The rats were kept on a 12-h light – dark cycle. Surgery. After arrival, the animals were kept in our animal house facility and were handled for several days before undergoing surgery. Rats were anesthetized with 2.5% sodium pentobarbital at an intraperitoneal dose of 50– 80 mg/kg. The head was positioned in a stereotaxic frame and a midline sagittal incision was made in the scalp. Two holes were drilled in the skull, and stainless steel guide cannulas (26 gauge) were lowered to a position just above the CA1 region (Fig. 1A). The stereotaxic coordinates with respect to bregma were: anterior – posterior, 3.8; medial – lateral, F1.5 from bregma, and dorso-ventral, 2.5 mm from the skull surface. Cannulas were lowered 1.0 mm below the surface of the skull and were then fixed on the skull with small screws and dental cement. Injectors were located 1.5 mm below the cannulas. A stainless steel wire stylet (33 gauge) was inserted into the guide cannula to prevent obstruction of the cannula. After surgery, animals were allowed to recover for 3 –5 days before behavioral experiments begun. Food restriction and habituation. Following a recovery period of 3 – 5 days, rats entered a food restriction and habituation period to prepare them for training in the holeboard spatial discrimination task, as described before (Pen˜a de Ortiz et al., 2000). This task is a hippocampal-dependent spatial task in which animals learn to discriminate between relevant (baited) and irrelevant (nonbaited) holes within a 16-hole arena using extra maze clues (a video camera, a photo, a poster, and the experimenter) (Oades and Isaacson, 1978; Pen˜a de Ortiz et al., 2000). Acquisition of the task is completed after 4 days of training. To enhance motivation for food searching, animals are food restricted until they reach 85% of their original body weight. During the food restriction period of our experiments, animals are exposed to 45-mg sugar pellets in addition to their regular lab chow. This is done to habituate the animals to the sugar pellets before training because these pellets are used as bait in the maze. In the habituation phase, animals are exposed to the maze in pairs with sugar pellets spread throughout the floor of the apparatus and in each of the 16 holes. Rats were allowed to consume the food pellets and freely explore the apparatus for 15 min. A second habituation session is given the following day in which animals are placed individually inside the maze, which this time contains food pellets in all 16 holes. Rats are also allowed to consume the food pellets

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subjected to surgery, food deprivation, and habituation to hippocampal microinfusions, as described above. The animals were then subjected to Pb2+ or Na+ acetate infusions into their ipsilateral and contralateral hippocampi, which were given once a day, on four alternate days. These rats were not subjected to acquisition training and were terminated 1 h after their last injection. For the dose-response study, additional doses of Pb2+, including 0.3, 0.7, and 3.0 nmol with an N = 11 each, in addition to a saline control (N = 7), were used. All treatment infusions consisted of saline plus Pb2+ or Na+ acetate.

Fig. 1. Effect of different doses of Pb+2 on acquisition and retention of the holeboard task. x – y plot showing the effect of different doses of Pb+2 acetate on the performance of the rats during acquisition and retention measured by the RMR. Error bars correspond to the SEM. The continuous line connects the experimental data points, while the dashed lines connect data obtained from acquisition training to the data obtained 7 days later during the retention test. While no significant differences between the doses were detected by repeated measures two-way ANOVA, a significant interaction between the sessions and the dose factor suggested that the perceptible effects of the doses between 0.7 and 3.0 nmol during the retention test might be indicative of a specific effect by Pb+2 on LTM. The saline group consisted of 7 animals, while the N for the Pb+2 doses of 0.7 and 3.0 nmol was of 11. The N for the 1.0-nmol dose was 12.

and to explore the maze for 15 min during this second habituation session. Drug infusions. To assess the effectiveness of the infusing pump system and to get the animals used to receiving intracerebral infusions, we subjected each animal to bilateral infusions (2 min at 0.5 Al/min) of 0.9% saline on the habituation days before the beginning of spatial training. Infusions were done by inserting 33-gauge stainless steel internal injectors into the guide cannulas so that they extended 1.5 mm beyond the tip of the guide right above the CA1 layer of pyramidal neurons of the hippocampus. The next day, animals were randomly separated into two groups that received Pb+2 acetate (Sigma, St. Louis, MO; N = 12) or Na+ acetate (Sigma; N = 12) at concentrations of 1 nmol. One infusion was given 20 min before the first training trial on each training day, except for the retention test. Thus, each animal received only one infusion per training day, which was given at the beginning of each acquisition session. Acquisition training was done on alternate days to avoid tissue damage from repeated intracerebral microinfusions. For the pathology studies, rats (N = 5) were

Spatial training. Training sessions were exactly as described before (Pen˜a de Ortiz et al., 2000; Va´zquez et al., 2000) and were given once a day on alternate days until completing a total of four sessions. For acquisition training, each animal was assigned a pattern of four baited holes. Several different patterns were used, but each animal always received the same pattern in each session. During the acquisition phase (sessions 1 to 4), animals received one training session per day consisting of five trials. At the start of each trial, each animal was placed in the start box and the door was opened to initiate the trial. The behavioral parameters recorded were the following: (1) the total time to complete each trial (i.e., gather all four pellets) measured by time watches, (2) the number of reference errors (visits to empty holes), (3) the number of working errors (repeat visits to baited holes), (4) the reference memory ratio (RMR), a measure of the preference of the rats for baited holes, the number of total errors (sum of reference and working errors); and (5) the search strategy (pattern of search behavior). The RMR was calculated as in Va´zquez et al. (2000): (number of total visits and revisits to baited holes) H (total number of visits). For the search strategy analysis, we examined the number of the different searching patterns (defined as the sequence of visits to baited holes) used by each rat per session. We also determined which searching pattern was most used in each training session by each animal and calculated the frequency of the most repeated searching pattern per session. All trials were recorded with a video camera, which also served as one of the visual cues. A maximum of 5 min (300 s) was allowed for a trial; when the animal did not find all the pellets during the assigned time, the trial was ended and the duration was recorded as 5 min. At the end of each trial, animals were returned to their home cages and allowed to rest for 1– 5 min before starting their next trial. The floor of the maze was cleaned with a 25% acetic acid solution at the end of each trial to remove possible odor cues left by the animals on the floor of the apparatus. Following acquisition, the rats rested for 7 days after which they were subjected to a retention test, as described (Pen˜a de Ortiz et al., 2000). No infusions were given before the retention test. The purpose of the retention test was to determine the effects of Pb+2, infused during acquisition training, on long-term memory (LTM). Brain cryosections and thionin staining were done on animals selected random-

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ly as previously described (Pen˜a de Ortiz et al., 2000) to confirm the position of the cannulas. For the biochemical studies, animals were terminated either just before training on day 3 (corresponding to the pretraining groups, which were terminated 20 min after hippocampal infusions) or 5 min after training on day 3. All animals, including controls, were sedated in an ether chamber immediately before terminated by decapitation. The brains of all animals were obtained and the dorsal hippocampi dissected and stored overnight at 80 jC. Immunohistochemistry. The brains of rats utilized in the pathology studies (see above) were obtained immediately after termination, placed on dry ice, and stored at 80 jC until used. Fresh frozen coronal sections (25 Am thick) were obtained in a cryostat at 20 jC, placed on positively charged glass slides, and stored at 80 jC. The sections were then used to assess hippocampal gliosis by detecting astrocytes with a monoclonal glial fibrillary acidic protein (GFAP) antibody (Clone G-A-5 from Sigma-Aldrich, Saint Louis, MO). Sections were allowed to air dry for 20 min and fixed with 2% paraformaldehyde for 20 min and washed twice with PBS 1, 5 min each time. Permeabilization was done with 0.1% Triton X-100 in 0.1% of sodium citrate for 5 min and washed as described above. The sections were then incubated with 0.3% H2O2 and methanol for 30 min and washed with PBS 1, which was followed by incubation with 5% goat serum in PBS 1 for 30 min. Primary anti-GFAP monoclonal antibody (Sigma-Aldrich, Saint Louis, MO) was diluted at 1:250 in 1% horse serum in PBS 1 (blocking buffer) and added to the sections, which were then incubated overnight at 4 jC in a moist chamber. The next day and following two PBS 1 washes, sections were incubated for 2 h at room temperature with a horse anti-mouse biotinylated secondary antibody diluted at 1:100 in 1% horse serum in PBS 1 and then washed with PBS 1 again. The sections were next incubated with ABC peroxidase reagent (avidin – biotin peroxidase complex, Pierce) for 1 h at room temperature and washed with PBS 1. Diaminobenzidene (DAB) was added to the sections for 10 min, followed by PBS 1 washing. The slides were mounted using permanent mounting medium (Vector Laboratories Inc.). DAB-oxidized brown precipitates were visualized with a binocular light microscope and photomicrographs were taken with a digital camera. GFAP-positive cells were counted within the CA1 hippocampal subregion around the microinfused area using the Image J program. Image J counting was done by a technician who was blind in terms of which sections corresponded to treated or control animals. The analysis compared the number of GFAP-positive cells between the Na+- and Pb+2-treated hippocampi obtained from a total of five rats. Preparation of protein extracts from cytosolic and particulate cellular fractions. For each homogenate, dissected hippocampi from two animals from each group were

pooled and subjected to the protein extraction procedure as previously described (Va´zquez et al., 2000). The hippocampi were macerated under liquid nitrogen and resuspended in homogenization buffer (20 mM Tris –HCl, pH 7.1, 2 mM ethylene diamine tetra-acetic acid, 50 mM mercaptoethanol, 0.2 mM phenylmethyl-sulfonyl fluoride, and 1 Ag/ml of protease inhibitors (aprotinin and leupeptin)). The homogenate was centrifuged at 14 000 rpm for 1 h at 4 jC and the resulting supernatant was obtained and diluted (as necessary) with homogenization buffer. The diluted supernatant was used as the cytosolic fraction in the kinase activity assays. The remaining pellet was resuspended in homogenization buffer and sonicated 4 times for 15 s at 4 jC. Sonication was followed by incubation with 0.2% Triton X-100 for 30 min at 4 jC followed by centrifugation at 14 000 rpm for 1 h at 4 jC. The resulting supernatant was obtained and diluted with homogenization buffer for later use as the particulate cellular fraction. The protein concentration in both cytosolic and particulate fractions was determined using the Bradford method. Protein fractions were kept at 4 jC and assayed for PKC activity on the same day. PKC activity assays. For these studies, a total of eight animals were used per condition. For each condition, the hippocampi from two animals were pooled to prepare the protein extracts. Thus, a total number of four separate protein extracts for the trained and pretrained groups were used and each extract was analyzed in triplicates. The activity assays were performed with reagents from the Signa(M)TECT PKC Activity Assay system Promega (Madison, WI). This assay measures the activity of all Ca+2/ phospholipid dependent PKC isotypes in the samples. Reactions were performed as previously described (Va´zquez et al., 2000). Briefly, activated PKC reactions were done in the presence of phospholipids by adding 5 Al of coactivation buffer (1.25 mM ethyleneglycol-bis(h-aminoethyl)-N,N,NV,NV-tetra-acetic acid, 2 mM CaCl2, 0.5 mg/ml bovine serum albumin), 5 Al of activation buffer (1.6 mg/ml phosphatidylserine, 0.16 mg/ml diacylglycerol, 100 mM Tris – HCl, pH 7.5, 50 mM MgCl2), 5 Al of substrate (100 AM biotinylated neogranin substrate), 42 AM of cold ATP, and 20 AM g 32P ATP (3000 Ci/mmol). Control reactions were prepared as described above except that control buffer (100 mM Tris –HCl, pH 7.5, 50 mM MgCl2) was added instead of the activation buffer. All reactions were done in triplicates for both cytosolic and particulate fractions from each treatment group. A 10-min preincubation at 30 jC was followed by addition of 4 Ag of protein from cytosolic or particulate hippocampal fractions. Reactions were then incubated at 30 jC for an additional 10 min and terminated by adding 12.5 Al of termination buffer (7.5 M guanidine hydrochloride). A 30-Al aliquot from each terminated reaction was spotted on a biotin-capture membrane. Membranes were then washed once for 30 s and 3 times for 2 min each in 2 M NaCl. These washes were followed by

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four additional washes of 2 min each in 2 M NaCl prepared in 1% H3PO4 and two washes with deionized water for 30 s each. The final wash was with 95% ethanol for 15 s. All the radioactive washes were disposed according to the regulations established by the Nuclear Regulatory Commission and the University of Puerto Rico. Membranes were dried for 15 min at room temperature and analyzed in a scintillation counter. To determine the specific activity of [g32P] ATP, we removed 5-Al aliquots from reactions tubes of each condition and spotted them onto individual membranes squares, which were then placed into individual scintillation vials for counting. The specific activity of [g32P] ATP was calculated in cpm/pmol of ATP as instructed by Promega. Statistics. For all experiments, we assumed statistical significance at P < 0.05. Repeated measures two-way ANOVA was used to analyze the dose response acquisition and retention data together. We also performed one-way ANOVA and Newman– Keuls posttests for detecting significant differences on rat performance among the doses of Pb+2 acetate and a saline control for the retention test. Repeated measures two-way ANOVA analysis was used for comparing performance of rats treated with the 1-nmol dose of Na+ and Pb+2 acetate for each learning measure (searching time, working, reference and total errors, reference memory ratio). Bonferroni posteriori tests were performed when significance was reached ( P < 0.05). The data for the diversity of searching strategies were subjected to two-way ANOVA and Bonferroni posttesting as well as linear regression using the minimum-sum-of-squares method. The standard error of the slopes was obtained by calculating the 95% confidence intervals, including analysis of covariance to compare the slopes of the learning curves to evaluate the learning rates in the different groups. The linear relationship between the number of training sessions and each behavioral parameter was determined by an F test to determine whether the slopes of the learning curves were significantly different from zero and whether the elevation of the learning curves was different. Finally, one-way ANOVA coupled to Newman – Keuls analysis was used for comparing PKC activity levels in the cytosolic and particulate extracts of control and treated groups. Student’s t test analysis was used to assess differences in the translocation index (as defined by Va´zquez et al., 2000) and differences in GFAP-labeled cells in the CA1 hippocampal subregion between control and Pb+2 treatments.

Results Dose response study The focus of our study was on the possible effects of Pb+2 on hippocampal-dependent LTM. We initially tested several doses of intrahippocampal Pb+2 to determine the

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appropriate dose to use in these studies. Previous reports using intrahippocampal Pb+2 to study its effects in learning used the 0.1-nmol concentration (Jett et al., 1996). Thus, our initial analysis focused on the effects of Pb+2 concentrations from 0 to 3 nmol on reference memory, as measured by the RMR. Higher doses were not used to avoid effects due to pathology. Fig. 1 shows the results of the different Pb+2 concentrations on reference learning as measured by the RMR on each of the four sessions of acquisition training and on long-term retention. Repeated measures two-way ANOVA of the RMR data from session 1 to 4, corresponding to acquisition training, and the retention data, corresponding to long-term memory testing, showed a significant effect of the sessions ( F(4, 200) = 25.46, ***P < 0.0001), but not by the dose ( F(4, 200) = 1.746, P > 0.05) factor. However, a significant interaction was detected between these two factors ( F(16, 200) = 1.911, *P < 0.05), suggesting that the effects of the various Pb+2 doses were not consistent throughout the different sessions. When visually inspecting the plotted data (Fig. 1), we noted an apparent effect by some doses on Pb+2 on the retention, but not on the acquisition data, as measured by the RMR. Thus, we analyzed the retention data independently from the acquisition data using one-way ANOVA. The results supported our interpretation that Pb+2 caused a significant effect in retention ( F(4, 54) = 3.131, *P < 0.05), although post hoc analysis did not identify specific differences among the doses. Based on the data shown in Fig. 1 for the retention test, we decided to proceed with further studies using the 1-nmol dose, a mid-value between 0.7 and 3 nmol, which was the range of doses that appeared to affect LTM in this task. For a more rigorous analysis of the effects of intrahippocampal Pb+2 on LTM, we decided to use Na+ acetate instead of saline as our control. Intrahippocampal Pb2+ impairs retention The results in Fig. 2 show the results of our analysis of different parameters of learning for rats trained in the holeboard task, both including acquisition and retention training. Fig. 2A shows the results of searching time for Na+ (white bars)- and Pb+2 (black bars)-injected rats. Repeated measures two-way ANOVA confirmed that both groups showed a significant reduction in total searching time or latency to complete the task throughout acquisition training (sessions factor: F(4, 55) = 20.11, ***P < 0.0001), indicating that animals in both groups showed learning. No difference in total searching time was detected between the two groups throughout the different training sessions (treatment factor: F(1, 55) = 0.6914, P > 0.05). On the other hand, we found significant differences between the groups when we analyzed the number of reference errors committed by the rats (Fig. 2B). Repeated measures two-way ANOVA revealed significant differences between the groups caused by the treatment ( F(1, 55) =

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Fig. 2. Effect of Pb+2 in the spatial discrimination holeboard task. Performance of Na+ (N = 12; white bars)- and Pb+2 (N = 12; black bars)-injected rats on each session of training and on a retention test (7 days after training) using searching time (A), reference errors (B), working errors (C), and RMR (D) as measures of learning and memory in the holeboard task. Two-way analysis of variance and posteriori analysis demonstrated significant results for reference errors (**P < 0.01) and RMR (***P < 0.001) learning parameters in the retention test.

7.036, *P < 0.05) and session ( F(4, 55) = 16.39, ***P < 0.0001) factors. Multiple comparisons posttesting showed that indeed the significant difference between the groups was only in the retention test (**P < 0.01), suggesting a specific impairment in LTM in the Pb+2-injected rats. Unlike reference errors, analysis of working errors (Fig. 2C) did not reveal significant differences between the groups (two-way ANOVA: treatment factor: F(1, 55) = 3.331, P > 0.05). Both groups, however, did show a learning-related decrease in the number of working errors throughout acquisition, as evidenced by a significant effect by the sessions factor ( F(4, 55) = 6.192, ***P < 0.0005). We next analyzed the effects on the RMR, a measure for the development of reference memory during the task (Fig. 2D). Similar to our results for reference errors and the results obtained in our dose – effect study (Fig. 1), repeated measures two-way ANOVA of RMR values demonstrated

significant differences between control and Pb+2-treated rats (treatment factor: F(1, 55) = 13.30, ***P < 0.001) and between sessions (sessions factor: F(4, 55) = 12.31, ***P < 0.0001). In addition, a significant interaction was found between the treatment and sessions factors (*P < 0.05), suggesting that differences between control and Pb+2-treated rats were not consistent among sessions. More specifically, the Bonferroni posttest analysis detected significant differences between the groups only in the retention test (***P < 0.001). Effects of intrahippocampal Pb2+ on the diversity of searching strategies used throughout acquisition and retention We next analyzed the possible effects of Pb2+ on the searching strategy of the animals during both acquisition

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and retention. We examined the number of different searching patterns used by each rat during each session and the frequency of the most repeated searching pattern for each rat per session. Fig. 3A shows the effects of intrahippocampal Pb2+ on the diversity of searching strategies during acquisition and retention. Repeated measures two-way ANOVA of the number of different searching patterns used per rat demonstrated significant differences between control and Pb+2-treated rats (treatment factor: F(1, 55) = 12.02, **P < 0.005) and between sessions (sessions factor: F(4, 55) = 3.73, **P < 0.01). Importantly, Bonferroni posttest analysis detected significant differences between the groups only in the retention test (*P < 0.05). Because the rate of decrease in the complexity of searching patterns during acquisition appeared to be similar between both groups, we performed linear regression analysis of the plotted data. Our analysis showed no significant differences between the slopes of the two curves ( P > 0.05), although the curve obtained from the Pb2+ animals was significantly more elevated than the curve obtained from the control rats ( P < 0.05). Correspondingly, when we examined the frequency of the most repeated searching strategy per rat per session, we found it to increase throughout acquisition for both groups (Fig. 3B). Repeated measures two-way ANOVA of the frequency data also demonstrated significant differences between control and Pb+2-treated rats (treatment factor: F(1, 55) = 5.513, *P < 0.05) and between sessions (sessions factor: F(4, 55) = 2.891, *P < 0.05). However, Bonferroni

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posttest analysis did not detect specific significant differences in any of the sessions. Histology analysis Fig. 4A presents a diagram identifying the approximate cannula positions in randomly selected rats used in our studies and trained in the spatial discrimination holeboard task. As expected, the injection site was found immediately above the hippocampal pyramidal cells in the CA1 region, thus confirming the estimated site of injection according to the Rat Brain Atlas (Paxinos and Watson, 1998). To ensure that the observed behavioral effects of intrahippocampal Pb+2 were not due to pathological tissue damage, we looked for gliosis in tissue sections prepared from additional rats injected with Na+ and Pb+2 acetate in either side of the brain. Animals (N = 5) were injected 4 times on alternate days, as with the previously treated animals, and were terminated 1 h after their injection. These animals were not subjected to behavioral training. According to Student’s t test analysis, we found no significant differences between the Na+- and Pb+2-injected hippocampi in terms of the number of astrocytes stained with a GFAP antibody present in the CA1 region (t8 = 0.4663, P > 0.05) (Figs. 4B and C). Thus, no evidence of gliosis was observed, suggesting that our Pb+2 acetate infusions did not cause major pathologic abnormalities in the hippocampus. This is also supported by the fact that the Pb+2injected animals were able to acquire the task fairly

Fig. 3. Effect of Pb+2 acetate on the diversity and predictability of searching strategies during acquisition and retention of the holeboard task. x – y plots depicting (A) the number of different searching patterns (diversity) and (B) the frequency by which the most repeated searching pattern (predictability) was used during acquisition and retention for the Pb2+ (black)- and Na+ (white)-treated animals (N = 12 for each group). Error bars correspond to the SEM and significant P values obtained by multiple comparisons posttesting are represented by an asterisk (*). The continuous line connects the experimental data points, while the dashed lines connect data obtained from acquisition training to the data obtained 7 days later during the retention test. Results showed significant differences between the groups in both diversity and predictability of searching strategies used as determined by one-way ANOVA.

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Fig. 4. Histopathology analysis of intrahippocampal Pb+2 acetate microinfusions. (A) Diagram of the estimated site of injection and trajectories of rats used in the holeboard task. Cannulas were positioned 1 mm below cranial surface; the injector was located 1.5 mm below the cannula. Thus, each circle represents the estimated region of the injection site. The analysis includes a total of nine rats. (B) Results of GFAP immunohistochemistry on brain sections of animals infused intrahippocampally with Pb2+ and Na+ acetate on either hemisphere. The top and bottom panels show 15 (scale bar = 0.15 mm) and 60 (scale bar = 0.6 mm) magnification photomicrographs, respectively, of results representative of our findings from five animals. The arrows depict the sites of GFAP-labeled astrocytes. (C) Bar graph showing the results of cell counting analysis. No differences in GFAP staining were observed between the Pb2+- and Na+-injected hippocampi (N = 5 for both groups).

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normally after every individual infusion given before training (Figs. 1 and 2). PKC activity assays As seen in Fig. 5A, training caused a moderate, yet nonsignificant, increase in the mean levels of PKC activity in cytosolic extracts of control, but not Pb+2-treated rats. No statistically significant differences in PKC activity in cytosolic extracts from dorsal hippocampi obtained from pretrained and trained rats treated with either Na+ or Pb+2 acetate were observed (one-way ANOVA: F(3, 44) = 2.072, P > 0.1). In contrast, data obtained from the PKC activity assays performed on particulate extracts (Fig. 5B) showed significant differences among groups ( F(3, 44) = 7.423, ***P < 0.0005). Newman –Keuls posttests showed significant differences in PKC activity within the particulate

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fractions between the pretrained Na+- and trained Na+treated rats (**P < 0.01) confirming our previous finding of learning-induced increases in hippocampal PKC activity (Va´zquez et al., 2000). Importantly, the post hoc analysis did not find significant differences in PKC activity in hippocampal particulate extracts of pretrained rats treated with Na+ or Pb+2 acetate ( P > 0.05). Significant differences in PKC activity were also found between trained Na+- and Pb+2-treated rats (###P < 0.001), indicating that Pb+2 effectively blocked the learning-induced increase in hippocampal PKC activity. In addition, differences were not detected between pretrained and trained animals of the Pb+2 groups ( P > 0.05). It is evident from these results that Pb+2 blocked the learning-induced increase in hippocampal PKC within particulate, but not cytosolic, cellular fractions. These findings suggested that Pb+2 interfered with the learning-related translocation of PKC. In support of this,

Fig. 5. Effect of Pb+2 acetate in learning-induced hippocampal PKC activity and translocation. PKC activity assays were obtained from cytosolic (A) and particulate (B) extracts from dorsal hippocampi from Na+ and Pb+2 acetate-treated rats. Results were obtained from a set of four protein samples that were run in triplicate. Each protein extract was obtained from pooling the hippocampi of two animals. The analysis includes a trained group (T), terminated immediately after training on day 3, and a pretrained group (PT), which did not receive training on day 3, but was terminated after injection on that day. Error bars represent the SEM. (C) Translocation index (ratio of PKC activity from particulate versus cytosolic extracts) from Na+ (white bars)- and Pb+2 (black bars)-injected rats as an indicator of the learning-induced redistribution of PKC. Post hoc analysis demonstrates different levels of significance among treatments in B (###P < 0.001) for trained Na+ versus trained Pb+2 comparison; and (**P < 0.01) for pretrained Na+ versus trained Na+ comparison. A t test comparison in C also shows significant differences between the trained groups (***P < 0.0001).

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the results shown in Fig. 5C indicate that Pb+2-injected rats had a lower hippocampal PKC translocation index than controls (t(22) = 6.692; ***P < 0.0001).

Discussion In the present study, we observed the effects of Pb+2 after intrahippocampal exposure of low levels of Pb+2 acetate in adult rats in acquisition and retention of the holeboard spatial discrimination task. During acquisition training, Pb+2 infused into the CA1 region of the hippocampus just before each session had no effect on searching time, working errors, or reference errors, but did interfere with the development of a predictable searching strategy. The results from the searching strategy analysis reveal that intrahippocampal Pb2+ impaired the acquisition of the task by causing the animals to diversify the searching strategies they used, not allowing them to focus in the most effective ones. Such an effect would certainly cause learning to be less efficient in the Pb2+ animals. Previous studies have shown that intrahippocampal microinjection of Pb+2 impairs acquisition of the Morris water maze (Jett et al., 1996), a task that is also dependent on the hippocampus (Eichenbaum et al., 1990; Gallager and Holland, 1992; Schenk and Morris, 1985), among other brain regions (de Bruin et al., 2001; Mura and Feldon, 2003; Vafaei and Rashidy-Pour, 2004). Acquisition of the Morris water maze has also been shown to be sensitive to the blockade of N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal subregion (Bannerman et al., 1995; Heale and Harley, 1990; Mc Lamb et al., 1990; Mc Namara and Skelton, 1993; Morris et al., 1986; Upchurch and Wehner, 1990). Interestingly, recent studies showed that Pb+2 blocks the NMDA-gated Ca+2 channel (Alkondon et al., 1990; Gavazzo et al., 2001) and NMDA receptor-dependent long-term potentiation (LTP) in the CA1 hippocampal subregion (Altmann et al., 1991; Gutowski et al., 1998; Nihei and Guilarte, 2001; Zaiser and Miletic, 1997), a cellular model for learning and memory in the brain (Silva, 2003). Hence, it is possible that blockade of NMDA receptors by low doses of Pb+2 is its main mechanism of learning impairment in the Morris water maze and is perhaps involved in the effects of Pb+2 on searching strategies during acquisition of the holeboard task (Fig. 3). Nevertheless, because of the potential multiplicity of molecular targets for Pb2+ in the central nervous system (CorySlechta, 1997; Marchetti, 2003), it is possible that other neurotransmitter and cellular signaling pathways are also associated to the effects of Pb2+ on spatial learning. On the other hand, the effects of Pb+2 on memory consolidation in spatial learning have not been addressed in the Morris water maze. Indeed, our study with the holeboard spatial discrimination task is the first to address the direct effects of Pb+2 on hippocampal-dependent LTM. We found that the effects of Pb+ administered during

acquisition had a robust effect on retention of reference, but not working, memory measured by the number of reference errors, the RMR, and the diversity of searching strategies used. The fact that Pb+2 had no effect on searching time during acquisition or retention argues against the possibility that the effects of intrahippocampal administration of this heavy metal ion could be due to impairment in visual acuity. Moreover, we found no evidence of reactive gliosis, an indicator of tissue pathology (O’ Callaghan and Miller, 1993), caused by intrahippocampal injections of Pb+2. It seems more likely that the effects of Pb+2 on hippocampal-dependent LTM are due to the possible interference of cellular cascades normally leading to long-term information storage. Previous studies have shown that acquisition of the holeboard task is accompanied by increased hippocampal expression of PKCg (Van der Zee et al., 1992). In addition, we previously showed that acquisition of the holeboard task is associated with increased hippocampal Ca2+/phospholipid-dependent PKC activity after training on day 3, but not on day 1 (Va´zquez et al., 2000), an effect confirmed by the studies presented here (Fig. 5). Protein kinases, including PKC, are thought to be important biochemical mediators of LTM in the brain (Abeliovich et al., 1993a, 1993b; Akers et al., 1986; Malinow et al., 1988; Mathis et al., 1992; Micheau and Riedel, 1999; Reymann et al., 1988; Selcher et al., 2002; Shobe, 2003; Wallestein et al., 2002). Several reports have demonstrated the role of PKC in neuronal signaling (see Haller et al., 1994), synaptic plasticity in hippocampal neurons (Kamphuis et al., 1995; Malinow et al., 1988), and in learning and memory processes in the brain (Bernabeu et al., 1995; Golski et al., 1995; Wehner et al., 1990). Interestingly, evidence suggests that Pb+2 interferes directly or indirectly with PKC in a dose-dependent fashion (Laterra et al., 1992; Long et al., 1994; Markovac and Goldstein, 1988b; Murakami et al., 1993; Sun et al., 1999). Moreover, Nihei et al. (2001) found a reduction in PKCg protein expression in both cytosolic and membranous extracts obtained from hippocampal tissue of adult rats exposed chronically to low-levels of Pb+2 in their drinking water throughout development. However, reduced levels of hippocampal PKCg did not result in reduced Ca2+-dependent or Ca2+-independent hippocampal PKC activity in Pb+2-exposed rats, a phenomenon that could be explained by compensational mechanisms involving upregulation of other PKCs in the hippocampus during development. Here, we found that intrahippocampal Pb+2 blocked the learninginduced increases in hippocampal Ca2+/phospholipid-dependent PKC activity on day 3 of acquisition of the holeboard task, while not affecting the pretraining activity levels of the kinase. Thus, the effects of Pb+2 were specific to PKC activity associated with learning mechanisms and not to constitutive PKC activation of this kinase because of training on days 1 and 2. Blockade of learning-related hippocampal PKC activation by Pb2+ could result in further

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alterations in gene expression, which are required for LTM (Pen˜a de Ortiz and Arshavsky, 2001; Silva, 2003). Interestingly, the time course of hippocampal PKC activation in the holeboard task in these and previous studies (Va´zquez et al., 2000) corresponded with the timing of the hippocampal induction of the genes such as hzf-3/nurr1 and akt, which encode a learning-related immediate – early transcription factor and a plasticity-associated kinase activated by phosphoinositol signaling, respectively (Ge et al., 2003; Pen˜a de Ortiz et al., 2000; Robles et al., 2003). Thus, it is possible that the increased hippocampal PKC activity is involved in the activation of learning-related genes, and that Pb2+mediated blockade of their induction results in LTM impairment. Overall, our findings suggest that hippocampal exposure to Pb2+ during active learning impairs formation of LTM, an effect that could be related to its blockade of the activation of Ca2+/phospholipid-dependent PKC during mid to late acquisition training.

Acknowledgments We would Dr. Carmen S. Maldonado-Vlaar for her assistance with the intracraneal surgeries. We would also like to thank Ms. Manhong Zou for her technical assistance in cryosectioning, and Dr. Mumna Al Banchaabouchi for collaboration with the immunohistochemistry studies. Finally, we thank Iva´n J. Santos and Lixmar Pereira for their assistance in cell counting using Image J. This work was supported by NIH (S.P.O. grants NIGMS-MBRS SOGGMO8102-26S1, NINDS-SNRP U54 NS39405, and IDEA-COBRE: NCRR-NIH 5P20 RR 15565-02), and the EPA (MAI STAR EPA Predoctoral Fellowship to A.V. (U91600901-0).

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