Nicotine attenuates spatial learning deficits induced in the rat by perinatal lead exposure

Brain Research 999 (2004) 142 – 147 www.elsevier.com/locate/brainres

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Nicotine attenuates spatial learning deficits induced in the rat by perinatal lead exposure Mingfu Zhou, Janusz B. Suszkiw * Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, P.O. Box 670576, Cincinnati, OH 45267-0576 USA Accepted 17 October 2003

Abstract Maternally lead (Pb)-exposed, juvenile rats exhibit significant deficits in spatial reference memory acquisition and working memory performance in the Morris water maze (MWM). Acute systemic application of nicotine reverses these deficits without affecting behavioral performance of the age-matched, lead-unexposed control animals. These results suggest that nicotinic agonist treatments can ameliorate learning and memory impairments, presumably by compensating for deficient nicotinic function in developmentally lead-exposed animals. D 2004 Elsevier B.V. All rights reserved. Theme: Disorder of the nervous system Topic: Neurotoxicity Keywords: Lead exposure; Nicotine; Spatial learning; Morris water maze

Exposure to environmental levels of inorganic lead (Pb) during early development has been implicated in neurobehavioral abnormalities, hyperactivity, and poor learning performance in children and in rodent models of lead neurotoxicity [6,15,17]. Rodent studies suggest that cognitive impairments including deficits in spatial learning and memory performance following lead exposure are related to disruption of long-term potentiation (LTP) in the hippocampus [2,8,11,16,23]. Impairment of hippocampal spatial memory processing in lead-exposed animals could be additionally exacerbated by diminished nicotinic acetylcholine facilitation of LTP [7,22] due to lead-induced loss of the septo-hippocampal cholinergic afferents [3,4,5,19] and possibly direct Pb inhibition of the nicotinic acetylcholine receptor (nAchR) function [13,24]. Support for cholinergic involvement in lead-induced behavioral deficits comes from recent observation that intrahippocampal transplants of cholinergic-rich septal and nucleus basalis tissue ameliorate the deficits [1]. Finally, there is a large body of literature documenting the effectiveness of nicotine in improving cognitive dysfunction and ameliorating memory impairments in various neurological disorders in humans as well * Corresponding author. Tel.: +1-513-558-3039; fax: +1-513-5585738. E-mail address: [email protected] (J.B. Suszkiw). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.10.068

as in animals with lesions to the hippocampus and/or basal forebrain cholinergic system [12,18]. In this context, it was of interest to determine if systemic application of nicotine would also ameliorate spatial learning impairments induced in the rat by developmental lead exposure. Rats (Charles River Sprague – Dawley) were maternally lead exposed via administration of 0.2% solution of lead acetate trihydrate in distilled drinking water to dams from gestational day 16 through weaning on postnatal day 21 (P21), with control dams receiving lead-free distilled drinking water during the same period. Litter size was culled on postnatal day 2 to eight pups balanced for gender. Following weaning, animals were maintained in pairs of same sex littermates per cage for the duration of behavioral testing. Animals were maintained on a 12:12-h light – dark cycle, and were provided a constant formula Teklad LM-485 rodent diet and drinking water ad libitum. Behavioral assessment was conducted beginning on postnatal day P28 and consisted of 6 days of spatial reference memory acquisition, followed by probe test on day 7 and 4 days of working memory tests in Morris water maze (MWM) [14]. The apparatus was a 145-cm-diameter black circular tank filled with tap water maintained at room temperature (20 jC) and containing a transparent, circular (d = 10 cm) Plexiglas escape platform which was submerged 2 cm below the surface of the water. The time to

M. Zhou, J.B. Suszkiw / Brain Research 999 (2004) 142–147

find the escape platform (escape latency), total swim time and swim distance were recorded and analyzed with the aid of a video-based, computer-assisted tracking system (Smart Video Tracking System, San Diego Instruments, CA). In the reference memory acquisition, the platform was located in a constant position in the middle of one of the four quadrants. A trial consisted of placing the rat in the water, facing the pool wall, in a randomly chosen quadrant and giving the subject 60 s to find the platform, after which time the animal was placed on the platform by the observer. After reaching or being placed on the platform, the subject was allowed to remain on the platform for 15 s before the start of the next trial. Each rat received one session of four trials per day, and the mean daily escape latency was calculated by averaging the latencies from each of the four daily trials and taken as a measure of acquisition. In the probe test, the platform was removed from the pool, and the time spent and distance traveled in each quadrant were recorded, and the percentage of total swim time or distance in the target quadrant that contained the platform during the preceding acquisition phase was calculated and taken as the measure of reference memory acquisition. Swimming speed obtained by dividing the total distance traveled by time was taken as a measure of motoric ability. In the test of working memory, rats were required to find the hidden platform that was placed in a new position each session [20]. The animals received four sessions per day, each session consisting of two 60-s trials to find the escape platform. In the first trial, the subject was placed in the water facing the wall of the pool at one of four start positions randomly and given 60 s to find the platform or was placed on the platform after unsuccessful 60-s swim time. After 10-s rest on the platform, trial 2 was started by placing the rat in the same starting position as during the trial 1 and recording the escape latency to find the platform. The latencies in trial 1 or trial 2 in each of the four sessions were averaged, and the trial 2 latency was taken as a measure of working memory. Cued learning (visible platform) test was performed on postnatal days P42 – P44 to ascertain that deficits in MWM learning tasks were not related to decreased visual acuity in Pb-exposed animals. The procedure was similar to that for the reference memory test except that the escape platform was made visible by raising it to the

Fig. 1. Reference memory acquisition. Latency to find the hidden platform as function of days (A) and mean latency over 6 days of training (B) in control (Con) and lead-exposed (Pb) rats. Results are means F S.E.M., n = 12 (6 females and 6 males per group, each pair derived from separate litter). Statistical significance between groups on each training day was analyzed by Student’s t-test and two-way RM-ANOVA followed by pairwise group comparisons by Student – Newman – Keuls (SNK) method. Two-way RM-ANOVA showed a significant overall difference among mean latencies over 6 days of testing [ F(3,287) = 3.23, p = 0.0314] and post hoc SNK analysis indicated significant increase in latency for Pb + Saline vs. Con + Saline, but no significant differences among the Con + Saline, Con + Nicotine and Pb + Nicotine groups. *p < 0.05 for group comparisons vs. Con + Saline.

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surface of the water and affixing to it a striped black and white polyethylene cylinder (6 cm in diameter and 10 cm in height) mounted on a rod 12 cm above the platform. There were two daily sessions of four trials each with the position of the platform remaining constant within each session but varied among each of the four pool quadrants between sessions. To assess the effects of nicotine on behavioral performance, half the littermates in the control and Pbexposure groups received a single intraperitoneal injection of either saline or ( )-nicotine-hydrogen-(+)-tartrate (Sigma; 0.35 mg/kg nicotine base in saline) 20 min prior to trials. Altogether, there were four treatment groups: control animals not exposed to lead and receiving saline injection (Con + Saline), control littermates injected with nicotine (Con + Nicotine), Pb-exposed animals injected with saline (Pb + Saline) and Pb-exposed littermates injected with nicotine (Pb + Nicotine). Each group contained 12 animals derived from six separate litters, one female and one male per litter. All groups were tested under identical conditions and on the same day, but the order of testing was varied everyday. Inasmuch as preliminary statistical analyses did not indicate significant differences between performance of male and female rats in behavioral assessments, data from both genders were combined. Behavioral assessments on Morris water maze confirmed the previously reported [9,10] impairments in performance of the water maze reference and working memory tasks by the lead-exposed rats. As shown in Fig. 1A, except for the first day of training when all subjects performed at chance level, the lead-exposed rats took significantly longer than the lead-unexposed control animals to find the hidden platform during the entire remaining period of reference memory acquisition. Administration of nicotine to the control animals prior to behavioral testing session had not significantly affected the escape latencies on any of the training days, but prevented the latency prolongation seen in Pb-exposed animals. Indeed, the escape latencies between days 3 and 6 of training were virtually indistinguishable among the control + saline, control + nicotine and Pb + nicotine groups. Two-way (group  day), repeated-measures analysis of variance (RM-ANOVA) revealed significant differences [ F(3,287) = 3.23, p = 0.0314] for group latencies averaged across all six training sessions. Post hoc pairwise group comparisons using Student –Newman –Keuls (SNK) analysis indicated that whereas mean latencies for the control + saline, control + nicotine and Pb + nicotine groups were not significantly ( p>0.05) different from each other, the mean escape latency of Pb + saline group was significantly different from that of either the control + saline, control + nicotine, or Pb + nicotine groups (Fig. 1B). Analysis of the swim time and swim distance in the probe trial (Fig. 2A) confirmed that Pb-exposed animals, which received saline only, expended a significantly smaller percentage of swim time or swim distance in the target quadrant than either the control + saline, control + nicotine, or Pb + nicotine groups. In contrast, swim speed,

Fig. 2. Probe test. Percentage of swimming time spent and swimming distance traveled in the quadrant where the platform was located during the preceding acquisition phase (A) and the average of swimming speed during probe test (B) in control and lead-exposed rats. Results are means F S.E.M., n = 12 (6 females and 6 males per group, each pair derived from separate litter). Data analyzed by one-way ANOVA and post hoc pairwise comparisons between groups using the SNK method. There are significant differences for swimming time [ F(3,47) = 4.19, p = 0.0108] and for swimming distance [ F(3,47) = 2.92, p = 0.0445] between Pb + Saline and Con + Saline, but not between Con + Saline and Con + Nicotine or Pb + nicotine groups. There is no significant difference among any of the treatment groups for swimming speed [ F(3,47) = 1.07, p = 0.3700].

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Fig. 3. Working memory. Latency (average of four daily sessions) to find the hidden platform as function of days (A, top: trial 1; bottom: trial 2) and mean latency over 4 days of training (B) in control and lead-exposed rats. Results are means F S.E.M. of n = 12 (6 females and 6 males per group, each pair derived from separate litter). Statistical significance between groups on each training day was analyzed by Student’s t-test and two-way RM-ANOVA was used to analyze responses over the entire period of training. There were no significant differences in overall means of 4 days of training for trial 1 [ F(3,191) = 0.421, p = 0.7389] and a significant differences in trial 2 [ F(3,191) = 3.676, p = 0.019]. Post hoc SNK analysis indicated significant difference between means of Pb + Saline and Con + Saline groups, but no significant differences among Con + Saline, Con + Nicotine, or Pb + nicotine groups. *p < 0.05 for group comparisons vs. control – saline.

on trial 2 did not significantly improve over the 4 days of training (Fig. 3A), confirming a relative absence of reference memory component in this paradigm. Two-way RMANOVA confirmed significant group differences in trial 2 [ F(3,191) = 3.676, p = 0.019] with post hoc SNK analysis indicating a significantly ( p < 0.05) longer mean latency to find the platform for the Pb-exposed rats in trial 2 than either the control + saline, control + nicotine, or Pb + nicotine groups, whereas there were no significant ( p > 0.05) differences among control + saline, control + nicotine, or Pb + nicotine groups (Fig. 3B). Thus, as in the case of

which was assessed as an index of motoric performance by the rats, did not significantly differ among any of the four groups (Fig. 2B). The results of working memory tests are illustrated in Fig. 3. As expected from random variation of platform placement and animal start positions between sessions, the latencies to find the hidden platform in trial 1 varied randomly within as well as among the groups over the 4 days of testing [ F(3,191) = 0.421, p = 0.7389]. The latencies to find the platform on the second trial were considerably shortened in each session, although the performance level

Table 1 Summary: effects of nicotine in control and Pb-exposed rats Groups

Reference memory Latency

Con + Saline Pb + Saline Con + Nicotine Pb + Nicotine

Probe test

Working memory Swimming speed

Latency

(s)

% of Con

(s)

Swimming time % of Con

(cm/s)

% of Con

(s)

% of Con

25.8 F 2.2 34.3 F 2.2* 25.8 F 2.2 27.9 F 2.2

NS 133 NS NS

38.5 F 2.9 27.8 F 1.1* 36.3 F 2.9 34.3 F 1.6

NS 72 NS NS

6.6 F 0.3 6.7 F 0.2 6.9 F 0.3 7.3 F 0.2

NS NS NS NS

12.5 F 2.0 20.7 F 2.0* 13.1 F 2.0 13.2 F 2.0

NS 166 NS NS

Means F S.E.M., n = 12. * p < 0.05 as compared to control group by two-way repeated-measures ANOVA (Reference memory, Working memory), or one-way ANOVA (probe test).

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Fig. 4. Cued learning test in control and lead-exposed rats. Latency (average of 4 trials/session) to find visible platform as function of sessions. Insert: mean latency over six sessions (2 sessions/day) of training. Results are means F S.E.M. from n = 8 animals (4 male and 4 female). No significant difference between control and lead-exposure groups by twoway RM-ANOVA [ F(1,95) = 0.859, p = 0.369]. Statistical significance between groups in each session was analyzed by Student’s t-test ( p > 0.1).

reference memory acquisition, Pb-exposure impaired the working memory performance and administration of nicotine reversed the effect of lead without having any effect on working memory performance in control rats that have not been exposed to lead. In summary (Table 1), perinatally lead-exposed rats exhibited approximately 30% increase in the latency to find hidden platform on the reference memory trials and 70% increase in the latency to find the hidden platform in the working memory trials, suggesting a more robust effect of lead on short- than long-term memory. The deficits in the spatial learning by the lead-exposed rats are not likely attributable to malnutrition. We have shown previously that identical lead-exposure paradigm used in these experiments did not have significant effect on body weight gain during the suckling period [4] and confirmed in the present study that the body weights of the offspring measured prior to the start of behavioral testing at P28 were not significantly different between the control and Pb-exposed animals: 89.3 F 7.4 vs. 90.2 F 7.9 g, respectively, for control and Pb-exposed female rats and 100.3 F 6.4 vs. 99.6 F 8.2 g, respectively, for control and Pb-exposed male rats (mean F S.D., n = 12; p > 0.5, Student’s t-test). Furthermore,

impaired performance of hidden platform tests by leadexposed rats cannot be attributed to motoric impairments because there were no significant differences between swimming speed in control and lead-exposed groups (Table 1, probe test) or to visual impairment, as both control and Pb-exposed rats performed equally well in cued learning with visible platform (Fig. 4). The reversal by nicotine of lead-induced spatial learning impairments is consistent with many observations that acute nicotine improves performance of memory tasks and suggests the involvement of hippocampal a7- and/or a4h2 nAChR subtypes which have been implicated in cognitive effects of nicotine [18]. Based on the recent report that nicotine enhances induction of long-term potentiation in the hippocampus by compensating for 192-IgG-saporin-induced loss of cholinergic function [24], it may be suggested that nicotine similarly reverses the spatial learning deficits in lead-exposed rats by compensating for diminished nicotinic activation of nAChRs arising from the lead-induced reduction of hippocampal ACh and, possibly, direct inhibition by Pb of nAChR function, to enhance nicotinic facilitation of hippocampal LTP. The effect may involve presynaptic receptor-mediated facilitation of acetylcholine, GABA and/or glutamate release [21] as well as direct activation of postsynaptic nicotinic receptors involved in modulation of LTP. However, additional experiments will be needed to characterize the cellular sites of the lead-induced cognitive impairments and their reversibility by nicotine.

Acknowledgements This work was supported in part by National Institute of Environmental Health Sciences grant ES09516.

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