Decreases in the number of microglia and neural circuit dysfunction elicited by developmental exposure to neonicotinoid pesticides in mice

Neonicotinoids are insecticides widely used in the world. Although neonicotinoids are believed to be toxic only to insects, their developmental neurotoxicity in mammals is a concern. Therefore, we examined the effects of developmental exposure to neonicotinoids on immune system in the brain and post‐developmental behaviors in this study. Imidacloprid or clothianidin was orally administered to dams at a dosage of 0.1 mg/kg/day from embryonic day 11 to postnatal day 21. Imidacloprid decreased sociability, and both imidacloprid and clothianidin decreased locomotor activity and induced anxiety, depression and abnormal repetitive behaviors after the developmental period. There was no change in the number of neurons in the hippocampus of mice exposed to imidacloprid. However, the number and activity of microglia during development were significantly decreased by imidacloprid exposure. Imidacloprid also induced neural circuit dysfunction in the CA1 and CA3 regions of the hippocampus during the early postnatal period. Exposure to imidacloprid suppressed the expression of csf1r during development. Collectively, these results suggest that developmental exposure to imidacloprid decreases the number and activity of microglia, which can cause neural circuit dysfunction and abnormal behaviors after the developmental period. Care must be taken to avoid exposure to neonicotinoids, especially during development.


| INTRODUCTION
Organophosphorus pesticides and carbamate pesticides have been used in the world; however, their use is limited at present because their efficacy has declined by organism's resistance and because they have toxicological considerations. 1 Recently, neonicotinoids are the most common and widely used insecticides in the world instead. 2 Neonicotinoids are often applied as seed coatings in addition to other applications including foliar sprays, trunk injections, and soil drench.
Neonicotinoids have a similar chemical structure to nicotine and thus work as agonists for nicotinic acetylcholine receptors (nAChRs) especially in invertebrates.The nAChRs are ligand-gated ion channels that allow small cations such as Na + , K + , and Ca 2+ to pass through when they are activated.Stimulation of nAChRs induces neuromuscular paralysis and ultimately causes death.Because the affinity of neonicotinoids for mammalian nAChRs is much lower than that for invertebrate, it is believed that neonicotinoid insecticides show less toxicity to mammalian compared with organophosphorus and carbamate pesticides. 3onicotinoids are persistent chemicals in water and soil owing to their relatively long half-lives ranging from 1 day to almost 19 years. 4Therefore, neonicotinoids present a potential environmental health concern.Because neonicotinoids have the small molecular weight and are soluble in water, they reach all parts of plants, which treated with neonicotinoids. 4Humans are continuously exposed to neonicotinoid insecticides by consuming food, drinking water, and through dermal contact.In fact, seven neonicotinoids, that is, acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam, were detected in urine in a Japanese population. 5 et al. estimated by Monte Carlo simulation that the maximum chronic daily intake of total neonicotinoids from water consumption in adults and children was 10.2 and 12.4 ng/kg/day, respectively. 6 described above, neonicotinoids are believed to be less toxic to mammals because of the structural difference in nAChRs between mammals and insects. 1 However, several reports suggest that neonicotinoids affect mammals, including humans.Neonicotinoid exposure may be associated with dizziness, headache, memory loss and finger tremor. 7Importantly, neonicotinoids are able to enter the brain in mammals. 8Clothianidin and its metabolites were found in the brain after intraperitoneal administration of clothianidin at a dose of 20 mg/kg. 9Imidacloprid was also detected in the brain at the concentrations of 0.3 and 0.5 μg/g after imidacloprid exposure for 28 days at doses of 0.8 and 2.25 mg/kg/day, respectively. 4e developing brain is vulnerable to chemicals because of immaturity of the blood-brain barrier.In addition, the effects of chemicals continue until after the developmental period as the cells in the developing central nervous system undergo differentiation and maturation.Burke et al. reported that after treatment of rats with 0.5 mg/kg/day imidacloprid during pregnancy and in the early postnatal stage, imidacloprid was detected at concentrations of 1.18 pmol/g in mothers and 0.044 pmol/g in offspring, respectively, 10 indicating that imidacloprid is transported to the fetal brain when administered during pregnancy.
There are several reports that developmental exposure to neonicotinoids induces abnormal behaviors after the developmental period.
Acetamiprid exposure during neonatal stage was found to interfere with the development of the neural circuit function required for sociosexual and anxiety-related behaviors. 11Developmental exposure to imidacloprid resulted in lower triglyceride levels, increased motor activity, enhanced social dominance, decreased depressive-like behavior, and reduction of social aggression in mice. 10croglia are immune cells in the brain that play crucial roles in neurological pathologies such as synaptic loss in Alzheimer's disease and neuroinflammation in secondary epilepsy. 12,13Microglia are also involved in neuronal circuit formation during development.Microglia are relatively activated in developmental stages and can engulf spines and synapses with low activity to refine neural circuit function. 14Oral administration of acetamiprid at a dose of 5 mg/kg/day from embryonic day (E) 6 to E18 to pregnant dams reportedly induced increases in the numbers of amoeboid-as well as M1-type microglia in offspring. 15Excess activation of microglia during development is able to interfere with the formation of neuronal circuit function and induce abnormal behaviors after the developmental period. 16On the basis of this knowledge, we assessed neonicotinoid-induced developmental neurotoxicity from the perspective of microglia.Pregnant ICR mice were purchased from Japan SLC (Shizuoka, Japan) and were maintained in a temperature-controlled animal facility on a 12-h light-dark cycle (light on from 8:00 a.m. to 8:00 p.m.).Pregnant female mice received imidacloprid (Wako Pure Chemical, Osaka, Japan) and clothianidin (Wako Pure Chemical) via drinking water at a dosage of 0.1 mg/kg/from E11 to postnatal day (P)21 (imidaclopridtreated and clothianidin-treated groups).These doses were determined by reference to the acceptable daily intake presented by the Food Safety Commission of Japan (0.057 mg/kg/day for imidacloprid and 0.097 mg/kg/day for clothianidin).Imidacloprid and clothianidin were dissolved in water at the appropriate concentration, which was determined based on each dam's body weight and drinking volume.

| MATERIALS AND METHODS
Maternal drinking water consumption was approximately 5 mL/day immediately after birth, increased as the pups grew larger, and was around 15 mL/day just before weaning.Vehicle controls received waters during experiments.Dams drank waters ad libitum.
Four to five mice were housed in the same cage from P22.We used a simple randomization method to divide the mice into groups. 17mple size of behavioral tests and other experiments was determined by our previous report. 16Twenty-nine dams and 108 male pups were used in this study.

| Behavioral tests
Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test, marble burying test and forced swim test from the age of 6 weeks and were conducted one test per week. 16In the open field test, the mice were released into an open field arena (90 Â 90 Â 50 cm) and then were monitored for 90 min.
The distance moved and the time spent in the center area were calculated by DIPP-Motion V/2D software (DITECT, Tokyo, Japan).The Y-maze test was performed using an apparatus with three arms (20 cm long Â 10 cm wide Â 20 cm high) at 120 angles.The mice were placed in the maze for 8 min.The percentage of alternations (entries into an arm that differed from the arm entered in the previous two entries) was calculated.A social affiliation test was performed in a rectangular apparatus (20 cm long Â 40 cm wide Â 40 cm high).
Empty wire containment cups were placed on the right and left sides 8 cm from the wall, and the mice were habituated to the apparatus for 5 min.A control mouse (stranger) was placed in a wire containment cup that was located on one of the sides of the apparatus, and then the mice were placed in the apparatus for 10 min.The amount of time spent around each cage (empty cage or stranger containing cage) was measured.The marble burying test was performed in a mouse cage (22 cm long Â 32 cm wide Â 15 cm high), in which 20 glass marbles (diameter 1.2 cm) were spaced evenly on a 2-cmdeep layer of shaved wood bedding.The mice were placed in the cage and left undisturbed for 30 min.After the test, the number of marbles buried (covered more than two-thirds with bedding) was counted.For the forced swim test, a clear glass cylinder with a diameter of 25 cm was filled with water (21-23 C) to a depth of 15 cm and then mice were placed in it.The mice were recorded for total 6 min in each test, and the immobility time was measured in the last 4 min of the test. 18e animals were euthanized after behavioral tests.

| Nissl staining
Mice were anesthetized with isoflurane.The brain was reperfused from the left ventricle with PBS and then 4% paraformaldehyde.The brain was isolated and put into 30% sucrose solution, and then was frozen in powdered dry ice.Brain sections (10 μm) including hippocampus area were prepared using a Cryostat (CM3050 S; Leica Biosystems, Nussloch, Germany).These sections were stained with 0.5% Cresyl violet acetate, rinsed in distilled water, dehydrated, differentiated and coverslipped with Permount™ Mounting Medium (Fisher Scientific Inc., Tokyo, Japan).Stained cells were counted under a light microscope.

| Immunohistochemistry
Mice were anesthetized with isoflurane.The brain was reperfused from the left ventricle with PBS and then 4% paraformaldehyde.The brain was isolated and cryoprotected in 30% sucrose, and then was frozen in powdered dry ice, and 50-μm-thick floating sections including hippocampus area were prepared using a Cryostat (CM3050 S; Leica Biosystems).The brain sections were stained with Iba1, CD68, and DAPI to calculate the number of microglia, the microglial soma area and CD68 expression in microglia, according to our previous reports. 19,20The antibodies used in this study were listed in Table 1.Stained sections were observed laser-scanning microscopy (LSM700; Carl Zeiss, Oberkochen, Germany).Images were analyzed with Zen (Carl Zeiss) and ImageJ software (National Institutes of Health, Bethesda, MD).

| Neural activity imaging using voltagesensitive dye
Neural activity was evaluated by voltage-sensitive dye (VSD) imaging according to our previous reports 16 .Acute hippocampal slices were loaded with di-4-ANEPPS (Thermo Fisher Scientific, Waltham, MA).
The changes in fluorescence (Ex: 530±10 nm, Em: >590 nm) were recorded by a C-MOS imager (MiCAM-02; Brainvision Co Ltd, Tokyo, Japan) when electrical stimulations under the condition of (40 V, bipolar and 200 μs duration) were applied to Schaffer collateral afferents in the CA3/CA1 border of CA1 region, in the granule cell layer to stimulate the mossy fiber pathway, or in the molecular layer of the upper blade of the dentate gyrus (DG).

| Evaluation of the microglial population and CD68 expression
Microglia were separated from macrophages to measure their CD68 expression using the method that we previously reported. 21Briefly, mouse hippocampus was isolated under isoflurane anesthesia and was minced and digested by the mixture of collagenase (Sigma-Aldrich) and DNase I (Sigma-Aldrich).Resulted cell suspension was separated with Percoll (GE Healthcare, Chicago, IL).The collected cells from the boundary between 37% and 70% Percoll were used for staining of propidium iodide, CD11b, CD45, and CD68, followed by flowcytometry analysis.Antibodies used in this study were listed in Table 1.Cells stained with propidium iodide were dead cells and thus were excluded from analysis.CD11b + /CD45 low cells were considered microglia, and CD11b + /CD45 high cells were considered myeloid infiltrates, macrophages.The CD68 fluorescence intensity in microglial

| Determination of imidacloprid concentration in the plasma and brain
Imidacloprid concentration was measured according to previous report. 23Plasma (50 μL) were mixed with 200 μL of acetonitrile.The mixtures were vortexed and centrifuged at 20 000 Â g for 10 min.
Resulting supernatants were dried under nitrogen gas and resuspended in 10% acetonitrile (90% water) containing 0.1% formic acid to prepare LC-MS/MS samples.Plasma added by 5 ng imidacloprid was used for calculating recovery rate.Mouse whole brains (100 mg) were homogenized in 1 mL of 85% methanol (15% water).
Homogenates were placed at À80 C for 20 min to easily remove fats derived from the brain tissue.The homogenates were centrifuged for 20 000 Â g for 15 min and then 500 μL of the supernatants were removed and dried under nitrogen gas.Resulting pellets were resuspended in 100 μL of 10% acetonitrile (90% water) containing 0.1% formic acid to prepare LC-MS/MS samples.Brain homogenates added by 10 ng imidacloprid was used for calculating recovery rate.
The samples were analyzed using ultra-performance liquid chromatography coupled to a tandem quadrupole mass spectrometer (Acquity TQD LC/MS/MS, Waters, Milford, MA) operated under positive ion mode.All compounds were separated using Acquity UPLC BEH C18 column (50 mm Â 2.1 mm, 1.7 μm, Waters).The analytes (5.0 μL) were injected and the following gradient method was used for analysis at a flow rate of 0.2 mL/min, using water (phase A) and acetonitrile (phase B) containing 0.1% formic acid: the initial conditions (90% A) decreased to 0% A by 5 min, holding until 6 min, and then returned to initial conditions and held for 1 min.Electron spray ionization was selected as mass spectrometry ion source.Other parameters of the mass spectrum were set as follows: the desolvation gas temperature was 350 C, the desolvation gas flow rate was 10 L/ min, the capillary voltage was set to 3000 V, and parent ion and product ion of imidacloprid were set to 256.1 and 175.1 (m/z), respectively.Imidacloprid standard for pesticide residue analysis (Wako Pure Chemical) was used for preparation of calibration curve.The limit of detection of imidacloprid in this system was determined as 5 pg (1 ng/mL).

| Abnormal behaviors induced by developmental exposure to imidacloprid and clothianidin
Imidacloprid and clothianidin are major neonicotinoids used in Japan, and the acceptable daily intakes were determined to be 0.057 and 0.097 mg/kg, respectively, by the Food Safety Commission of Japan.
Therefore, mice were exposed to these neonicotinoids at dosages of 0.1 mg/kg/day, a similar dose to the acceptable daily intakes, in this study.The locomotor activity of the offspring of dams orally administered 0.1 mg/kg/day imidacloprid or clothianidin from E11 to P21 was significantly decreased compared with that of the offspring of vehicle-treated animals at 6 weeks of age (Figure 1A).The time spent in the center was also decreased in the imidacloprid-and clothianidintreated groups (Figure 1B), suggesting increased anxiety in the mice.
Spatial learning memory was not affected by imidacloprid or clothianidin, as confirmed by the Y-maze test (Figure 1C).Administration of imidacloprid, but not clothianidin, impaired social interaction (Figure 1D).The number of buried marbles was decreased in the groups treated with imidacloprid and clothianidin (Figure 1E), indicating abnormal repetitive behaviors (Figure 1E).The increase in immobility time in the imidacloprid-and clothianidin-treated groups, as evaluated by the forced swim test, indicated increased depressive-like behaviors (Figure 1F).
The developmental neurotoxicity of imidacloprid was stronger than that of clothianidin, because developmental exposure to imidacloprid elicited abnormal sociability as well as anxiety and depression.5][26] Therefore, we evaluated the effect of developmental imidacloprid exposure on the hippocampus.

| Decreases in microglia and neural excitation during development induced by imidacloprid exposure
We first examined the number of neurons in the hippocampus of mice exposed to imidacloprid.The results of Nissl staining indicated that the number of neurons in the CA1, CA3, and DG regions of the hippocampus decreased with age (Figure 2).Developmental exposure to imidacloprid did not show any effect on neuronal number in the hippocampus (Figure 2).Then, we focused on brain immune cells, microglia.
Microglia Collectively, these results suggest that developmental exposure to imidacloprid decreases microglial number and activity in the hippocampus.
Recently, it was reported that there are several types of macrophages in the brain involving in brain homeostasis and disease. 27,28us, we separated microglia from macrophages by CD11b/CD45 staining to measure the number and activity of microglia and macrophages.The ratio of microglia to macrophages showed no change between the vehicle-and imidacloprid-treated groups at P10 (Figure 4A-C).CD68 expression decreased in the microglial

| Changes in Csf1r expression in the hippocampus of mice exposed to imidacloprid
Several genes have been reported to be involved in microglial proliferation and survival, such as trem2 and p2 Â 7r. 29,30Among these, Csf1r is one of the major factors that controls microglial proliferation and survival during development. 31Csf1r expression increased from P1 to P10 in the hippocampus (Figure 6).Imidacloprid exposure significantly suppressed csf1r expression at P5 and P10 (Figure 6).Therefore, decreases in csf1r expression might be involved in the loss of microglia during development in the hippocampus of mice treated with imidacloprid.

| Imidacloprid concentration in the brain of pups and maternal plasma
Finally, imidacloprid concentration in the brain of male pups (1, 5, 10, and 70 days after birth) and the plasma of dams (day 18 of pregnancy) was measured using LC-MS/MS.Imidacloprid concentration of maternal plasma was approximately 140 ng/mL (Figure 7).The imidacloprid concentration in the brains of male pups was maintained at approximately 100 ng/g brain during lactation, which was similar to that in maternal plasma.However, imidacloprid concentration in the brain of pups decreased to below the detection limit 70 days after birth (Figure 7).In this study, developmental exposure to imidacloprid and clothianidin induced loss of locomotor activity after the developmental period.
Abd-Elhakim et al. revealed that adult rats orally administered 1 mg/kg/day imidacloprid for 60 days showed less exploratory activity. 32Adult mice exposed to 5 mg/kg clothianidin have been reported to show decreased locomotor activity, as measured by the open field test. 33Therefore, a decrease in locomotor activity is considered to be a common phenomenon in animals exposed to neonicotinoids.Nicotine is a cholinergic agonist and is reported to cause a decrease in, but not complete loss of, locomotor activity. 34Because nAChRs on dopaminergic neurons regulate motor behaviors, neonicotinoids might act on mammalian nAChRs to elicit similar behaviors as nicotine.However, mice were exposed to imidacloprid and clothianidin from E11 to P21 in our study, decreased locomotor activity was detected at the age of 6 weeks old, indicating a time lag between neonicotinoid exposure and the consequent effects.Because nAChRs can be desensitized by sustained stimulation, 35 relationships between nAChR dynamics and neonicotinoid action should be investigated.We showed here that prenatal exposure to imidacloprid and clothianidin indued anxiety-like behaviors evaluated by open field tests.Acute oral exposure to clothianidin and in utero and lactational exposure to acetamiprid are reported to elicit anxiety-related behaviors in mice. 11,36Importantly, while nicotine exposure at a dose of 0.5 mg/kg from E10 to birth reduced anxiety-like behaviors in male offspring, 37 mice whose mothers were exposed to nicotine by drinking water (0.2 mg/mL) throughout gestation or from E14 to birth increased anxiety-related behaviors, 38 suggesting that nicotine-induced anxietylike behaviors can be affected by experimental conditions, such as the dose, exposure duration and time windows.On the other hand, when the ratio of total distance traveled and time spent at the center was calculated, there is no difference among vehicle, imidacloprid and clothianidin groups.Therefore, the results of open field test may reflect reduced motor functioning rather than a direct effect on emotional behavior.Future studies should examine the effects of neonicotinoids on locomotion and emotion, including the role of nAChRs.
In this study, the number of buried marbles decreased in the groups treated with imidacloprid and clothianidin, suggesting that imidacloprid-treated group, the number of buried marbles decreased by approximately 90% in imidacloprid-treated group compared with vehicle-treated group.Therefore, the reduction rates were not consistent.In this regard, results in marble burying tests cannot be explained solely by a decrease in locomotor activity.We used forced swim test to analyze depressive-like behaviors.Recently, some reports mentioned that the forced swim test reflect adaptive ability for the forced swim stressor. 39,40Therefore, mice prenatally exposed to imidacloprid or clothianidin might have defects in adaptive ability.However, because stress coping ability can be involved in depression, more analysis was needed to reveal the involvement of prenatal exposure to imidacloprid or clothianidin in adaptive/depressive behaviors.Collectively, neonicotinoid exposure in fetus can cause several behavioral abnormalities after growth.
Microglia are necessary to form proper neuronal networks.They engulf synapses and spines that do not receive inputs from synaptic contacts during postnatal development, and then, eliminate redundant neurons that do not establish functional circuits. 41Considering these important roles of microglia during development, decreases in the number of microglia can critically impair brain development.Microglial depletion by intracerebroventricular infusion of clodronate liposomes in the neonatal period was shown to increase novelty exploration and social avoidance, and induce learning disability after growth. 42Specific depletion of cerebellar microglia by genetic Csf1 depletion in neural progenitor cells was found to produce defects of motor learning and social interaction in juvenile period. 43In this study, imidacloprid decreased sociability and induced anxiety, depression and abnormal repetitive behaviors.In addition, imidacloprid decreased the number of microglia during development.In this regard, the abnormal behaviors induced by imidacloprid might be due to a decreased number of microglia.Importantly, microglia depletion during late postnatal development, from P23 to P45, in mice does not affect social interaction in adults. 44In addition, imidacloprid concentration in the brain of pups was reduced after weaning and was not detected in 70 days old pups, indicating that developmental exposure to imidacloprid affects behaviors after growth.Nakayama et al showed that mice administered 5 mg/kg/ day imidacloprid or nicotine by oral gavage from P12 to P26 presented an increment of the number of amoeboid-and M1-type microglia. 45erefore, the effects of imidacloprid on microglia and behaviors, at least in part, can be affected by the exposure period and dose.
CSF-1R is expressed at higher levels in monocytes and tissue macrophages, including microglia. 46The known ligands for CSF-1R are CSF-1 and IL-34, which can activate kinase signaling via MEK and PI3K pathways downstream of CSF-1R, which contribute to cellular proliferation.In this study, csf1r was downregulated by developmental exposure to imidacloprid.This might be a mechanism by which the number of microglia was decreased by imidacloprid administration.
DNA methylation was found to be significantly increased in human neuroblastoma SH-SY5Y cells exposed to imidacloprid at a concentration of 100 μM. 47Exposure to imidacloprid was also found to induce DNA methylation in mouse embryonic stem cells. 48Therefore, epigenetic modification might be involved in changes in csf1r expression by developmental exposure to imidacloprid.
The nAChRs transfer signals between neurons in the central nervous system and peripheral nerves via synaptic transmission at vertebrate neuromuscular junctions.The nAChR forms a pentamer composed of various subunits, such as α1 to α10 and β1 to β4. 49 Expression of α7 nAChR is detected in the hippocampus and frontal cortex, which are related to cognition and memory, and intereatingly α7 nAChR is reported to be expressed microglia. 50Microglial α7 nAChRs are considered to have anti-inflammatory and neuroprotective effects. 51Activation of α7 nAChR stimulates the Jak2/STAT3 pathway, which inhibits nuclear translocation of NF-κB and activates the master regulator of oxidative stress Nrf-2 to suppress inflammatory reactions, while elevation of intracellular Ca 2+ levels and evoked Ca 2+ signals can reportedly activate microglia. 52Loser et al. reported that acetamiprid, imidacloprid, clothianidin and thiacloprid at the concentrations of 10-100 μM triggered Ca 2+ signaling in an α7 nAChR-dependent manner. 53However, a much lower dose was detected in the brains of fetuses exposed to neonicotinoids during gestation. 4Collectively, it is important to carefully consider the involvement of nAChRs in neonicotinoid-induced developmental neurotoxicity by evaluating their ADME and effect on Ca 2+ flux.
A recent report indicated that neonicotinoid insecticides, including imidacloprid, were detected in human blood. 54They examined 115 blood samples and interestingly, the neonicotinoid insecticide with the highest detection rate in red blood cells was thiamethoxam, F I G U R E 6 Downregulation of Csf1r expression by developmental exposure to imidacloprid.Imidacloprid (IMI) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P10.RNA was extracted from the hippocampus of P1, P5, and P10 male pups, and then qPCR was performed to determine Csf1r mRNA levels.The reported values are the mean ± S.D. (n = 5 male pups from 2 dams in each group).The data were analyzed using Student's t test.
which was found in 19.1% of samples; however, the neonicotinoid with the highest detection rate in plasma was imidacloprid, which was found in 26.1% of samples.This result indicates that the distribution of neonicotinoids in the body is dependent on their chemical structures.A metabolite of imidacloprid, olefin-imidacloprid, was also detected in human blood and urine. 55Further study is needed to reveal the mechanism by which neonicotinoids and their metabolites are transported into the brain.
In conclusion, we have shown in this study that sustained exposure to low doses of neonicotinoids during development decreases the number and activity of microglia, which can cause neural circuit dysfunction and abnormal behaviors after the developmental period.
Neonicotinoid pesticides are used worldwide, and humans are exposed to neonicotinoids via water, foods, soil and air.Special attention should be given to the effects of exposure to neonicotinoids during fetal and infancy.
F I G U R E 7 Imidacloprid concentration in the brain of pups and maternal plasma.Imidacloprid (IMI) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P21.The whole brain was collected from male pups at the age of 1, 5, 10, and 70 days old.Plasma was isolated from dams (day 18 of pregnancy).Imidacloprid was extracted from these samples and then imidacloprid concentration was determined by LC-MS/MS.The reported values are the mean ± S.D. (n = 3 animals in each group).Total 12 male pups and 3 dams were used in this experiment.N.D: not determined.

2. 1 |
Neonicotinoid exposure to mice All animal procedures were performed in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the Jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan.The Animal Care and Use Committee of Hiroshima University approved the experimental protocols (No.C22-13).
figure legend, and the p value is indicated in each figure; differences were considered significant when p values were less than .05.

T A B L E 2
Primers used for qPCR.Name Sequence (5 0 -3 0 ) Mouse β-Actin-For CTAGGCACCAGGGTGTGATG Mouse β-Actin-Rev GGGGTACTTCAGGGTCAGGA Mouse Csf1r-For ATGTGTGGTCCTACGGCAT Mouse Csf1r-Rev GGTAGGGGTTCAGACCAAGC play an important role in the formation and maturation of neural circuits during development.Microglia increase in number and are relatively activated during development and then contribute to neuronal proliferation and differentiation, and then pruning of excess synapses to induce remodeling of neural circuits.Immunohistochemical staining for Iba1, a marker for microglia, indicated that the number of microglia increased from P1 to P10 in the CA1, CA3, and DG regions of the hippocampus (Figure3A-D).Imidacloprid administration significantly decreased the number of hippocampal microglia, especially at P10 (Figure3A-D).Microglial activity, which was evaluated by Iba1/CD68 staining, was significantly decreased in the CA1 and CA3 regions in P10 mice exposed to imidacloprid (Figure3A, E-J).

F I G U R E 1
Abnormal behaviors induced by developmental exposure to imidacloprid or clothianidin.Imidacloprid (IMI) or clothianidin (CLO) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P21, and male pups were applied to behavior tests.(A) Locomotor activity was measured by the open field test at the age of 6 weeks.The reported values are the mean ± S.D. (n = 10 male pups from 3 dams in each group).The area under the curve (AUC) was calculated, and the AUC data were analyzed using one-way ANOVA [F(2, 27) = 10.87,p = .0003]followed by Dunnett's multiple comparisons test.The P values for the vehicle versus imidacloprid groups and vehicle versus clothianidin groups were .0005and .0015,respectively.(B) The time spent in the center area was evaluated by the open field test.The reported values are the mean ± S.D. (n = 10 male pups from 3 dams in each group).The data were analyzed using one-way ANOVA [F(2, 27) = 4.177, p = .0263]with Dunnett's corrected multiple comparison test.(C-E).The number of alternations, the exploration time and the number of buried marbles were evaluated by the Y-maze, social affiliation and marble burying tests, respectively, at the age of 7-9 weeks.The reported values are the mean ± S.D. (n = 10 male pups from 3 dams in each group).(C) and (E) The data were analyzed using one-way ANOVA [C: F(2, 27) = 0.003, p = 0.997, E: F(2, 27) = 38.08,p < .0001]with Dunnett's corrected multiple comparison test.(D) The data were analyzed using Student's t test.(F) Depressive-like behavior was evaluated by the forced swim test at the age of 10 weeks.The reported values are the mean ± S.D. (n = 10 male pups from 3 dams in each group).The data were analyzed using one-way ANOVA [F(2, 27) = 14.57, p < .0001]with Dunnett's corrected multiple comparison test.population (Figure 4D) but not in the macrophage population (Figure 4E) after imidacloprid exposure, confirming microglial inactivation by imidacloprid.The hippocampal neuronal responses of the three major synaptic pathways, Schaffer collateral afferents projecting from CA3 to CA1, mossy fibers projecting from the DG to CA3, and the perforant pathway connecting the EC to the DG, were measured upon electrical stimulation.There was no difference in the time to peak signal between the vehicle-and imidacloprid-treated groups (Figure 5A,B).Developmental imidacloprid exposure significantly elevated excitatory activity, especially by electrical stimulation of Schaffer collaterals and mossy fibers, in P10 mice (Figure 5A,C).Collectively, these results suggest that imidacloprid exposure during development causes dysfunction of neural circuit as well as behavioral disorders after development.

F I G U R E 2
Evaluation of the number of neurons in mice exposure to imidacloprid during development.Imidacloprid (IMI) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P21.Brains were isolated from male pups at the ages of 1, 5, 10, and 70 days, followed by Nissl staining.(A) Representative images of the hippocampal CA1, CA3, and DG regions of 70-day-old mice.(B-D) The number of neurons in the CA1, CA3, and DG regions was measured by Nissl staining.The reported values are the mean ± S.D. (n = 3 male pups from 2 dams in each group).
repetitive behavior was suppressed by administration of imidacloprid and clothianidin.According to the results of the open field test, locomotor activity in the groups of imidacloprid and clothianidin decreased 30 min after putting them in the field.Therefore, it is possible that the reduction of the locomotor activity is involved in decreases in the number of buried marbles.However, while the total distance traveled was reduced by approximately 30% in F I G U R E 3 Effects of developmental imidacloprid exposure on the number and activity of microglia.Imidacloprid (IMI) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P21.Brains were isolated from male pups at the ages of 1, 5, 10, and 70 days, followed by immunohistochemical staining of Iba1 and CD68.(A) Representative images of the hippocampal CA1 region of 1-, 5-, 10-, and 70-day-old mice.(B-D) Time-dependent changes in the number of microglia in the CA1, CA3, and DG regions, as determined by Iba1/DAPI staining.(E-G) Time-dependent changes in the cell bodies of microglia in the CA1, CA3, and DG regions, as measured by images stained by Iba1/ DAPI.(I-K).Time-dependent changes in CD68 expression in microglia in the CA1, CA3, and DG regions, as evaluated by Iba1/CD68 staining.The reported values are the mean ± S.D. (n = 5 male pups from 2 dams in each group).The data were analyzed using Student's t test.

F I G U R E 4
Analysis of microglia in mice exposed to imidacloprid by flow cytometry.Imidacloprid (IMI) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P10.Brains were isolated from male pups at the age of 10 days old, and then dispersed brain cells were analyzed by flow cytometry.(A) Representative flow cytometry plots of CD11b and CD45.The left panel shows data from vehicletreated pups, and the right panel shows data from imidacloprid-treated pups.(B) and (C) The ratio of microglia to macrophages was compared between vehicle-and imidacloprid-treated groups.(D) and (E) The levels of CD68 expression in the microglial (D) and macrophage (E) fractions were measured in the vehicle-and imidacloprid-treated groups.The reported values are the mean ± S.D. (n = 4 male pups from 2 dams in each group).The data were analyzed using Student's t test.

F I G U R E 5
Neuronal excitation-inhibition imbalance induced by developmental exposure to imidacloprid.Imidacloprid (IMI) was administered to dams via drinking water at a dosage of 0.1 mg/kg/day from E11 to P10.Hippocampus was isolated from 10-day-old male pups and acute slices were prepared.The electrical stimulation was applied to Schaffer collateral afferents at the CA3/CA1 border in the CA1 region, the granule cell layer to stimulate the mossy fiber pathway, and the molecular layer of the upper blade of the DG.(A) Representative pseudocolored images of neuronal activity in the CA1 region.(B) Measurement of the time to peak signals.(C) Quantification of the neural response.The reported values are the mean ± S.D. (n = 10 male pups from 3 dams in each group).The data were analyzed using Student's t test.
Antibodies used in this study.
2.7 | RNA extraction and quantitative PCRof primers used in this study are listed in Table2.Csf1r mRNA levels were normalized to the β-actin mRNA levels to calculate the relative mRNA expression.