A single dose of clothianidin exposure induces varying sex-specific behavioral changes in adulthood depending on the developmental stage of its administration

Abstract

Clothianidin (CLO), a neonicotinoid that is widely used in forests and agricultural areas, was recently reported to cause toxicity in mammals. Although sensitivity to chemicals varies between sexes and developmental stages, studies that comprehensively evaluate both males and females are limited. Therefore, in this study we utilized murine models to compare the sex-specific differences in behavioral effects following CLO exposure at different developmental stages. We orally administered CLO to male and female mice as a single high-dose solution (80 mg/kg) during the postnatal period (2-week-old), adolescence (6-week-old), or maturity (10-week-old), and subsequently evaluated higher brain function. The behavioral battery test consisted of open field, light/dark transition, and contextual/cued fear conditioning tests conducted at three and seven months of age. After the behavioral test, the brains were dissected and prepared for immunohistochemical staining. We observed behavioral abnormalities in anxiety, spatial memory, and cued memory only in female mice. Moreover, the immunohistochemical analysis showed a reduction in astrocytes within the hippocampus of female mice with behavioral abnormalities. The behavioral abnormalities observed in female CLO-treated mice were consistent with the typical behavioral abnormalities associated with hippocampal astrocyte dysfunction. It is therefore possible that the CLO-induced behavioral abnormalities are at least in part related to a reduction in astrocyte numbers. The results of this study highlight the differences in behavioral effects following CLO exposure between sexes and developmental stages.

INTRODUCTION

Insecticides are indispensable tools in everyday life, with many types of insecticides having been developed over the years. However, certain types of insecticides, such as DDT, have been pointed out for their toxicity toward mammals and are prohibited by law in many countries, including Japan.

Neonicotinoid insecticides (neonicotinoids) are a class of insecticides developed in the 1980s that are widely used worldwide. Neonicotinoids act as agonists of the insect nicotinic acetylcholine receptor (nAChR), inducing excessive excitatory transmission and leading to insect death (Calas-List et al., 2013). Neonicotinoids are considered safe insecticides because of their markedly higher affinity for insect nAChRs than for reports have indicated that neonicotinoids can cross the mammalian blood-brain barrier and demonstrate a higher affinity for mammalian nAChRs than originally thought (Ford and Casida, 2006; Kimura-Kuroda et al., 2012; Li et al., 2011). Although nAChRs are expressed in various organisms, many studies have suggested that exposure to neonicotinoids has adverse effects on the mammalian central nervous system, resulting in behavioral abnormalities (Albuquerque et al., 2009; Abreu-Villaça and Levin, 2017). Clothianidin (CLO), a neonicotinoid commonly sprayed over forests and agricultural areas, is known for its neurotoxic effects on mammals (Ikenaka et al., 2019). Recent research revealed that acute CLO exposure induces neurotoxicity as a behavioral abnormality (Kubo et al., 2022). Overall, the interest in neonicotinoid neurotoxicity, including that of CLO, has been steadily gaining increased attention.

Brain plasticity differs greatly at different developmental stages (Kolb and Gibb, 2011). A representative example of a change in brain structure is synapse elimination, which occurs from fetal development through the early postnatal stages. Neurotransmitter inputs play a significant role (Kano and Hashimoto, 2009), and CNS development is sensitive to environmental insults (Rice and Barone, 2000). Therefore, the disruption of synaptic transmission during developmental stages can disturb the normal development of brain functions (Rice and Barone, 2000). In regard to neonicotinoids, imidacloprid exposure during development reportedly impairs learning and memory in adulthood (Saito et al., 2023). Considering that CLO is used as a wood preservative, as well as in food crops, and given that multiple generations of people are exposed to CLO, understanding the differences in the effects of CLO on behavior based on developmental stages is essential.

However, most studies on the neurotoxicity of CLO have targeted only males, with limited insights regarding the neurodevelopmental toxicity in females. There are significant sex differences in brain development due to hormonal, genetic, epigenetic, and other sex-specific factors (McCarthy and Arnold, 2011). Moreover, there are also a number of sex-based differences in the prevalence of developmental disorders, such as attention deficit hyperactivity disorder, and autism spectrum disorder (Ramtekkar et al., 2010; Loomes et al., 2017), with susceptibility to neurological disorders being as much as 2- to 5-fold greater in one sex than in the other (Weiss, 2011). Additionally, many reports have indicated varying behavioral toxicities due to chemical exposure depending on sex (Weiss, 2011; Caldarone et al., 2008). Given these findings and the high susceptibility of the brain to chemical exposure during the developmental period, it is possible that CLO exposure during development may cause differences between males and females. Therefore, it is imperative to observe the toxic effects of CLO in females as well.

In this study, we aimed to compare the differences in the behavioral effects following CLO exposure between males and females and the developmental stage by administering a single dose of CLO to male and female mice during the postnatal period, adolescence, or maturity, and subsequently evaluating higher brain functions.

MATERIALS AND METHODS

Animals and treatments

An outline of the experiment is shown in Fig. 1. Male and female C57BL/6N mice at postnatal day (P)-10 were purchased from Japan SLC (Shizuoka, Japan). To prepare the CLO solution, we dissolved CLO (Wako, Osaka, Japan) in 0.5% (w/v) methyl cellulose (MC; Wako) in acetone (MC and acetone at a 4:1 ratio). To eliminate the risk of adverse effects associated with acetone, only the minimal amount of acetone was used. The dose of CLO was set at 80 mg/kg, based on the NOAEL (ICR female mice: 65.1 mg/kg/day) for chronic administration, anticipating scenarios where exposure to high concentrations of CLO might be a possibility due to accidents (Food and Agriculture Organization of the United Nations, 2011). CLO solution was treated at two weeks of age (2w-CLO I), six weeks of age (6w-CLO I), and ten weeks of age (10w-CLO I), respectively. The 2w-CLO II, 6w-CLO II, and 10w-CLO II groups received the same treatments. Mice in both the vehicle control group I (VC I) and vehicle control group II (VC II) were treated with solvent at 2, 6, and 10 weeks of age by gavage. The mouse behavioral battery test (BBT) was conducted at 3 months of age in group I and at 7 months of age in group II. We attempted to control the noise levels in the animal room and surrounding areas to reduce animal stress. Housing conditions were maintained at a consistent temperature of 24 ± 1°C and humidity at 60 ± 10% with a 12 hr light/dark cycle. Mice were provided free access to food (MF; Oriental Yeast Co., Ltd. Tokyo, Japan), and water. All animal care and experimental procedures followed the protocols approved by the Tohoku University Institutional Animal Care and Use Committee (2019Nodo-004-01).

Fig. 1

Experimental design. Gray: administration of vehicle; Red: administration of CLO-dissolved solution.

Mouse behavioral battery test

We conducted the BBT consisting of open field test (OF), light/dark transition test (LD), and contextual/cued fear conditioning test (FZ). Our OF, LD, and FZ methods were based on a previous study (Sasaki et al., 2021). A concise summary of the behavioral tests is presented below:

OF test: Locomotor activity was measured for 10 min using an open-field apparatus made of white plastic and measuring 50 × 50 × 30 (H) cm. The LED light system was positioned approximately 50 cm above the center of the field, providing 25 lx at the center. To monitor the mice’s behavior, we used a charge-coupled device camera positioned centrally above the apparatus. In the OF test, we measured the total distance traveled by the mice, total number of movement episodes, and the time spent at the center area (30 cm × 30 cm).

LD test: The apparatus consisted of a cage (21 cm × 42 cm × 25 (H) cm) divided into two chambers by a partition with an opening. One chamber was brightly illuminated (250 lx, light box) and was made of white plastic, whereas the other chamber was dark (5 lx, dark box) and was made of black plastic. The behavior was recorded using a CCD camera positioned above each chamber. Mice were allowed to move freely between the two chambers through an opening for 5 min, beginning in a dark box. In the LD test, we measured the total distance traveled in a light box, the latency to enter the light box for the first time, and the transition number between the light and dark boxes.

FZ test: We used a conditioning chamber (test chamber: 17 cm × 10 cm × 10 (H) cm) made of clear plastic with a ceiling. The chamber floor had stainless steel rods (2 mm in diameter) spaced 5 mm apart, which delivered an electric foot shock to the mice. The inner walls of the chamber were covered with black and white plastic strips. The LED light system was positioned approximately 50 cm above the chamber, providing 50 lx at the center of the floor. To monitor behavior, we used a CCD camera positioned above the center of the chamber. During the conditioning trial, mice were placed individually in the conditioning chamber and given three tone-shock pairings (30 sec of tone at 65 dB, followed by 2 sec of 0.15 mA electric shock), each separated by 120 sec. The mice were returned to their home cages. The following day, we conducted a contextual fear test by returning the mice to the conditioning chamber for 6 min without a tone or shock. The following day, we conducted a cued fear test by placing the mice in a novel chamber (with a different design and lacking plastic black and white stripes and stainless-steel rods). After 3 min, a conditioning tone (with no shock) was presented for 3 min. We measured the freezing response of mice using Image FZ2 as a consecutive 2-second period of immobility. The freezing rate (%) was calculated as follows: (freezing/session time) × 100.

We used the public domain ImageJ software for image analysis (Image OF2, Image LD2, and Image FZ2; O’Hara & Co., Ltd., Japan). All experimental tests were performed between 9:00 and 13:00, with the background noise level maintained at approximately 50 dB during the BBT. After each trial, the equipment was rinsed thoroughly with water and cleaned.

Tissue collection

Body weights were measured before and after BBT. After BBT, the mice were euthanized using a mixed anesthetic (medetomidine, midazolam, and butorphanol) at 3 months, and 7 months of age, for group I and II, respectively. The brains were surgically removed, fixed with methacarn solution (methanol, chloroform, and acetic acid at a 6:3:1 ratio), and subsequently treated with ethanol and xylene. The specimens were then embedded in paraffin and sectioned into 10 μm sagittal slices.

Histological analysis

The sections were deparaffinized with xylene and rehydrated with ethanol (100%, 95%, 90%, 80%, and 70%). After rinsing with distilled water, the nuclei were stained with hematoxylin and the cytoplasm was stained with eosin. Subsequently, sections were dehydrated with ethanol, clarified with xylene, and mounted for examination. Images were acquired using a BX50 polarization microscope and analyzed using the CellSens software (OLYMPUS, Tokyo, Japan).

Immunohistochemical analysis

Sections were deparaffinized with xylene, rehydrated with ethanol, rinsed in distilled water, and then incubated with HistoVT One (Nacalai Tesque, Kyoto, Japan) at 90°C for 30 min. The sections were incubated with Blocking One (Nacalai Tesque) at 4°C for 1 hr then incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: rabbit monoclonal anti-SRY-related HMG-box 2 (SOX2; Abcam, ab92494; diluted 1:300), rabbit polyclonal anti-doublecortin (DCX; Abcam, ab18723; diluted 1:300), rabbit monoclonal anti-neuronal nuclei (NeuN; NOVUS; NBP1-77686; diluted 1:600), rabbit monoclonal anti-glial fibrillary acidic protein (GFAP; NOVUS; NB100-53809; 1:400). After rinsing with phosphate-buffered saline, immunoreactive elements were visualized with Alexa Fluor 488-labeled anti-rabbit and Alexa Fluor 555-labeled anti-rabbit or anti-goat secondary antibodies (Invitrogen, Walthan, MA, USA; diluted 1:2000) by treating at 4°C for 90 min. Nuclei were stained with Hoechst 33342 (Nacalai Tesque; diluted 1:5000). For the immunohistochemical negative control, tests were conducted without the primary antibody and the lack of immunoreactivity was verified.

Image quantification

Using a confocal microscope (FLUOVIEW FV3000, OLYMPUS), stained images of the dentate gyrus (DG) in the hippocampus were assessed. The DCX-positive (immature neuron marker), SOX2-positive (neural stem cell [NSC] marker), and GFAP-positive (astrocyte marker) cells were counted. The average of two sampling areas (on the upper and lower sides of the DG) was used to calculate the number of SOX2-, DCX-, and GFAP-positive cells. To quantify astrocytes (GFAP-immunoreactive cells), the number of GFAP-positive cells was calculated from sampling areas in the hilar region (100 × 100 μm/field) as well as the subgranular zone (SGZ) and granule cell layer (GCL) of the DG. To quantify NSC, the number of SOX2-positive cells in the SGZ of the DG was counted. NeuN was used to delineate the boundary between the SGZ and the GCL. To quantify the number of immature neurons (DCX-immunoreactive cells), we counted DCX-positive cells in the GCL of the DG.

Statistical analyses

Statistical analyses were performed using one-way ANOVA, assuming equal variances, with the Dunnett’s post-hoc test for multiple comparisons. The results were considered statistically significant when the P-value was less than 0.05 (**P < 0.01, *P < 0.05). In addition, a P-value < 0.1 was considered a statistically significant trend (^†P < 0.1). All results are presented as means ± standard errors (S.E.). All statistical analyses were performed using the KyPlot 6.0 version 6.0.2 (KyensLab Inc., Tokyo, Japan).

RESULTS

General observations

After CLO administration, we observed acute peripheral neurotoxicity in our mouse models, such as tremors in the limbs. However, these symptoms were transient in nature and did not last more than 24 hr. Mice which showed extreme weight loss due to hydrocephalus or died before CLO treatment were not used in BBT. In addition, no significant differences were found regarding the body weight between the CLO-treated and the VC groups before and after BBT (Table 1).

Table 1. Body weights of mice before and after the BBT.

Treatment groups		Body weight (g)
		Before the behavioral test battery			After the behavioral test battery
Male I	VC I	29.03	±	0.32	30.16	±	0.32
	2w-CLO I	29.04	±	0.65	29.75	±	0.71
	6w-CLO I	26.86	±	1.04	27.76	±	0.01
	10w-CLO I	27.80	±	0.67	29.03	±	0.63
Female I	VC I	21.04	±	0.40	20.60	±	0.33
	2w-CLO I	21.23	±	0.62	21.24	±	0.62
	6w-CLO I	21.66	±	0.28	21.50	±	0.28
	10w-CLO I	20.83	±	0.30	21.05	±	0.28
Male II	VC II	33.06	±	1.22	34.01	±	1.18
	2w-CLO II	31.22	±	1.31	32.44	±	1.23
	6w-CLO II	30.69	±	1.59	31.18	±	1.52
	10w-CLO II	34.07	±	1.04	34.42	±	0.95
Female II	VC II	25.06	±	0.32	25.41	±	0.36
	2w-CLO II	25.67	±	0.64	25.85	±	0.62
	6w-CLO II	25.22	±	0.58	25.60	±	0.65
	10w-CLO II	26.44	±	0.75	26.15	±	0.61

Data analyzed by one-way ANOVA with Dunnett’s multiple comparison pos-hoc test and are expressed as the mean ± S.E.. The number of mice used was as follows: Male I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 7), and 10w-CLO I (n = 8). Female I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 8), 10w-CLO I (n = 8). Male II: VC II (n = 9), 2w-CLO II (n = 8), 6w-CLO II (n = 6), 10w-CLO II (n = 8). Female II: VC II (n = 10), 2w-CLO II (n = 8), 6w-CLO II (n = 8), 10w-CLO II (n = 8).

OF test results

The total distance traveled did not change significantly in the CLO-treated group compared to that of the VC group (Fig. 2A). The total number of movement episodes also did not significantly change between the CLO-treated and control groups (Fig. 2B). In contrast, regarding the anxiety-like behavior, the time spent in the center region by female mice in the 2w-CLO II group significantly decreased, whereas we did not observe a similar reduction in time among the other CLO-treated groups (Fig. 2C).

Fig. 2

Results of the OF test. A: Total distance traveled (cm) during the test period (total test time: 600 sec). B: Total number of movement episodes. C: Total time spent at the central area (sec). Data are expressed as mean ± S.E., and were analyzed by one-way ANOVA using Dunnett’s multiple comparison post-hoc test (*P < 0.05, compared with the VC group). The number of mice in each group was as follows: Male I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 7), and 10w-CLO I (n = 8). Female I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 8), 10w-CLO I (n = 8). Male II: VC II (n = 9), 2w-CLO II (n = 8), 6w-CLO II (n = 6), 10w-CLO II (n = 8). Female II: VC II (n = 10), 2w-CLO II (n = 8), 6w-CLO II (n = 8), 10w-CLO II (n = 8).

LD test results

In the LD test, the distance traveled in the light box by the female mice in the 6w-CLO I group significantly decreased (Fig. 3Ab). A similar decrease was not observed in the other CLO-treated groups (Fig. 3A). In terms of the latency to enter the light box for the first time, male mice in the 2w-CLO II group entered the chamber earlier compared with their counterparts in the VC II group, while female mice in 2w-CLO II group entered the chamber with a notable delay relative to female mice of the VC II group, although such differences were not statistically significant (Fig. 3B). The number of transitions between the light and dark boxes was also comparable for the CLO-treated and VC groups (Fig. 3C).

Fig. 3

Results of the LD test. A: Total distance traveled in light boxes (cm). B: Latency to enter the light box for the first time (sec). C: Transition number between light and dark boxes. Data are expressed as mean ± S.E., and were analyzed by one-way ANOVA using Dunnett’s multiple comparison post-hoc test (*P < 0.05, compared with the VC group). The number of mice in each group was as follows: Male I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 7), and 10w-CLO I (n = 8). Female I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 8), 10w-CLO I (n = 8). Male II: VC II (n = 9), 2w-CLO II (n = 8), 6w-CLO II (n = 6), 10w-CLO II (n = 8). Female II: VC II (n = 10), 2w-CLO II (n = 8), 6w-CLO II (n = 8), 10w-CLO II (n = 8).

FZ test results

In the FZ test, as the conditioning cycles repeated, the freezing response percentage increased in all groups. This time-dependent change in freezing rate confirmed that learning was established in both the CLO-treated and VC groups. In the contextual fear test, the contextual freezing rate significantly decreased in 6w-CLO II female mice (Fig. 4Bd), while in contrast, that was significantly increased in 2w-CLO II male mice (Fig. 4Bc). We did not observe any similar changes in the contextual freezing rate among the other CLO-treated groups (Fig. 4B). In the cued fear test, the freezing rate significantly decreased among 2w-CLO II and 10w-CLO II group female mice (Fig. 4Cd). We also noted a decrease in the cued freezing rate for the female 6w-CLO II mice although this difference was not significant (Fig. 4Cd). The cued freezing rate was similar between the other CLO-treated groups (Fig. 4C).

Fig. 4

Results of the FZ test. A: Average total freezing scores (%) during the conditioning test (FZ1). B: Average total freezing scores (%) during the contextual test (FZ2). C: Average total freezing scores (%) during the cued test (FZ3). Data are expressed as mean ± S.E., and were analyzed by one-way ANOVA using Dunnett’s multiple comparison post-hoc test (*P < 0.05 and **P < 0.01, compared to the VC group). The number of mice used was as follows: Male I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 7), and 10w-CLO I (n = 8). Female I: VC I (n = 10), 2w-CLO I (n = 8), 6w-CLO I (n = 8), 10w-CLO I (n = 8). Male II: VC II (n = 9), 2w-CLO II (n = 8), 6w-CLO II (n = 6), 10w-CLO II (n = 8). Female II: VC II (n = 10), 2w-CLO II (n = 8), 6w-CLO II (n = 8), 10w-CLO II (n = 8).

Hippocampal morphology

In the H&E-stained hippocampus, no pathological abnormalities were observed, such as neuronal cell death, neuronal degeneration, and structural disorganization (data not shown).

Immunohistochemical analysis

The brains of group II mice, which demonstrated prominent behavioral differences between males and females, were obtained for further immunohistochemical analysis, to assess the number of NSCs, immature neurons, and astrocytes in the DG. The number of GFAP-positive astrocytes in male mouse brain samples did not significantly differ between the CLO-treated and the VC groups (Fig. 5A, B, C). By contrast, we observed a reduction in GFAP-positive astrocytes in the hilus of the DG in female mice of the 2w-CLO II group compared to the astrocyte number in female mice of the VC II group (Fig. 5D, E). A decreased number of GFAP-positive astrocytes in the GCL + SGZ of the DG was observed in 2w-CLO II and 6w-CLO II female mice (Fig. 5D, F). By comparison, the immunohistochemical staining did not reveal any significant differences regarding DCX- and SOX2-positive cells between the CLO-treated and VC groups for either sex (Fig. 6C, D, G, H).

Fig. 5

Immunohistochemical images and analysis of GFAP expression. A: GFAP expression in the dentate gyrus of 7-month-old male mice (Male II). B, E: Number of GFAP-positive cells in the hilus per brain section in each group (number of GFAP-positive cells / 100 μm×100 μm in hilus). C, F: Number of GFAP-positive cells within the subgranular zone and granule cell layer region per brain section in each group. D: GFAP expression in the dentate gyrus of 7-month-old female mice (Female II). Scale bars, 100 µm (A, D). Data are expressed as mean ± S.E., and were analyzed by one-way ANOVA using Dunnett’s multiple comparisons post-hoc test (n = 3 per group; *P < 0.05 and ^†P < 0.1, compared to the VC II group).

Fig. 6

Immunohistochemical images and analysis for Sox2 and DCX expression. A: SOX2 expression in the dentate gyrus of Male II mice. B: DCX expression in the dentate gyrus of Male II mice. C, G: Number of SOX2-positive cells in the subgranular zone per brain section in each group. D, H: Number of DCX-positive cells in the granule cell layer per brain section in each group. E: SOX2 expression in the dentate gyrus of Female II mice. F: DCX expression in the dentate gyrus of Female II mice. Scale bars, 100 µm (A, B, E, F). Data are expressed as mean ± S.E., and were analyzed by one-way ANOVA using Dunnett’s multiple comparisons post-hoc test (n = 3 per group; *P < 0.05 and ^†P < 0.1 compared to the VC II group).

DISCUSSION

In this study, we conducted behavioral tests in adulthood to evaluate the impact of transient CLO administration during the postnatal period, adolescence, and maturity, regarding lasting and sex-dependent differences in behavior between male and female mice.

In the OF test, the time spent at the center area by female mice of the 2w-CLO II group was significantly decreased, although the total distance traveled and total number of movement episodes did not change. This result was attributed to the excessive anxiety of these mice after introduction to a novel environment. Despite a tendency for increased behaviors related with anxiety in unfamiliar environments among female 2w-CLO II mice, we did not observe similar changes in 2w-CLO II male mice. These results suggest that CLO administration during the early postnatal period induces anxiety-like behaviors only in female mice, which is consistent with the findings of a previous study reporting that CLO exposure in male mice during the early postnatal period did not affect the time spent in the central region of the OF test (Shoda et al., 2023).

In regard to the FZ test, the freezing rate during the contextual fear test significantly decreased among female mice in the 6w-CLO II group. This suggests that CLO exposure during adolescence may lead to spatial memory abnormalities only in female mice. We noted similar responses during the cued fear test, as the freezing rate among female mice in the 2w-CLO II and 10w-CLO II groups significantly decreased. Moreover, the freezing rate of the 6w-CLO II female mice was lower than that of their VC II group counterparts, although not by a statistically significant degree. In contrast, the freezing rate response of male CLO II mice during the cued fear test, was comparable to that of control males. These results suggest that cued memory retrieval impairments occur only in the female CLO II groups.

Our findings from the immunohistochemical analyses did not indicate any distinct sex-specific differences regarding the number of NSCs and immature neurons, despite previous studies reporting a decrease of NSCs in adult male mice after chronic administration of the neonicotinoid compound, imidacloprid, during the developmental period (Saito et al., 2023). However, we observed a reduction of the GFAP-positive astrocytes in the DG of 2w-CLO II female mice, and also in 6w-CLO II females to a lesser extent. This result is similar to that of Saito et al., who demonstrated that imidacloprid exposure during early life, led to a decrease in astrocyte number in adult male mice. In our work, we did not observe a reduction in astrocytes in male mice. The variation in the impact of CLO on neuronal cells and astrocyte populations compared to previous studies, may be at least partly attributed to the different types of neonicotinoids used in each work, and the single-dose administration protocol followed in this experiment. Many studies have reported a reduction in astrocyte numbers and activity in adulthood, following chemical exposure during the developmental stages (Singha et al., 2021; Saito et al., 2017), and astrocytes within the hippocampus undergo a rapid increase during early postnatal development, particularly around postnatal day 14 (Schneider et al., 2022). As such, the decrease in the number of astrocytes may have been influenced by CLO exposure during the early postnatal period. However, the mechanisms underlying the changes in astrocyte numbers, and the potential reasons as to why such changes may seemingly be specific to females, remain unclear.

Astrocytes in the hippocampus are largely involved in mammalian behavior. Astrocytes actively contribute to information processing and the establishment of the blood-brain barrier (Ballabh et al., 2004; Eroglu and Barres, 2010). Several neurological disorders, including anxiety and fear-related disorders, are thought to be associated with astrocyte dysfunction (Bergink et al., 2004, Cortese and Phan, 2005). Moreover, hippocampal astrocytes modulate anxiety-related behavior (Cho et al., 2022), and also contribute to cognitive functions, in conjunction with neuronal activity (Banaclocha, 2007; Hassanpoor et al., 2014). The increased anxiety-related behaviors displayed by the 2w-CLO II group, together with the limited spatial associative memory capabilities of the 6w-CLO II group during behavioral tests, were consistent with the typical behavioral abnormalities observed when hippocampal astrocyte function is compromised. Therefore, the reduced number or reduced activity of astrocytes in the DG of 2w-CLO II and 6w-CLO II adult female mice may impede normal information transmission, and could be related to these behavioral abnormalities. In classical fear conditioning, the amygdala and hippocampus play significant roles in associating conditioned and unconditioned stimuli (Wilensky et al., 2006). The α4β2 nAChRs are widely distributed in the amygdala (Philip et al., 2010), and nicotine activates amygdala neurons (Iha et al., 2017). Considering these studies, and the fact that the FZ test is a memory test that uses aversive stimuli, it is possible that the abnormalities in memory retrieval observed in the FZ test could be related to functional changes not only in the hippocampus but also in the amygdala. As such, it is necessary to also investigate the functionality of the amygdala, so as to understand the differences in cued memory retrieval ability between males and females.

The results of our study highlight the behavioral differences between males and females after a single-dose CLO administration across multiple developmental stages, although dose-response relationships could not be established. In addition, the observed female-specific behavioral abnormalities underscore the importance of evaluating the effects of chemicals in both sexes, not just in males. This study may contribute to the accumulation of knowledge regarding the risks associated with high levels of neonicotinoid exposure due to accidents, while also providing insights for potential treatment.

ACKNOWLEDGEMENTS

We thank A. Hasegawa and Q. Liu for their help regarding the mouse dissection. This study was supported in part by the JSPS KAKENHI (Grant Number 19H01142). We would like to thank Editage (www.editage.com) for English language editing.

Conflict of interest

The authors declare that there is no conflict of interest.

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