Effect of Vinyl Acetate, Glass Fibers Contents, and Buffer Space on EVA's Mechanical Property and Shock Absorption Ability.

OBJECTIVES: The aim of the study was to evaluate the mechanical properties and impact absorption capacity of prototype materials comprising ethylene vinyl acetate (EVA) of different hardness reinforced using different amounts of glass fibers (GFs), considering a buffer space. MATERIALS AND METHODS: Six prototype materials were made by adding E-GFs (5 and 10 wt%) to EVA with vinyl acetate (VA) contents of 9.4 wt% ("hard" or HA) and 27.5 wt% ("soft" or SO). Durometer hardness and tensile strength tests were performed to evaluate the mechanical properties of the materials. Moreover, an impact test was conducted using a customized pendulum impact tester to assess the impact absorption capacity (with or without a buffer space) of the specimens. RESULTS: The mechanical properties of the prototypes, namely, durometer hardness, Young's modulus, and tensile strength, were significantly higher in the HA group than in the SO group, regardless of the presence or added amount of GFs. The addition of GFs, particularly in a large amount (10 wt%), significantly increased these values. In terms of the impact absorption capacity, the original hardness of the EVA material, that is, its VA content, had a more substantial effect than the presence or absence of GFs and the added amount of GFs. Interestingly, the HA specimens with the buffer space exhibited significantly higher impact absorption capacities than the SO specimens. Meanwhile, the SO specimens without the buffer space exhibited significantly higher impact absorption capacities than the HA specimens. Moreover, regardless of the sample material and impact distance, the buffer space significantly improved impact absorption. In particular, with the buffer space, the impact absorption capacity increased with the added amount of GFs. CONCLUSION: The basic mechanical properties, including durometer hardness, Young's modulus, and tensile strength, of the EVA prototype were significantly increased by reducing the amount of VA regardless of the presence or added amount of GFs. Adding GFs, particularly in large amounts, significantly increased the values of aforementioned mechanical properties. Impact absorption was significantly affected by the hardness of the original EVA material and enhanced by the addition of the buffer space. The HA specimen had a high shock absorption capacity with the buffer space, and the SO specimen had a high shock absorption capacity without the buffer space. With the buffer space, impact absorption improved with the amount of added GFs.

Maternal immunoglobulins are distributed in the offspring's brain to support the maintenance of cortical interneurons in the postnatal period.

It is known that maternal immunoglobulins (Igs) are transferred to the offspring across the placenta. However, receiving maternal Igs, especially before the blood-brain barrier (BBB) is formed in the offspring's brain, carries the risk of transferring some brain-reactive Igs. It is thus hypothesized that there may be some unknown benefit to the offspring's brain that overweighs this risk. In this study, we show that the Ig detected in the embryonic/perinatal mouse brain is IgG not produced by the pups themselves, but is basically transferred from the mother across the placenta using the neonatal Fc receptor (FcRn) during embryonic stages. The amount of IgG in the brain gradually decreases after birth, and almost disappears within 3 weeks postnatally. IgG is detected on axon bundles, microglia, and some meningeal cells, including border-associated macrophages (BAMs), endothelial cells, and fibroblasts. Using Fcer1g knock-out (KO) mice, we show that BAMs and microglia receive maternal IgG in an Fc receptor γ chain (FcRγ)-dependent manner, but IgG on other meningeal cells and axon bundles is received independently of the FcRγ. These results suggest that maternal IgG may be used in multiple ways by different mechanisms. In maternal IgG-deficient mice, the number of interneurons in the cerebral cortex is not altered around birth but is reduced postnatally, suggesting that receipt of maternal IgG is necessary for the maintenance of cortical interneurons in the postnatal period. These data suggest that maternal IgG has an important function in the developing brain, where neither obvious inflammation nor infection is observed.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Interactions between neural cells and blood vessels in central nervous system development.

The sophisticated function of the central nervous system (CNS) is largely supported by proper interactions between neural cells and blood vessels. Accumulating evidence has demonstrated that neurons and glial cells support the formation of blood vessels, which in turn, act as migratory scaffolds for these cell types. Neural progenitors are also involved in the regulation of blood vessel formation. This mutual interaction between neural cells and blood vessels is elegantly controlled by several chemokines, growth factors, extracellular matrix, and adhesion molecules such as integrins. Recent research has revealed that newly migrating cell types along blood vessels repel other preexisting migrating cell types, causing them to detach from the blood vessels. In this review, we discuss vascular formation and cell migration, particularly during development. Moreover, we discuss how the crosstalk between blood vessels and neurons and glial cells could be related to neurodevelopmental disorders.

Depletion of macrophages deteriorates bisphosphonate-related osteonecrosis of the jaw-like lesions in mice.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a potentially intractable disease with no definitive pathophysiology and no treatment and prevention strategies. This study aimed to investigate whether time-selective depletion of macrophages worsens BRONJ-like lesions in mice. A murine model of high-prevalence BRONJ-like lesions in combination with zoledronate/chemotherapeutic drug administration and tooth extraction was created according to the methods of our previous studies. Daily intra-oral submucosal administration of clodronate-loaded liposomes, which temporarily depletes systemic macrophages, was performed immediately after tooth extraction. Spleens, femora, tibiae, and maxillae were dissected 2 weeks after extraction to evaluate BRONJ-like lesions and systemic conditions by micro-computed tomography analysis, histomorphometric and immunofluorescent analyses, and serum chemistry with ELISA. Depletion of macrophages significantly decreased the numbers of local and systemic macrophages and osteoclasts on the bone surface, which markedly worsened osseous healing, with increased necrotic bone and empty lacunae in the existing alveolar bone and newly formed bone in the extraction sockets, and soft tissue healing, with decreased collagen production and increased infiltration of polymorphonuclear cells. Interestingly, the depletion of macrophages significantly shifted macrophage polarization to M1 macrophages through an increase in F4/80+CD38+ M1 macrophages and a decrease in F4/80+CD163+ M2 macrophages, with decreases in the total number of F4/80+ macrophages. These data demonstrated that severe inhibition of osteoclasts in bone tissue and polarization shifting of macrophages in soft tissue are essential factors associated with BRONJ.

Diagnostic accuracy of a predictive scoring tool for patients who are eligible for home discharge from a palliative care unit.

BACKGROUND: Staying at a preferred place, principally at home, is of great value for dying patients, and palliative care units (PCUs) have an important role in providing adequate support so that patients can be discharged and go home. We attempted to create and validate a scoring tool to predict whether a cancer patient admitted to a PCU will be discharged home. METHODS: All 369 cancer patients admitted to the PCU of a 533-bed general hospital in Japan from October 2016 to October 2019 were enrolled. As outcomes, we recorded whether patients were discharged to home, died in hospitals, or were discharged to other hospitals. Attending physicians recorded 22 potential scale items at admission, including (I) demographic variables, (II) patient general conditions, (III) vital signs, (IV) medications, and (V) patient symptoms. Training-testing procedure to develop a screening score was performed. RESULTS: Among 369 cancer patients admitted to the PCU, we excluded 10 cases for whom a death location could not be identified. Among the remaining 359 patients, 180 were analyzed in the development phase and 179 in the validation phase. Multivariate logistic regression analysis identified five items as independent factors associated with discharge to home, and a prediction equation was created using the regression coefficients: sex (female, 4 points), calorie intake (520 kcal or more, 19 points), availability of daytime caregivers (11 points), family's preferred place of care (home, 139 points), and symptoms that resulted in hospitalization (not fatigue, 7 points). Using a cutoff point of 155, the area under the curve (AUC) value was 0.949 with 95% confidence intervals of 0.918 to 0.981. In the validation sample, the sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and error rate were 75.3%, 86.3%, 82.2%, 80.6%, and 18.4%, respectively. CONCLUSIONS: Whether a patient admitted to a PCU can discharge to home could be predicted using the simple clinical tool. Further validation and outcome studies are warranted.

Heterozygous Dab1 Null Mutation Disrupts Neocortical and Hippocampal Development.

Loss-of-function mutations in Reelin and DAB1 signaling pathways disrupt proper neuronal positioning in the cerebral neocortex and hippocampus, but the underlying molecular mechanisms remain elusive. Here, we report that heterozygous yotari mice harboring a single autosomal recessive yotari mutation of Dab1 exhibited a thinner neocortical layer 1 than wild-type mice on postnatal day (P)7. However, a birth-dating study suggested that this reduction was not caused by failure of neuronal migration. In utero electroporation-mediated sparse labeling revealed that the superficial layer neurons of heterozygous yotari mice tended to elongate their apical dendrites within layer 2 than within layer 1. In addition, the CA1 pyramidal cell layer in the caudo-dorsal hippocampus was abnormally split in heterozygous yotari mice, and a birth-dating study revealed that this splitting was caused mainly by migration failure of late-born pyramidal neurons. Adeno-associated virus (AAV)-mediated sparse labeling further showed that many pyramidal cells within the split cell had misoriented apical dendrites. These results suggest that regulation of neuronal migration and positioning by Reelin-DAB1 signaling pathways has unique dependencies on Dab1 gene dosage in different brain regions.

Zoledronate/Anti-VEGF Neutralizing Antibody Combination Administration Increases Osteal Macrophages in a Murine Model of MRONJ Stage 0-like Lesions.

The pathophysiology, pathogenesis, histopathology, and immunopathology of medication-related osteonecrosis of the jaw (MRONJ) Stage 0 remain unclear, although 50% of MRONJ Stage 0 cases could progress to higher stages. The aim of this study was to investigate the effects of zoledronate (Zol) and anti-vascular endothelial cell growth factor A (VEGFA) neutralizing antibody (Vab) administration on polarization shifting of macrophage subsets in tooth extraction sockets by creating a murine model of MRONJ Stage 0-like lesions. Eight-week-old, female C57BL/6J mice were randomly divided into 4 groups: Zol, Vab, Zol/Vab combination, and vehicle control (VC). Subcutaneous Zol and intraperitoneal Vab administration were performed for 5 weeks with extraction of both maxillary first molars 3 weeks after drug administration. Euthanasia was conducted 2 weeks after tooth extraction. Maxillae, tibiae, femora, tongues, and sera were collected. Structural, histological, immunohistochemical, and biochemical analyses were comprehensively performed. Tooth extraction sites appeared to be completely healed in all groups. However, osseous healing and soft tissue healing of tooth extraction sites were quite different. The Zol/Vab combination significantly induced abnormal epithelial healing, and delayed connective tissue healing due to decreased rete ridge length and thickness of the stratum granulosum and due to decreased collagen production, respectively. Moreover, Zol/Vab significantly increased necrotic bone area with increased numbers of empty lacunae compared with Vab and VC. Most interestingly, Zol/Vab significantly increased the number of CD169+ osteal macrophages (osteomacs) in the bone marrow and decreased F4/80+ macrophages, with a slightly increased ratio of F4/80+CD38+ M1 macrophages compared to VC. These findings are the first to provide new evidence of the involvement of osteal macrophages in the immunopathology of MRONJ Stage 0-like lesions.

Head injuries caused by contact with teeth during sports and exercise activities in Japanese schools during the period 2012-2018.

BACKGROUND/AIM: During sports activities, teeth-related contact can cause injury to both ally and opponent players, which can lead to potential infections and aesthetic problems. However, the extent of such injuries remains unclear. This study aimed to clarify the frequency and situation of head injuries caused by teeth (HICBT) occurring under the supervision of schools in Japan. MATERIAL AND METHODS: HICBT records were extracted from the Japan Sport Council data on head injuries occurring reported during the 7-year period from 2012 to 2018 under the supervision of schools in Japan. RESULTS: Of the total 463,527 head injury cases during the study period, 4495 cases (approximately 1%) were HICBT. Of the HICBT cases, 3650 (81.20%) were related to sports and athletic activity. Such injuries were reported to occur most often during basketball with a rate of 57.07% and 50.43%; soccer/futsal was the next most common sport with a rate of 13.38% and 24.01% in junior high school and high school students. Tag games were responsible for a similar number of HICBT cases at 22.73% and 39.03% in kindergartens and elementary school students. CONCLUSIONS: A total of 4495 cases of HICBT were identified, accounting for about 1% of all head injuries under the supervision of schools in Japan during the study period. This result reminds us that our teeth could be the weapon against the players during sports events. HICBTs occurring during basketball and soccer/futsal, in which mouthguards are not mandatory, were conspicuous among junior and senior high school students. Active use of mouthguards in various sports will protect players as well as their teammates and opponents. Sports dentists should encourage the revision of rules, such as mandating the use of mouthguards, in popular sports with a high incidence of HICBT, such as basketball and soccer/futsal.

A Unique "Reversed" Migration of Neurons in the Developing Claustrum.

The claustrum (CLA) is a cluster of neurons located between the insular cortex and striatum. Many studies have shown that the CLA plays an important role in higher brain function. Additionally, growing evidence suggests that CLA dysfunction is associated with neuropsychological symptoms. However, how the CLA is formed during development is not fully understood. In the present study, we analyzed the development of the CLA, especially focusing on the migration profiles of CLA neurons in mice of both sexes. First, we showed that CLA neurons were generated between embryonic day (E) 10.5 and E12.5, but mostly at E11.5. Next, we labeled CLA neurons born at E11.5 using the FlashTag technology and revealed that most neurons reached the brain surface by E13.5 but were distributed deep in the CLA 1 d later at E14.5. Time-lapse imaging of GFP-labeled cells revealed that some CLA neurons first migrated radially outward and then changed their direction inward after reaching the surface. Moreover, we demonstrated that Reelin signal is necessary for the appropriate distribution of CLA neurons. The switch from outward to "reversed" migration of developing CLA neurons is distinct from other migration modes, in which neurons typically migrate in a certain direction, which is simply outward or inward. Future elucidation of the characteristics and precise molecular mechanisms of CLA development may provide insights into the unique cognitive functions of the CLA.SIGNIFICANCE STATEMENT The claustrum (CLA) plays an important role in higher brain function, and its dysfunction is associated with neuropsychological symptoms. Although psychiatric disorders are increasingly being understood as disorders of neurodevelopment, little is known about CLA development, including its neuronal migration profiles and underlying molecular mechanisms. Here, we investigated the migration profiles of CLA neurons during development and found that they migrated radially outward and then inward after reaching the surface. This switch in the migratory direction from outward to inward may be one of the brain's fundamental mechanisms of nuclear formation. Our findings enable us to investigate the relationship between CLA maldevelopment and dysfunction, which may facilitate understanding of the pathogenesis of some psychiatric disorders.

Improving light-cured intermediate resin for hard and space mouthguard using a glass fiber.

BACKGROUND/AIMS: A light-cured intermediate material is useful for fabricating a hard insert and a buffer space mouthguard (H&SMG). However, it requires improvement in its mechanical properties and shock-absorbing capacity. The aim of this study was to evaluate the mechanical properties of two prototype light-cured intermediate materials reinforced with glass fibers, and the impact absorption capacity and durability of H&SMGs made with the prototype intermediate materials. MATERIALS AND METHODS: Two prototype materials containing long and microlength glass fibers in a light-cured intermediate material, Innerframe LC®, for H&SMG, were fabricated and tested. A three-point bending test was performed for evaluation of the mechanical properties. In addition, a shock absorption test was conducted using a customized pendulum impact testing machine to evaluate the H&SMGs' impact absorption capacity and durability. RESULTS: Long and microlength glass fibers significantly improved flexural modulus and strength. H&SMGs made with these two glass fiber-containing materials had high impact absorption capacity against both low and high impact forces, while the mouthguards made with long glass fiber materials had the best results. CONCLUSION: Long and microlength glass fibers with the prototype materials improved the mechanical properties of Innerframe LC® and the impact absorption capacity and durability of H&SMGs. H&SMGs made with the long glass fiber prototype materials had the best performance.

Effects of Mouthguards on Skin Damage In Vitro Study.

OBJECTIVE: Mouthguards can prevent and reduce orofacial sports traumas, which occur to the players themselves. However, the effect of mouthguards on skin damage has not been clarified. The present study's purpose was to examine whether the mouthguard can reduce or prevent skin damage caused by teeth (including the difference in mouthguard thickness). MATERIALS AND METHODS: Pigskins, artificial teeth, and Ethylene-vinyl acetate (EVA) mouthguard blanks with 1.5- and 3.0-mm thickness were employed. Each of the two type mouthguards was produced in 10 replicates. Mouthguard incisal thickness and collision touch angle were measured on a PC using imaging software. A pendulum-type machine was used to apply impact. Strain gauges attached to the tooth and impacted plate were used to measure mouthguards' effect on impact stress. Also, a microscope was used to observe the after impacted skin condition, and the extent of damage was assessed as a score. RESULTS: The pigskin was ruptured in without mouthguard (NOMG) with presenting the highest damage score, whereas the complete rupture was not seen in the 1.5 mm MG, but the damage of the skin (defeat) was observed. No tissue change was found with the 3 mmMG. In both the flat plate and impact tooth strain, no significant difference was observed between NOMG and 1.5 mmMG. However, 3 mmMG had a significantly smaller value than the other two conditions. These results are likely to be strongly influenced by the mouthguard incisal thicknesses and collision touch angles differences. CONCLUSION: The present study results clarified that two different thickness mouthguards reduced the skin damage, and the thicker mouthguard showed more effectiveness. Therefore, mouthguards may prevent the wearer's stomatognathic system's trauma and avoid damage to the skin of other athletes they are playing with. This effect seems to be an essential basis for explaining the necessity of using mouthguards for others besides full-contact sports.

Jaw-Clenching Intensity Effects on Masseter Oxygen Dynamics and Fatigue: A NIRS Oximetry Study.

The purpose of this study was to clarify the effects of jaw-clenching intensity on masseter muscle oxygen dynamics during clenching and recovery and masseter muscle fatigue using the spatially resolved method of near-infrared spectroscopy. Pulse rate, mean power frequency from electromyography in the masseter and visual analogue scale for masseter fatigue were also examined as related items. The 25% and 50% maximum voluntary contractions were determined using electromyography before the experiment and used as visual feedback on the screen. Twenty-three healthy adult male subjects volunteered for this study. Clenching decreased oxygen and oxygenated haemoglobin, and increased deoxygenated haemoglobin in the masseter muscle. The higher the intensity of clenching, the more prominent the effect. The oxygen dynamics tended to return to normal after clenching, but the change was slower with higher clenching intensity. Pulse rate increased with clenching, and the increment was more prominent with higher clenching intensity. Clenching caused a shift of mean power frequency to a lower range, an increase in subjective fatigue, an early appearance of a breakpoint appearance time and a prolongation of a 1/2 recovery time. All of these effects were more evident with increasing clenching intensity. In conclusion, clenching intensity influenced the oxygen dynamics of the masseter muscle and fatigue state during clenching and recovery. The higher the intensity, the greater the impact.

Activation of Sonic Hedgehog Signaling Promotes Differentiation of Cortical Layer 4 Neurons via Regulation of Their Cell Positioning.

Neuronal subtypes in the mammalian cerebral cortex are determined by both intrinsic and extrinsic mechanisms during development. However, the extrinsic cues that are involved in this process remain largely unknown. Here, we investigated the role of sonic hedgehog (Shh) in glutamatergic cortical subtype specification. We found that E14.5-born, but not E15.5-born, neurons with elevated Shh expression frequently differentiated into layer 4 subtypes as judged by the cell positioning and molecular identity. We further found that this effect was achieved indirectly through the regulation of cell positioning rather than the direct activation of layer 4 differentiation programs. Together, we provided evidence that Shh, an extrinsic factor, plays an important role in the specification of cortical superficial layer subtypes.

Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex.

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin ß1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

Development of intensiometric indicators for visualizing N-cadherin interaction across cells.

N-cadherin (NCad) is a classical cadherin that mediates cell-cell interactions in a Ca2+-dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.

TPT1 Supports Proliferation of Neural Stem/Progenitor Cells and Brain Tumor Initiating Cells Regulated by Macrophage Migration Inhibitory Factor (MIF).

One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.

Dab1-deficient deep layer neurons prevent Dab1-deficient superficial layer neurons from entering the cortical plate.

The mammalian neocortex has a 6-layered cytoarchitecture, where early- and late-born neurons are positioned deeply and superficially, respectively. Inverted lamination has been observed in mice defective in the Reelin/Disabled-1 (Dab1) pathway. Considering that Dab1-deficient superficial layer neurons can migrate into the Dab1 +/+ cortical plate and that Dab1 is thought to function cell-autonomously, it is unclear why superficial layer neurons are positioned below deep layer neurons in Reelin/Dab1-deficient mice. Here, we reconfirmed that Dab1 -/- superficial layer neurons enter the cortical plate using in utero electroporation on embryonic day (E) 14.5 Dab1-floxed mice. Electroporation in E12.5 Dab1-floxed mice reconfirmed that many deep layer neurons were mispositioned below the subplate. We also found an accumulation of Dab1-deficient superficial layer neurons below the cortical plate in many of these brains, in which deep layer neurons below the subplate showed high cell density. These phenotypes were rescued by decreasing the knockout probability and by expressing Dab1 in deep layer neurons. These observations suggest that cell-dense Dab1 -/- deep layer neurons prevent Dab1 -/- superficial layer neurons from entering the cortical plate. This reflects a non-cell-autonomous function of Dab1 and may explain the preplate splitting failure and outside-in lamination observed in Reelin/Dab1-deficient mice.