Edited by: Tod Edward Kippin, University of California, Santa Barbara, United States
Reviewed by: Jared Robert Bagley, Binghamton University, United States; Kenji Hashimoto, Chiba University, Japan
This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience
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As a series of high effectiveness and low mammalian toxic insecticide, pyrethroids have been widely used in residential and agricultural areas (
Type I pyrethroids induce an extended opening of the channels (mainly sodium channel and calcium channel), and type II pyrethroids cause an even longer prolongation than type I pyrethroids (
Brain derived neurotrophic factor (BDNF) plays an important role in neurodevelopment, synapse regulation, learning, and memory through the activation of NMDAR in hippocampus (
Taking into account the possible neurotoxic effects of pyrethroids, this study investigated the potential cognitive impairment in offspring prenatally exposed to deltamethrin. In the present study, pregnant SD rats were administered daily with different doses of deltamethrin, co-administration of deltamethrin and memantine, or memantine alone. On postnatal day 21 (PND 21), Morris water maze (MWM) were utilized to observe the cognitive abilities in the offspring. The protein expressions of GluN1, GluN2A, GluN2B, BDNF, phosphorylated tyrosine kinase B (pTrkB) receptor, and phosphorylated CREB (pCREB), in the hippocampus were quantified by western blotting analysis. The results of the present study suggested that a high dose of deltamethrin (9 mg/kg) exposure during pregnancy resulted in cognitive impairment and altered expression of GluN1, GluN2A, GluN2B, BDNF, pCREB, and pTrkB in offspring on PND 21. The observed cognitive dysfunction was ameliorated by memantine treatment.
Male and female Sprague-Dawley (SD) rats weighing 220–250 g (7–9 weeks of age) obtained from Kunming laboratory animal center (Kunming, China) were used in this investigation. The animals were housed in plastic cases in a ventilated room with a controlled environment (22 ± 2°C, 55 ± 10% humidity, and 12-h light/dark cycle). The rats were provided with free access to food and water throughout the study. Animal handling and procedures followed the guideline of Chinese Academy of Sciences. As F0-generation, 42 female rats were evenly divided into seven groups: a control group, groups treated daily with deltamethrin (≥98% pure; Sigma-Aldrich, St. Louis, MO, United States) at a dose of 0.54 mg/kg, 1.35 mg/kg, 2.7 mg/kg, or a 9 mg/kg bw (equivalent to 1/250, 1/100, 1/50, 1/15 of the LD50), a group treated with deltamethrin (9 mg/kg) mixed with memantine (10 mg/kg; H. Lundbeck A/S, Valby, Denmark), and a group treated with memantine alone. Female rats were mated overnight at the proportion of two females to one male. Pregnant rats were administered with different those compounds dissolved in coin oil (Yuanye, Shanghai, China) by intragastric administration daily beginning on the first day of pregnancy (vaginal smear positive) throughout the gestation period. Control animals were given the vehicle alone (coin oil) in the same way as deltamethrin-treated animals. Maternal weight was monitored daily during pregnancy. Pregnant rats were observed after deltamethrin treatment for clinical signs of pyrethroid toxicity such as seizures, salivation, and tremors. All pregnant rats were allowed to deliver and nurture their offsprings normally. The day of birth was determined to be postnatal day 0 (PND 0). Pups were weighed at PND 0, PND 7, PND 14, and PND 21. All six litters were used in each group. Variables such as litter size and the number of alive pups were assessed. At weaning (PND 21), male and female pups were housed separately. To control specific litter effects, four animals (half male and half female) were randomly selected from each litter for following tests.
Twelve pups of each group (one male and one female from each litter) were selected randomly at PND 21, and MWM test was used to evaluate spatial discrimination and memory abilities in offspring through multiple attempts in searching a fixed hidden platform in the pool (90 cm of diameter), to which the water mixed with ink at 25°C was added. The pool was divided into four quadrants and the platform was put into the middle of the quadrant III. In place navigation test, rats were put into the water in a certain order. Duration from dropping into the water to reaching the hidden platform was observed and recorded as escape latency. If the rats could not find the platform within 120 s, it was recorded as 120 s. The test was conducted for five consecutive days. On day 6, the place probe trial was performed to determine the familiarity of the rats with the platform in the water maze. Each rat was subjected to a special probe trial (60 s) initially after removing the platform. Spatial memory ability was evaluated by the percentage of time that rats spent in the target zone (quadrant III) within 60 s. Data were recorded on computer with a video tracking system and analyzed with the SMART 3.0 software (Panlab/Harvard Apparatus, Barcelona, Spain).
At 21 days of age, another set of 12 pups was randomly selected from each group (one male and one female from each litter). One side of the brain was used for western blot and the other side of brain was used for immunofluorescence assay. To examine protein expression levels of GluN1, GluN2A, GluN2B, BDNF, pTrkB, and pCREB, hippocampal tissues were homogenized in RIPA (Beyotime, Shanghai, China), and protein concentrations were measured using BCA assay kit (Beyotime, Shanghai, China). Protein (20 μg) was separated by SDS–PAGE at 80 V for 2 h, and then transferred onto PVDF membranes (Millipore, Billerica, MA, United States). The membranes were then blocked in 5% non-fat milk for 1 h at room temperature. Then, blocked membranes were incubated with primary antibody anti-GluN1 (dilution, 1:1,000; Abcam, Cambridge, MA, United States), anti-GluN2A (dilution, 1:1,000; Abcam, Cambridge, MA, United States), anti-GluN2B (dilution, 1:1,000; Abcam, Cambridge, MA, United States), anti-BDNF (dilution, 1:1,000; Abcam, Cambridge, MA, United States), anti-CREB (dilution, 1:1,000; Abcam, Cambridge, MA, United States), anti-phosphorylated (p)-CREB (dilution, 1:10,000; Abcam, Cambridge, MA, United States), anti-TrkB (dilution, 1:500; Abcam, Cambridge, MA, United States), anti-phosphorylated (p)-TrkB (dilution, 1:1,000; Abcam, Cambridge, MA, United States), and anti-β-actin (dilution, 1:1,000; Abcam, Cambridge, MA, United States) primary antibodies overnight at 4°C, followed by incubation with the secondary antibodies (dilution, 1:5,000; goat anti-rabbit IgG; ZSGB-BIO, Beijing, China) for 1 h at room temperature. The membranes were developed with enhanced chemiluminescence (ECL) kit and pictures were captured in a Bio-Gel Imagining System (Bio-Rad, Hercules, CA, United States). Protein levels in the different groups were presented as percentages of the control group.
For immunofluorescence assay, brains tissues from each group were harvested and then immersed in 4% formaldehyde solution for 24 h followed by gradient of dehydration in 10, 20, and 30% of sugar solution. The brain tissues were OCT-embedded and sliced into coronal sections by a frozen microtome (Leica CM1900, Wetzlar, Hesse-Darmstadt, Germany) at a thickness of 10 μm through the dorsal hippocampus. After the sections were permeabilized with PBS/0.1% Triton × 100, the sections were incubated in the mixture anti-NeuN (mouse, Abcam, 1:1000) and anti-BDNF (rabbit, Abcam, 1:750) at 4°C overnight, and then followed by fluorescence secondary antibody, including DyLight 488 affiniPure goat anti-mouse IgG (green) and DyLight 594 affiniPure goat anti-rabbit IgG (red). Then, the sections were washed with PBS and mounted by DAPI. Finally, the pictures were captured by a Nikon A1 confocal laser scanning microscope system (Nikon Corp., Tokyo, Japan).
All data were presented as means ± standard deviation (SD) and analyzed with SPSS 17.0. To assess statistical differences among groups in escape latency and swimming distance, data were analyzed by two-way repeated measures ANOVA (day × group). One-way ANOVA was utilized to analyze data obtained from multiple groups. Least significant difference (LSD)
Deltamethrin and/or memantine did not exhibit any sign of maternal toxicity. There were no death of female rats in any of the treated groups. And the female rats treated with deltamethrin did not show any clinical signs of toxicity such as seizures, salivation, and tremors. The maternal weight gain was similar in all groups (
Weight gain of dams and parameters of rat pups following prenatal exposure to deltamethrin and/or memantine.
Control | 9 mg/kg DM | 2.7 mg/kg DM | 1.35 mg/kg DM | 0.54 mg/kg DM | 9 mg/kg DM +MEM | MEM | |
---|---|---|---|---|---|---|---|
Days 0–7 | 14.1 ± 3.08 | 13.71 ± 2.95 | 13.31 ± 2.79 | 13.95 ± 2.35 | 13.71 ± 2.97 | 13.32 ± 2.42 | 13.51 ± 2.42 |
Days 7–14 | 14.76 ± 5.54 | 13.84 ± 5.51 | 13.45 ± 4.75 | 14.53 ± 5.26 | 13.01 ± 4.48 | 14.40 ± 4.47 | 14.26 ± 5.33 |
Days 14–21 | 65.39 ± 15.2 | 63.68 ± 15.59 | 64.09 ± 14.76 | 64.33 ± 13.8 | 65.55 ± 16.48 | 65.68 ± 13.27 | 66.7 ± 16.48 |
Overall weight gain during pregnancy (%) | 40.31 ± 6.17 | 39.08 ± 6.18 | 38.77 ± 6.03 | 39.76 ± 5.49 | 39.36 ± 4.88 | 39.96 ± 4.4 | 40.32 ± 5.98 |
Number of litters | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
Litter size | 10.0 ± 1.41 | 8.5 ± 1.38 | 9.2 ± 1.47 | 9.7 ± 1.97 | 9.5 ± 1.87 | 9.0 ± 1.41 | 9.8 ± 1.17 |
Live birth index (%)a | 98.3 | 98 | 100 | 98.3 | 96.5 | 98.1 | 100 |
Weaning index (%)b | 96.7 | 98 | 96.4 | 98.2 | 100 | 100 | 96.7 |
PND 0 | 5.67 ± 0.41 | 5.98 ± 0.38∗∗ | 5.78 ± 0.43 | 5.61 ± 0.43 | 5.53 ± 0.45 | 5.54 ± 0.48 | 5.72 ± 0.41 |
PND 7 | 13.53 ± 0.78 | 13.74 ± 0.75 | 13.65 ± 0.71 | 13.32 ± 0.75 | 13.53 ± 0.67 | 13.57 ± 0.74 | 13.50 ± 0.77 |
PND 14 | 27.62 ± 1.37 | 28.12 ± 1.50 | 27.78 ± 1.49 | 27.48 ± 1.38 | 27.32 ± 1.37 | 27.38 ± 1.19 | 28.09 ± 1.62 |
PND 21 | 57.10 ± 4.21 | 52.67 ± 4.72 | 49.73 ± 4.87 | 50.48 ± 4.40 | 49.46 ± 4.91 | 51.24 ± 4.48 | 52.00 ± 4.39 |
The present study examined whether prenatal exposure to deltamethrin impairs cognitive abilities in the offsprings. Two-way repeated ANOVA analyses of the data from escape latency revealed that there were statistically significant differences for day [
The evaluation of learning and memory ability in MWM test. The results of MWM test are shown:
Data from swimming distance indicated that there were significant differences for day [
The spatial probe task was performed on day 6. The percentage of time spent in the target quadrant differed significantly between the seven groups [
Oral administration of different doses (9, 2.7, 1.35, and 0.54 mg/kg) of deltamethrin to pregnant rats produced a dose-dependent changes in GluN1 [
Western blot of protein expression of hippocampus GluN1, GluN2A, GluN2B
The expression of BDNF in neurons of CA1 region is shown in
Immunofluorescence analysis of BDNF expression in hippocampus. Representative confocal micrographs of BDNF expression in the CA1 region (BDNF staining shown in red, neurons shown in green, and DAPI counterstain in blue). Immunofluorescence staining for protein expression of BDNF in the CA1 area of hippocampus in different groups: control group, 9 mg/kg deltamethrin treated group, 2.7 mg/kg deltamethrin treated group, 1.35 mg/kg deltamethrin treated group, 0.54 mg/kg deltamethrin treated group, co-administered of 9 mg/kg deltamethrin and memantien group, memantine group. Scale bar = 200 μm. Data are mean ± SD.
In the present study, we investigate the effects of prenatal exposure to different doses of deltamethrin on cognitive abilities in F1-rats and the expression of NMDARs, BDNF, pCREB/CREB, and pTrkB/TrkB in hippocampus. Consistent with previous studies, we found that deltamethrin has a dose-dependent effect on cognitive functions of F1-rats born to deltamethrin-treated rats (
In this study, maternal weight gain was not affected by deltamethrin and/or memantine treatment, and no obvious adverse effects on pregnant rats were observed. These results are in good agreement with previous studies suggesting absence of maternal toxicity induced by deltamethrin exposure (
Morris water maze is a reliable test to evaluate cognitive functions in rodent animals and can objectively assess changes in spatial learning and memory abilities in offspring. Previous studies have suggested that prenatal exposure to deltamethrin induced locomotor effects in offspring (
It has been reported that the half life for deltamethrin in rat hippocampus was 38.50 h (
The NMDA receptor, a ligand-gated and voltage-dependent glutamate receptor, is composed of an obligatory subunit GluN1 and at least one of the regulatory subunits which include GluN2 (GluN2A, GluN2B, GluN2C, and GluN2D subtypes) and GluN3 (GluN3A and GluN3B subtypes). The different functional attributes of the NMDA receptors are decided by various NMDAR isoforms (
Brain derived neurotrophic factor, which plays a critical role in synaptic plasticity, neurodevelopment, and neuroprotection through NMDA receptor activation in the hippocampus, binds to tropomyosin-related kinase B (TrkB) receptor, activates intracellular signal transduction pathways, and strengthen synapses and thus improve learning and memory (
Memantine, a non-competitive, partial NMDAR antagonist, inhibits NMDA glutamate receptors to regulate the glutamatergic system and relieve cognitive and memory deficits in several diseases, such as Alzheimer’s disease, Parkinson’s disease, and ischemia (
In conclusion, we have found that exposure to a relatively high does of deltamethrin (9 mg/kg) alters cognition in offspring on postnatal day 21 and that this cognitive dysfunction can be ameliorated by memantine treatment. Moreover, NMDAR/BDNF signaling may be associated with the effects of prenatal exposure to deltamethrin on cognitive ability in offspring.
This study was carried out in accordance with the recommendations of Chinese Academy of Sciences. The protocol was approved by the Committee on the Ethics of Animal Experiments of Kunming Medical University.
CZ and YL formulated the research questions and designed the study. CZ, QX, and XZ conducted the study. WL and QK carried out the data analysis. CZ wrote the manuscript. TW and XX provided critical review and comments. YL was the supervisor of this research.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.