Sex-Difference in Olfactory Interhemispheric Malformation Caused by Pax6 Haploinsufficiency

Abstract

Sex differences are increasingly recognized as critical factors influencing the onset and manifestation of various neurodevelopmental disorders, including autism spectrum disorder (ASD). Among ASD-associated genes, Pax6, which encodes a transcription factor, has been implicated in sex-dependent phenotypes when mutated. The anterior commissure (AC), a major commissural pathway involved in higher cognitive and social functions, exhibits structural abnormalities both in individuals with ASD and in those carrying Pax6 mutations. Anatomically, the AC consists of two principal fiber bundles: the anterior limb (aAC), connecting the olfactory bulbs and anterior olfactory nucleus, and the posterior limb (pAC), connecting the piriform cortex and amygdaloid regions, both converging at the midline. In this study, we investigated sex-specific structural abnormalities of the AC in Pax6 mutant mice. Analysis of horizontal and sagittal AC sections revealed that Pax6 haploinsufficiency in mice induces abnormal de-fasciculation in the aAC and aberrant intermingling of aAC and pAC fibers at the midline. Notably, Pax6 mutant females exhibited more diverse phenotypes. These females showed a pronounced reduction in overall AC size and disruptions in pAC shape regularity, likely due to a more severe disruption of the boundary between the aAC and pAC axon bundles. These sex-dependent alterations in AC morphology may contribute to the sexually dimorphic phenotypes observed in various neurodevelopmental disorders, including autism spectrum disorder.

Introduction

Properly integrating neural circuits between the two hemispheres is essential for higher brain functions such as sensory processing and motor control. These circuits are formed by commissural fibers, including the corpus callosum (CC) linking the cerebral cortex, the hippocampal commissure connecting the hippocampi, and the anterior commissure (AC). The AC connects the olfactory and limbic regions across the hemispheres through two distinct bundles. The anterior limb (aAC) connects the olfactory bulb (OB) to the anterior olfactory nucleus (AON), while the posterior limb (pAC) links the piriform cortex (PIR), amygdaloid region, and related areas (Haberly and Price 1978; Suarez et al. 2014; Fenlon et al. 2021). Although some neurons project to the contralateral nucleus accumbens (NAc) via both pathways (Tian et al. 2024), AC neurons are typically classified based on their predominant projections to either the aAC or pAC—a classification scheme used in the present study.

Brain development is regulated by numerous transcription factors, including Pax6, which is expressed in neural stem/progenitor cells and plays a crucial role in early neural tube patterning and neurogenesis (Osumi 2001; Haubst et al. 2004; Osumi et al. 2008; Manuel et al. 2015). In humans, PAX6 mutations cause aniridia (Hill et al. 1991; Jordan et al. 1992) and are often associated with structural abnormalities in interhemispheric pathways, including the absence or hypoplasia of the AC and CC (Bamiou et al. 2004; Abouzeid et al. 2009). Magnetic resonance imaging (MRI) analyses have identified AC malformations and deficits in auditory processing in individuals carrying PAX6 mutations (Sisodiya et al. 2001; Bamiou et al. 2004, 2007; Abouzeid et al. 2009). In rodent studies, the AC has been linked to cognitive flexibility, social behavior, and processing of aversive information (Huang et al. 2014; Tian et al. 2024). Additionally, a recent study has reported structural abnormalities of the AC in individuals with autism spectrum disorder (ASD) (Kilroy et al. 2022), further highlighting the significance of interhemispheric connectivity in neurodevelopmental phenotypes.

Our previous study on Pax6 mutant rats also revealed notable AC abnormalities and sex-dependent phenotypes. MRI analysis demonstrated that a Pax6 mutation leads to volume reduction in multiple brain regions, including the neocortex, hippocampal formation, amygdala, major white matter tracts (e.g., CC, AC, cingulum and internal capsule), diencephalon and olfactory system, with more pronounced reductions in females (Hiraoka et al. 2016). Intriguingly, the AC was the only region showing a significant genotype-by-sex interaction. While postmortem studies of human brains suggest that the AC is more prominent in females than in males (Allen and Gorski 1991, 1992), rodent research has reported the opposite trend (Noonan et al. 1998). These findings highlight the need for further investigation into the sex-specific effects of Pax6 mutations on AC formation, which are still poorly understood.

The present study aims to investigate sex-specific phenotypes caused by Pax6 haploinsufficiency by examining structural abnormalities in the AC of Pax6 mutant mice. Our analysis reveals a range of phenotypic abnormalities, from AC morphology nearly identical to that of wild-type (WT) mice to significant reductions in the AC area and disruptions in the boundary between the aAC and pAC. Notably, female Pax6 mutant mice displayed a more pronounced decrease in the AC area and reduced pAC circularity, likely due to the boundary disruptions. Collectively, these findings underscore a sex-dependent vulnerability to AC malformations in Pax6 mutant mice.

Materials and Methods

Animals

All mice were bred and housed in a pathogen-free facility at Tohoku University School of Medicine. Pax6-heterozygous mice (Pax6^Sey/+) (Hill et al. 1991) were maintained on an inbred C57BL/6J background under a 12-h light/dark cycle with ad libitum access to food and water. All experimental procedures were approved by the Ethics Committee for Animal Experiments at Tohoku University Graduate School of Medicine (2023 Medical Research and Development −032-05).

Pax6 genotyping

Genomic DNA was extracted from tail tips or ear notches of adult mice using standard protocols for polymerase chain reaction (PCR) analysis. The primers used to distinguish between the WT and Sey alleles were as follows: SP1 (5'-GAGAACACCAACTCCATCAGTTCTAAGT-3'), SP2 (5'-AGCAACAGGAAGGAGGGGGAACGAACACCAACTCCATCAGTTCTTACG-3'), and MC130 (5'-CTTTCTCCAGAGCCTCAATCTG-3') (Swanson et al. 2005). The PCR conditions were as follows: an initial denaturing at 95°C for 2 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min; followed by 30 cycles of 95°C for 40 sec, 55°C for 40 sec, and 72°C for 40 sec, with a final extension at 72°C for 10 min.

Tissue collection

Male Pax6^Sey/+ mice were mated with WT females to produce Pax6^Sey/+ and WT offspring. At 8 weeks of age, the mice were deeply anesthetized with isoflurane and then perfused with PBS, followed by 4% paraformaldehyde (PFA) in PBS. Brain tissues were then collected and post-fixed with the same fixative overnight at 4°C. For sagittal sectioning, the brains were bisected along the longitudinal cerebral fissure using a razor blade. After cryoprotection in 30% sucrose/PBS, the brains were embedded in O.C.T. compound (Tissue-Tek, SAKURA) and sectioned at 60 µm (horizontal) or 20 µm (sagittal). The 60 µm-thick sections were collected in PBS-filled well plates, while the 20 µm-thick sections were mounted onto glass slides.

Luxol Fast Blue (LFB) staining

Both 60-µm free-floating sections and 20-µm slide-mount sections were processed for LFB staining. Sections were first washed with PBS and then delipidized by incubating in 70% ethanol for 24 h. They were subsequently incubated in the solution, containing 0.1% LFB (Solvent Blue 38, MP Biomedicals) dissolved in 95% ethanol and 0.5% acetic acid, at 56°C overnight. After rinsing with distilled water and PBS, the excess dye was removed by differentiation with 0.05% lithium carbonate solution, followed by 70% ethanol. Following final washes with PBS and distilled water, the free-floating sections were mounted onto glass slides and air-dried at room temperature. Prior to imaging, slides were rehydrated with a series of 70%, 80%, 96%, and 100% ethanol and cleared with xylene. Images were acquired using an all-in-one fluorescence microscope (BZ-X700, KEYENCE).

Statistics

ImageJ software (NIH) was used to analyze the morphology of LFB-stained sagittal AC sections. Regions of interest (ROIs) for the whole AC, aAC and pAC were defined by outlining their perimeters with the Polygon selection tool. The area, perimeter, and circularity of each ROI were then measured using the Measure command. Two sections per individual were analyzed, one from the right hemisphere and the other from the left. The length of the aAC-pAC boundary was calculated using the formula:

The circularity of the aAC and pAC was calculated using the following formula:

This calculation was automatically performed by ImageJ based on the area and perimeter measurements. For statistical analysis, the normality of residuals and homogeneity of variance were assessed using the Shapiro-Wilk test and Levene’s test, respectively. Group differences were evaluated using the Kruskal-Wallis test, followed by pairwise comparisons with the Wilcoxon rank-sum test. The Brown-Forsythe test was used to compare variances between groups. Statistical significance was set at p < 0.05 (*^†p < 0.05, **^††p < 0.01, ***^†††p < 0.001). All data processing and analyses were carried out using R software (version 4.4.1; R Core Team, 2024).

Results

Abnormal branching of Pax6^Sey/+ aAC prior to target innervation

The AC traverses the ventral brain, establishing interhemispheric connections. In this pathway, the aAC projects from the AON to the contralateral OB, while the pAC extends from the PIR to the contralateral PIR and the amygdaloid complex (Haberly and Price 1978; Huang et al. 2014; Fenlon et al. 2021) (Fig. 1A). The aAC axon bundle emerges from the AON, extends caudally, makes a sharp turn at the midline, and projects to the opposite OB. In contrast, the pAC originates from the ipsilateral PIR and follows a gently arching trajectory in a rostral direction towards the contralateral PIR. At the midline region, just anterior to the third ventricle (3V), the aAC and pAC axon bundles converge into a single tract (Fig. 1B,C).

To assess abnormalities in AC formation associated with Pax6 mutation, we performed LFB staining on horizontal AC sections to visualize myelinated fibers. In these images, the aAC appears dark blue, while the pAC is light blue (Cheng et al. 1998). Three regions within the AC were examined in both WT and Pax6^Sey/+ mice (outlined in Fig. 2A). At the midline, where the aAC and pAC axon bundles converge, WT mice exhibited a clear boundary between the two pathways (Fig. 2B,C). In contrast, Pax6^Sey/+ mice showed intermingling of the aAC and pAC bundles, resulting in an indistinct boundary (Fig. 2D,E). In regions where the aAC and pAC diverge into two separate pathways, the aAC in WT mice maintained a consolidated, single-bundle structure (Fig. 2F,G). In Pax6^Sey/+ mice, however, the aAC deviated from its typical trajectory and split into multiple branches as it turned sharply towards the OB (Fig. 2H,I). Furthermore, while the aAC in WT mice remained consolidated in both the AON and OB regions (Fig. 2J,K), those in Pax6^Sey/+ mice exhibited aberrant branching into two to three branches in the AON region before reaching the OB (Fig. 2L,M). Collectively, these findings suggest that Pax6 mutation disrupts the standard bundling of aAC and pAC axons and induces abnormal branching of the aAC prior to target innervation.

Fig. 1.

The structure of the AC in the mouse brain.

(A) The overall structure of the AC is shown as 3D image. (B,C) The sagittal and horizontal view of the AC, respectively. All images were generated and modified from the Allen Brain Explorer^® Beta (https://x.gd/PClbt). AC, the anterior commissure; aAC, anterior limb of AC; pAC, posterior limb of AC; OB, olfactory bulb; AON, anterior olfactory nucleus; NAc, nucleus accumbens; PIR, piriform area; BLA, basolateral amygdala; ENT, entorhinal area; 3V, the third ventricle.

Fig. 2.

Pax6^Sey/+ aAC axons exhibit abnormal branching prior to target innervation.

(A) Luxol fast blue-stained horizontal section of the entire AC, with enlarged views of the boxed areas shown in (B-E), (F-I), and (J-M). In WT mice, the aAC and pAC exhibit a well-defined boundary at the midline (B,C). In contrast, Pax6^Sey/+ mice display axonal intermingling as highlighted in the insets (white arrows, D,E). Near the midline, where the aAC and pAC diverge, WT aAC remains consolidated (F,G), whereas, in Pax6^Sey/+mice, it deviates from its typical trajectory (white arrows, H,I). WT aAC remains unified before reaching the OB (J, K), whereas in Pax6^Sey/+ mice it splits into branches (white arrows, L,M). Scale bars, 1 mm (A) and 300 µm (B-M). AC, the anterior commissure; aAC, anterior limb of AC; pAC, posterior limb of AC; AON, anterior olfactory nucleus; PIR, piriform area; 3V, the third ventricle.

Sex differences in morphological abnormalities in sagittal AC sections of Pax6^Sey/+ mice

To examine the details of axon bundle formation abnormalities in the aAC and pAC of Pax6^Sey/+ mice, we analyzed LFB-stained sagittal sections of the AC. The entire AC was first delineated, with the darker-stained region designated as the aAC and the lighter-stained region as the pAC, following a previous report (Cheng et al. 1998) (Fig. 3A). In sagittal sections from WT brains, the aAC and pAC bundles were tightly aligned and merged into a single tract, with a distinct vertical boundary separating the two components (Fig. 3B,C).

Analysis of Pax6^Sey/+ AC sections revealed a spectrum of morphological phenotypes resulting from Pax6 haploinsufficiency. Some Pax6^Sey/+ samples displayed the whole AC, aAC, and pAC dimensions comparable to WT, with a well-defined boundary between the aAC and pAC (Fig. 3D,E). However, in other sections, the entire AC appeared markedly smaller than in WT (Fig. 3F,G). In these cases, both the aAC and pAC areas were proportionally reduced, although the aAC-pAC boundary remained distinct (Fig. 3F,G). More severe cases exhibited pronounced disruption of the aAC-pAC boundary, characterized by an abnormally jagged interface and irregular, convoluted structures in both the aAC and pAC (Fig. 3H,I). In the most extreme instances, the axonal bundles that typically coalesce into a unified tract in WT brains were dispersed into multiple clusters, with fibers from the aAC and pAC frequently invading each other’s domains (Fig. 3J,K).

To quantitatively assess these phenotypic variations across genotype and sex, we defined the whole AC, aAC, and pAC as ROIs and measured their respective areas (Fig. 4A). The whole AC area was significantly reduced in both sexes in Pax6 mutants. In Pax6^Sey/+ males, the median whole AC area was 0.104 [0.098-0.111] mm², representing a 14.0% reduction compared to WT males. In Pax6^Sey/+ females, the median whole AC area was 0.097 [0.091-0.099] mm², a 14.2% reduction compared to WT females. Notably, the reduction in the entire AC area was significantly more pronounced in females than males. Furthermore, both aAC and pAC sizes were significantly diminished in Pax6^Sey/+ mice of both sexes (Fig. 4A), indicating that Pax6 haploinsufficiency affects the development of both components.

Given the evident shape disruptions in the aAC and pAC regions, we first measured the perimeter for each ROI. The whole AC perimeter was significantly reduced in Pax6^Sey/+ mice of both sexes, consistent with the decrease in the entire AC area. However, despite the reduction in AC size, neither the aAC nor pAC perimeter showed a significant decrease (Fig. 4B). To account for differences in variance between Pax6^Sey/+ and WT mice, we performed the Brown-Forsythe test, which revealed significant differences in the variances of the aAC (p = 0.03) and pAC (p = 0.02) perimeters only in Pax6^Sey/+ females. This suggests that abnormal aAC and pAC perimeter lengths occur more frequently in Pax6^Sey/+ females.

Considering that irregular aAC and pAC perimeters may reflect disruptions in the aAC-pAC boundary, we estimated the length of this boundary using the perimeter measurements from each ROI (Fig. 4C). Although the aAC-pAC boundary length tended to be greater in Pax6^Sey/+ mice, the differences did not reach statistical significance in either males (p = 0.84), or females (p = 0.20). This may be attributed to the overall smaller AC area in Pax6^Sey/+ mice, leading to similar perimeters and boundary lengths between WT and Pax6^Sey/+ mice. However, variance in the aAC-pAC boundary length was significantly different in Pax6^Sey/+ females (p = 0.02), indicating that abnormal aAC-pAC boundary lengths are more common in Pax6^Sey/+ females. Overall, these results indicate that abnormal aAC and pAC perimeters, which are observed in Pax6^Sey/+ females, are likely the result of disruptions in the aAC-pAC boundary.

To assess potential morphological disruptions in the aAC and pAC, we evaluated their circularity, which reflects the ratio of perimeter to area. Lower circularity values indicate a more irregular shape, suggesting a disturbance of the aAC-pAC boundary. In Pax6^Sey/+ females, aAC circularity was reduced by 9.0%. Although this difference did not reach statistical significance, the variances differed significantly (p = 0.00499) (Fig. 4D). In contrast, pAC circularity tended to decrease in both male and female Pax6^Sey/+ mice (Fig. 4E). Notably, Pax6^Sey/+ females exhibited a median pAC circularity of 0.36 [0.27-0.51], representing a significant 41.3% reduction compared to WT females. Furthermore, the median pAC circularity in Pax6^Sey/+ females was significantly lower than that of Pax6^Sey/+ males, whose median was 0.56 [0.46-0.62] (p = 0.00478). These findings suggest that the pAC morphology is more severely disrupted in Pax6^Sey/+ females, likely reflecting a greater disturbance of the aAC-pAC boundary than in males.

Fig. 3.

Sagittal sections of Pax6^Sey/+ AC show diverse morphological phenotypes.

(A) Regions of whole AC, aAC and pAC were defined based on the color intensity of luxol fast blue staining. In some Pax6^Sey/+ samples, the AC closely resembles that of WT mice (B-C, D-E). Other Pax6^Sey/+ samples exhibit a reduction in AC area (F-G) or disruptions in the aAC-pAC boundary (H-K). Scale bar, 100 µm. AC, the anterior commissure; aAC, anterior limb of AC; pAC, posterior limb of AC.

Fig. 4.

Sex differences in AC area reduction and pAC shape disruptions in Pax6^Sey/+ mice.

(A) Both male and female Pax6^Sey/+ mice show reduced areas in all regions of interest (ROIs), with Pax6^Sey/+ females showing smaller overall AC size than Pax6^Sey/+ males. (B) Although the whole AC perimeter is significantly shorter in Pax6^Sey/+ mice, no significant differences are observed in the aAC and pAC perimeters. (C) In some Pax6^Sey/+ samples, the boundary between the aAC and pAC is extended. (D) The aAC circularity does not significantly differ by genotype or sex. (E) In contrast, pAC circularity is notably reduced specifically in Pax6^Sey/+ females. Boxplots depict the interquartile range (IQR), median, and whiskers; each dot representing an individual section measurement. Histograms show the data distribution using the Gaussian kernel density estimation, with bar heights representing probability density. Overlaid density curves provide a smoothed approximation of the distribution, and dashed vertical lines indicate median values. Statistical comparisons of medians were performed using the Kruskal-Wallis test followed by Wilcoxon rank-sum pairwise comparisons (*p < 0.05; **p < 0.01; ***p < 0.001), while the Brown-Forsythe test was used to assess the differences in variance among groups (^†p < 0.05; ^††p < 0.01; ^†††p < 0.001). WT male, n = 20; WT female, n = 20; Pax6^Sey/+ male, n = 18; Pax6^Sey/+ female, n = 20. AC, anterior commissure; aAC, anterior limb of AC; pAC, posterior limb of AC.

Discussion

In this study, we characterized morphological phenotypes of the AC in Pax6 heterozygous mutant mice, with a particular focus on sex differences. Our analysis demonstrates that Pax6 haploinsufficiency disrupts normal axon bundling, leading to intermingling between the aAC and pAC axons, as well as inducing abnormal branching within the aAC. Detailed morphological analysis on sagittal AC sections revealed an overall reduction in the AC size in both sexes, with Pax6^Sey/+ females exhibiting a more pronounced decrease than males. Additionally, a significant reduction in pAC circularity, likely reflecting disruption in the aAC-pAC boundary, was explicitly observed in Pax6 mutant females.

The navigation and/or segregation of the AC axonal trajectories is regulated by the variety of repulsive and attractive activities from guidance molecules both in contact-dependent and independent manners. For example, the loss of the aAC attractant Semaphorin 3B (Sema3B) and its receptor component Nrcam leads to premature branching of the aAC before it reaches the OB (Falk et al. 2005). Similarly, null mutations in the repulsive cues EphA4 and EphB2 cause aberrant de-fasciculation of the aAC and intermingling of aAC and pAC axons at the midline (Ho et al. 2009). Notably, AC axon extension begins at embryonic day 13.5 (Martin-Lopez et al. 2018), coinciding with the period when Pax6 regulates the expression of Nrcam, Nrp2, and EphA4 (Walcher et al. 2013; Kikkawa et al. 2019; Manuel et al. 2022; Ochi et al. 2024). Thus, Pax6 haploinsufficiency may impair the regulation of these axon guidance molecules, contributing to the observed malformation in aAC axon bundling.

AC axons originate near the pallial-subpallial boundary (PSB) (Martin-Lopez et al. 2018). The loss of Pax6 is known to disrupt this boundary (Stoykova et al. 2000; Carney et al. 2009; Cocas et al. 2011), potentially leading to a reduction of AC neurons and, consequently, a smaller overall AC size compared to WT mice. Furthermore, alterations in PSB pattering due to Pax6 haploinsufficiency may further compromise the formation of a distinct aAC-pAC boundary. Although Pax6 is predominantly expressed in neural stem/progenitor cells, some PSB-derived neurons continue to express Pax6 even after differentiation (Mi et al. 2013). Additional studies are required to determine whether Pax6 functions primarily within neural stem/progenitor cells or within differentiated neurons to ensure proper formation in the aAC-pAC boundary.

Previously, our group reported that in Pax6 mutant rats, the AC was the only brain structure showing a significant volume decrease in female mutants, as revealed by MRI-based analysis (Hiraoka et al. 2016). In the current study, histological analysis detected a reduction in AC size in both sexes, with a more pronounced effect observed in female Pax6 mutant mice. A comprehensive three-dimensional analysis using whole-brain clearing techniques (Mano et al. 2018) could provide further insights into the overall phenotype of the AC in Pax6 mutant mice.

Sex-specific phenotypes resulting from Pax6 haploinsufficiency remain underexplored. In rodents, the sizes of the AC and CC tend to be larger in males, potentially influenced by sex hormones (Fitch et al. 1990; Noonan et al. 1998). Intriguingly, EphB2, a repulsive guidance cue implicated in AC axon guidance, has been linked to sex-dependent phenotypes; heterozygous mutations in EphB2 result in behavioral abnormalities and altered cortical excitability only in females (Assali et al. 2021). Our findings, which show more severe AC malformations in Pax6 mutant females, suggest that females may be more vulnerable to developmental disruptions without proper regulation by AC guidance molecules. Although the interplay between Pax6 and EphA4/B2 signaling remains unclear, these data raise the possibility that hormonal factors may exacerbate the impact of Pax6 haploinsufficiency on AC formation in females.

Structural abnormalities in interhemispheric commissures are a hallmark of ASD. Reduced connectivity and morphological alterations in the AC and CC have been reported in both ASD patients and animal models (Huang et al. 2014; Fenlon et al. 2015; Kilroy et al. 2022). The PAX6 gene was originally found within the locus of WAGR syndrome (Wilms tumor, Aniridia, Genital ridge defect and mental Retardation) (Hanson 2003). Patients with WAGR frequently exhibit ASD phenotypes and mutations in PAX6 have also been reported in ASD patients (Davis et al. 2008; Maekawa et al. 2009; Yamamoto et al. 2014), leading to its classification as a syndromic ASD gene in the SFARI gene database (SFARI, https://gene.sfari.org/).

We have previously reported a female-specific reduction in maternal separation-induced ultrasonic vocalization, a behavior considered indicative of ASD-like deficits (Umeda et al. 2010), suggesting a potential connection between Pax6 mutations and ASD-related phenotypes, particularly in females. The pronounced malformations of the AC observed in Pax6^Sey/+ female mice observed in this study may contribute to ASD-like behaviors in these animals. Another intriguing issue is how Pax6 deficiency induces phenotypic diversity, as also observed in the Pax6^Sey/+ eyes in the C57BL6 background but less in 129S1/SvImJ background (sex not examined) (Kanakubo et al. 2006; Hickmott et al. 2018). Further studies on projection patterns and behavioral outcomes are needed to clarify the role of PAX6 haploinsufficiency in ASD pathophysiology, particularly in females.

Acknowledgments

We would like to thank Drs. Yoshio Wakamatsu, Hitoshi Inada, and Shohei Ochi for their technical advice and valuable comments. We are also grateful to Ms. Sayaka Makino for her assistance with animal care and technical support. Additionally, we thank all other members of the Osumi laboratory for their insightful comments and discussions.

Author Contributions

Conceptualization: N.J., T.K., and N.O.; Methodology & Investigation: N.J., T.I. and T.K.; Writing- Original Draft: N.J., T.K. and N.O.; Writing- Review & Editing: N.J., T.K., T.I. and N.O.; Supervision: T.K. and N.O.; Funding acquisition: T.K., and N.O.

Funding

This work was supported by JSPS KAKENHI funding (#24K02203) and AMED (#JP21wm0425003) to N.O and AMED (#JP24wm0625311) to T.K. A part of this study was supported by the Support System for Young Researchers to use research equipment, instruments, and devices at Tohoku University.

Conflict of Interest

The authors declare no conflict of interest.

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