Long-term changes in brain cholinergic system and behavior in rats following gestational exposure to lead: protective effect of calcium supplement

Abstract

Our earlier studies showed that lactational exposure to lead (Pb) caused irreversible neurochemical alterations in rats. The present study was carried out to examine whether gestational exposure to Pb can cause long-term changes in the brain cholinergic system and behavior of rats. The protective effect of calcium (Ca) supplementation against Pb toxicity was also examined. Pregnant rats were exposed to 0.2% Pb (Pb acetate in drinking water) from gestational day (GD) 6 to GD 21. The results showed decrease in body weight gain (GD 6-21) of dams, whereas no changes were observed in offspring body weight at different postnatal days following Pb exposure. Male offspring treated with Pb showed marginal alterations in developmental landmarks such as unfolding of pinnae, lower and upper incisor eruption, fur development, eye slit formation and eye opening on postnatal day (PND) 1, whereas significant alterations were found in the righting reflex (PNDs 4-7), slant board behavior (PNDs 8-10) and forelimb hang performance (PNDs 12-16). Biochemical analysis showed decrease in synaptosomal acetylcholinesterase (AChE) activity and an increase in acetylcholine (ACh) levels in the cortex, cerebellum and hippocampus on PND 14, PND 21, PND 28 and in the four-month age group of rats following Pb exposure. Significant deficits were also observed in total locomotor activity, exploratory behavior and open field behavior in selected age groups of Pb-exposed rats. These alterations were found to be maximal on PND 28, corresponding with the greater blood lead levels observed on PND 28. Addition of 0.02% Ca to Pb reversed the Pb-induced impairments in the cholinergic system as well as in behavioral parameters of rats. In conclusion, these data suggest that gestational exposure to Pb is able to induce long-term changes in neurological functions of offspring. Maternal Ca administration reversed these neurological effects of Pb later in life, suggesting a protective effect of calcium in Pb-exposed animals.

Keywords: behavior; blood Pb levels; calcium supplementation; cholinergic system; lead toxicity; long-term effects.

Figure 1

Study design: A schematic representation of the exposure period and the parameters determined in different age groups of rats.

Figure 2

Effect of Pb-exposure and calcium supplementation on maternal body weight gain (%) of rats. Rats were exposed to either deionized drinking water (control) or Pb-acetate (0.2%) or calcium together with Pb in deionized water from gestational day 6 (GD 6) to GD 21 through drinking water. Values are mean ± SD of eight (n=8) observations. The values marked with “asterisk” are significantly different from corresponding controls as evaluated by the ANOVA followed by Student-Newman-Keuls (SNK) post hoc test (p<0.05).

Figure 3

Effect of Pb-exposure and calcium supplementation on righting reflex (time in seconds) behavior in rats. Rats were exposed to either deionized drinking water (control) or Pb-acetate (0.2%) or calcium together with Pb in deionized water from gestational day 6 (GD 6) to GD 21 through drinking water. Values are mean ± SD of eight (n=8) observations. The values marked with “asterisk” are significantly different from corresponding controls as evaluated by the ANOVA followed by Student-Newman-Keuls (SNK) post hoc test (p<0.05).

Figure 4

Effect of Pb-exposure and calcium supplementation on slant board behavior (time in seconds) of rats. Rats were exposed to either deionized drinking water (control) or Pb-acetate (0.2%) or calcium together with Pb in deionized water from gestational day 6 (GD 6) to GD 21 through drinking water. Values are mean ± SD of eight (n=8) observations. The values marked with “asterisk” are significantly different from corresponding controls as evaluated by the ANOVA followed by Student-Newman-Keuls (SNK) post hoc test (p<0.05).

Figure 5

Effect of Pb-exposure and calcium supplementation on forelimb hang performance (time in seconds) of rats. Rats were exposed to either deionized drinking water (control) or Pb-acetate (0.2%) or calcium together with Pb in deionized water from gestational day 6 (GD 6) to GD 21 through drinking water. Values are mean ± SD of eight (n=8) observations. The values marked with “asterisk” are significantly different from corresponding controls as evaluated by the ANOVA followed by Student-Newman-Keuls (SNK) post hoc test (p<0.05).

Figure 6

(a,b,c) Effect of Pb-exposure and calcium supplementation on AChE activity in cortex, cerebellum and hippocampus. Rats were exposed to either deionized drinking water (control) or Pb-acetate (0.2%) or calcium together with Pb in deionized water from gestational day 6 (GD 6) to GD 21 through drinking water. AChE activity was determined in the synaptosomal fractions of brain regions. Values are mean ± SD of six separate experiments. The 0.05 level of probability was used as the criterion for significance.

Figure 7

(a,b,c) Effect of Pb-exposure and calcium supplementation to Pb on ACh levels in cortex, cerebellum and hippocampus. Rats were exposed to either deionized drinking water (control) or Pb-acetate (0.2%) or calcium together with Pb in deionized water from gestational day 6 (GD 6) to GD 21 through drinking water. The neurotransmitter levels were determined in the synaptosomal fractions of brain regions. Values are mean ± SD of six separate experiments. The 0.05 level of probability was used as the criterion for significance.

Figure 8

Effect of Pb-exposure and calcium supplementation on exploratory behavior in different age groups of rats. The values are Mean ± SD of six separate experiments. The 0.05 level of probability was used as the criterion for significance.

Figure 9

Effect of Pb-exposure and calcium supplementation on total locomotor activity in different age groups of rats. The activity was measured with OPTO-VARIMEX, Columbus Instruments, Ohio, USA on the designated PNDs and was presented as counts/min. The values are Mean ± SD of six separate experiments. The 0.05 level of probability was used as the criterion for significance.

Figure 10

Effect of Pb-exposure and calcium supplementation on open-field behavior in different age groups of rats. The values are Mean ± SD of six separate experiments. The 0.05 level of probability was used as the criterion for significance.