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.

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.

Rhythmic activation of excitatory neurons in the mouse frontal cortex improves the prefrontal cortex-mediated cognitive function.

The prefrontal cortex (PFC) plays essential roles in cognitive processes. Previous studies have suggested the layer and the cell type-specific activation for cognitive enhancement. However, the mechanism by which a temporal pattern of activation affects cognitive function remains to be elucidated. Here, we investigated whether the specific activation of excitatory neurons in the superficial layers mainly in the PFC according to a rhythmic or nonrhythmic pattern could modulate the cognitive functions of normal mice. We used a C128S mutant of channelrhodopsin 2, a step function opsin, and administered two light illumination patterns: (i) alternating pulses of blue and yellow light for rhythmic activation or (ii) pulsed blue light only for nonrhythmic activation. Behavioral analyses were performed to compare the behavioral consequences of these two neural activation patterns. The alternating blue and yellow light pulses, but not the pulsed blue light only, significantly improved spatial working memory and social recognition without affecting motor activity or the anxiety level. These results suggest that the rhythmic, but not the nonrhythmic, activation could enhance cognitive functions. This study indicates that not only the population of neurons that are activated but also the pattern of activation plays a crucial role in the cognitive enhancement.

Comprehensive characterization of migration profiles of murine cerebral cortical neurons during development using FlashTag labeling.

In the mammalian cerebral neocortex, different regions have different cytoarchitecture, neuronal birthdates, and functions. In most regions, neuronal migratory profiles are speculated similar based on observations using thymidine analogs. Few reports have investigated regional migratory differences from mitosis at the ventricular surface. In this study, we applied FlashTag technology, in which dyes are injected intraventricularly, to describe migratory profiles. We revealed a mediolateral regional difference in the migratory profiles of neurons that is dependent on developmental stage; for example, neurons labeled at embryonic day 12.5-15.5 reached their destination earlier dorsomedially than dorsolaterally, even where there were underlying ventricular surfaces, reflecting sojourning below the subplate. This difference was hardly recapitulated by thymidine analogs, which visualize neurogenic gradients, suggesting a biological significance different from the neurogenic gradient. These observations advance our understanding of cortical development and the power of FlashTag in studying migration and are thus resources for future neurodevelopmental studies.

Human neocortical development as a basis to understand mechanisms underlying neurodevelopmental disabilities in extremely preterm infants.

AIM: Recent advances in perinatal and neonatal medicine have resulted in marked improvements in the survival rates of extremely preterm infants (born before 28 gestational weeks) around the world, and Japan is among the countries with the highest reported survival rates of extremely preterm infants. However, it remains a major concern that many survivors develop neurodevelopmental disabilities, including cognitive dysfunctions and neurodevelopmental disorders later in life. In order to understand the pathophysiological mechanisms underlying the neurodevelopmental disabilities observed in the survivors of extremely preterm births, we reviewed recently reported findings about the development of the human neocortex. METHODS: First, we have summarized the current knowledge about the development of the neocortex, including recently reported human- and/or primate-specific developmental events. Next, we discussed the possible causal mechanisms underlying the development of neurodevelopmental disabilities in extremely preterm infants. RESULTS: Around the birth of extremely preterm infants, neurogenesis and succeeding neuronal migrations are ongoing in the neocortex of human brain. Expansion and maturation of the subplate, which is thought to reflect the axonal wiring in the neocortex, is also prominent at this time. CONCLUSION: Brain injuries that occur around the birth of extremely preterm infants are presumed to affect the dynamic developmental events in the neocortex, such as neurogenesis, neuronal migrations and maturation of the subplate, which could underlie the neurodevelopmental disabilities that often develop subsequently in extremely preterm infants. These possibilities should be borne in mind while considering maternal and neonatal care to further improve the long-term outcomes of extremely preterm infants.

Increased densities of white matter neurons as a cross-disease feature of neuropsychiatric disorders.

While neurons of the human cerebral cortex are mainly distributed in the gray matter, the white matter (WM) also contains some excitatory and inhibitory neurons, so-called WM neurons. Studies on the cytoarchitectural alterations in the brains of patients with neuropsychiatric disorders have repeatedly reported increased densities of the WM neurons in a proportion of patients with schizophrenia and autism spectrum disorder. Although some studies have demonstrated increased densities of superficial WM neurons, others have demonstrated increased densities of deep WM neurons and increased WM neuron densities can be considered as one of the cross-disease features of neuropsychiatric disorders. Nevertheless, what actually causes the increase in the densities of the WM neurons still remains under debate, and several hypothetical mechanisms have been proposed. The WM neurons in normal brains are considered as remnants of the subplate neurons, which represent a transient cytoarchitectural zone present during development of the mammalian neocortex; it has been suggested that increased densities of the WM neurons could result from inappropriate apoptosis of the subplate neurons in the brains of patients with neuropsychiatric disorders. On the other hand, recent experimental studies have demonstrated that genetic and environmental factors that enhance the risk of development of neuropsychiatric disorders could cause altered distribution of neurons in the WM. To understand the pathophysiology underlying the increased densities of the WM neurons, it is important to investigate the cellular characteristics of the WM neurons in the brains of both normal subjects and patients with neuropsychiatric disorders.

Both excitatory and inhibitory neurons transiently form clusters at the outermost region of the developing mammalian cerebral neocortex.

During development of the mammalian cerebral neocortex, postmitotic excitatory neurons migrate toward the outermost region of the neocortex. We previously reported that this outermost region is composed of densely packed relatively immature neurons; we named this region, which is observed during the late stage of mouse neocortical development, the "primitive cortical zone (PCZ)." Here, we report that postmigratory immature neurons spend about 1-1.5 days in the PCZ. An electron microscopic analysis showed that the neurons in the PCZ tend to be in direct contact with each other, mostly in a radial direction, forming "primitive neuronal clusters" with a height of 3-7 cells and a width of 1-2 cells. A time-course analysis of fluorescently labeled neurons revealed that the neurons took their positions within the primitive clusters in an inside-out manner. The neurons initially participated in the superficial part of the clusters, gradually shifted their relative positions downward, and then left the clusters at the bottom of this structure. GABAergic inhibitory interneurons were also found within the primitive clusters in the developing mouse neocortex, suggesting that some clusters are composed of both excitatory neurons and inhibitory interneurons. Similar clusters were also observed in the outermost region of embryonic day (E) 78 cynomolgus monkey occipital cortex and 23 gestational week (GW) human neocortices. In the primate neocortices, including human, the presumptive primitive clusters seemed to expand in the radial direction more than that observed in mice, which might contribute to the functional integrity of the primate neocortex.

Drebrin-like (Dbnl) Controls Neuronal Migration via Regulating N-Cadherin Expression in the Developing Cerebral Cortex.

The actin cytoskeleton is crucial for neuronal migration in the mammalian developing cerebral cortex. The adaptor protein Drebrin-like (Dbnl) plays important roles in reorganization of the actin cytoskeleton, dendrite formation, and endocytosis by interacting with F-actin, cobl, and dynamin. Although Dbnl is known to be expressed in the brain, the functions of this molecule during brain development are largely unknown. In this study, to examine the roles of Dbnl in the developing cerebral cortex, we conducted experiments using mice of both sexes with knockdown of Dbnl, effected by in utero electroporation, in the migrating neurons of the embryonic cortex. Time-lapse imaging of the Dbnl-knockdown neurons revealed that the presence of Dbnl is a prerequisite for appropriate formation of processes in the multipolar neurons in the multipolar cell accumulation zone or the deep part of the subventricular zone, and for neuronal polarization and entry into the cortical plate. We found that Dbnl knockdown decreased the amount of N-cadherin protein expressed on the plasma membrane of the cortical neurons. The defect in neuronal migration caused by Dbnl knockdown was rescued by moderate overexpression of N-cadherin and αN-catenin or by transfection of the phospho-mimic form (Y337E, Y347E), but not the phospho-resistant form (Y337F, Y347F), of Dbnl. These results suggest that Dbnl controls neuronal migration, neuronal multipolar morphology, and cell polarity in the developing cerebral cortex via regulating N-cadherin expression.SIGNIFICANCE STATEMENT Disruption of neuronal migration can cause neuronal disorders, such as lissencephaly and subcortical band heterotopia. During cerebral cortical development, the actin cytoskeleton plays a key role in neuronal migration; however, the mechanisms of regulation of neuronal migration by the actin cytoskeleton still remain unclear. Herein, we report that the novel protein Dbnl, an actin-binding protein, controls multiple events during neuronal migration in the developing mouse cerebral cortex. We also showed that this regulation is mediated by phosphorylation of Dbnl at tyrosine residues 337 and 347 and αN-catenin/N-cadherin, suggesting that the Dbnl-αN-catenin/N-cadherin pathway is important for neuronal migration in the developing cortex.

ApoER2 Controls Not Only Neuronal Migration in the Intermediate Zone But Also Termination of Migration in the Developing Cerebral Cortex.

Neuronal migration contributes to the establishment of mammalian brain. The extracellular protein Reelin sends signals to various downstream molecules by binding to its receptors, the apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor and exerts essential roles in the neuronal migration and formation of the layered neocortex. However, the cellular and molecular functions of Reelin signaling in the cortical development are not yet fully understood. Here, to gain insight into the role of Reelin signaling during cortical development, we examined the migratory behavior of Apoer2-deficient neurons in the developing brain. Stage-specific labeling of newborn neurons revealed that the neurons ectopically invaded the marginal zone (MZ) and that neuronal migration of both early- and late-born neurons was disrupted in the intermediate zone (IZ) in the Apoer2 KO mice. Rescue experiments showed that ApoER2 functions both in cell-autonomous and noncell-autonomous manners, that Rap1, integrin, and Akt are involved in the termination of migration beneath the MZ, and that Akt also controls neuronal migration in the IZ downstream of ApoER2. These data indicate that ApoER2 controls multiple processes in neuronal migration, including the early stage of radial migration and termination of migration beneath the MZ in the developing neocortex.

Impaired dendritic growth and positioning of cortical pyramidal neurons by activation of aryl hydrocarbon receptor signaling in the developing mouse.

The basic helix-loop-helix (bHLH) transcription factors exert multiple functions in mammalian cerebral cortex development. The aryl hydrocarbon receptor (AhR), a member of the bHLH-Per-Arnt-Sim subfamily, is a ligand-activated transcription factor reported to regulate nervous system development in both invertebrates and vertebrates, but the functions that AhR signaling pathway may have for mammalian cerebral cortex development remains elusive. Although the endogenous ligand involved in brain developmental process has not been identified, the environmental pollutant dioxin potently binds AhR and induces abnormalities in higher brain function of laboratory animals. Thus, we studied how activation of AhR signaling influences cortical development in mice. To this end, we produced mice expressing either constitutively active-AhR (CA-AhR), which has the capacity for ligand-independent activation of downstream genes, or AhR, which requires its ligands for activation. In brief, CA-AhR-expressing plasmid and AhR-expressing plasmid were each transfected into neural stems cells in the developing cerebrum by in utero electroporation on embryonic day 14.5. On postnatal day 14, mice transfected in utero with CA-AhR, but not those transfected with AhR, exhibited drastically reduced dendritic arborization of layer II/III pyramidal neurons and impaired neuronal positioning in the developing somatosensory cortex. The effects of CA-AhR were observed for dendrite development but not for the commissural fiber projection, suggesting a preferential influence on dendrites. The present results indicate that over-activation of AhR perturbs neuronal migration and morphological development in mammalian cortex, supporting previous observations of impaired dendritic structure, cortical dysgenesis, and behavioral abnormalities following perinatal dioxin exposure.

Association of impaired neuronal migration with cognitive deficits in extremely preterm infants.

Many extremely preterm infants (born before 28 gestational weeks [GWs]) develop cognitive impairment in later life, although the underlying pathogenesis is not yet completely understood. Our examinations of the developing human neocortex confirmed that neuronal migration continues beyond 23 GWs, the gestational week at which extremely preterm infants have live births. We observed larger numbers of ectopic neurons in the white matter of the neocortex in human extremely preterm infants with brain injury and hypothesized that altered neuronal migration may be associated with cognitive impairment in later life. To confirm whether preterm brain injury affects neuronal migration, we produced brain damage in mouse embryos by occluding the maternal uterine arteries. The mice showed delayed neuronal migration, ectopic neurons in the white matter, altered neuronal alignment, and abnormal corticocortical axonal wiring. Similar to human extremely preterm infants with brain injury, the surviving mice exhibited cognitive deficits. Activation of the affected medial prefrontal cortices of the surviving mice improved working memory deficits, indicating that decreased neuronal activity caused the cognitive deficits. These findings suggest that altered neuronal migration altered by brain injury might contribute to the subsequent development of cognitive impairment in extremely preterm infants.

Reelin transiently promotes N-cadherin-dependent neuronal adhesion during mouse cortical development.

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized ß-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.

Excessive activation of AhR signaling disrupts neuronal migration in the hippocampal CA1 region in the developing mouse.

The aryl hydrocarbon receptor (AhR) avidly binds dioxin, a ubiquitous environmental contaminant. Disruption of downstream AhR signaling has been reported to alter neuronal development, and rodent offspring exposed to dioxin during gestation and lactation showed abnormalities in learning and memory, emotion, and social behavior. However, the mechanism behind the disrupted AhR signaling and developmental neurotoxicity induced by xenobiotic ligands remains elusive. Therefore, we studied how excessive AhR activation affects neuronal migration in the hippocampal CA1 region of the developing mouse brain. We transfected constitutively active (CA)-AhR, AhR, or control vector plasmids into neurons via in utero electroporation on gestational day 14 and analyzed neuronal positioning in the hippocampal CA1 region of offspring on postnatal day 14. CA-AhR transfection affected neuronal positioning, whereas no change was observed in AhR-transfected or control hippocampus. These results suggest that constitutively activated AhR signaling disrupts neuronal migration during hippocampal development. Further studies are needed to investigate whether such developmental disruption in the hippocampus leads to the abnormal cognition and behavior of rodent offspring upon maternal exposure to AhR xenobiotic ligands.

Reelin and Neuropsychiatric Disorders.

Proper neuronal migration and laminar formation during corticogenesis is essential for normal brain function. Disruption of these developmental processes is thought to be involved in the pathogenesis of some neuropsychiatric conditions. Especially, Reelin, a glycoprotein mainly secreted by the Cajal-Retzius cells and a subpopulation of GABAergic interneurons, has been shown to play a critical role, both during embryonic and postnatal periods. Indeed, animal studies have clearly revealed that Reelin is an essential molecule for proper migration of cortical neurons and finally regulates the cell positioning in the cortex during embryonic and early postnatal stages; by contrast, Reelin signaling is closely involved in synaptic function in adulthood. In humans, genetic studies have shown that the reelin gene (RELN) is associated with a number of psychiatric diseases, including Schizophrenia (SZ), bipolar disorder (BP) and autistic spectrum disorder. Indeed, Reln haploinsufficiency has been shown to cause cognitive impairment in rodents, suggesting the expression level of the Reelin protein is closely related to the higher brain functions. However, the molecular abnormalities in the Reelin pathway involved in the pathogenesis of psychiatric disorders are not yet fully understood. In this article, we review the current progress in the understanding of the Reelin functions that could be related to the pathogenesis of psychiatric disorders. Furthermore, we discuss the basis for selecting Reelin and molecules in its downstream signaling pathway as potential therapeutic targets for psychiatric illnesses.

SUMOylation of DISC1: a potential role in neural progenitor proliferation in the developing cortex.

DISC1 is a multifunctional, intracellular scaffold protein. At the cellular level, DISC1 plays a pivotal role in neural progenitor proliferation, migration, and synaptic maturation. Perturbation of the biological pathways involving DISC1 is known to lead to behavioral changes in rodents, which supports a clinical report of a Scottish pedigree in which the majority of family members with disruption of the DISC1 gene manifest depression, schizophrenia, and related mental conditions. The discrepancy of modest evidence in genetics but strong biological support for the role of DISC1 in mental conditions suggests a working hypothesis that regulation of DISC1 at the protein level, such as posttranslational modification, may play a role in the pathology of mental conditions. In this study, we report the SUMOylation of DISC1. This posttranslational modification occurs on lysine residues where small ubiquitin-related modifier (SUMO) and its homologs are conjugated to a large number of cellular proteins, which in turn regulates their subcellular distribution and protein stability. By using in silico, biochemical, and cell biological approaches, we now demonstrate that human DISC1 is SUMOylated at one specific lysine 643 (K643). We also show that this residue is crucial for proper neural progenitor proliferation in the developing cortex.

In Utero Bisphenol A Exposure Induces Abnormal Neuronal Migration in the Cerebral Cortex of Mice.

Bisphenol A (BPA) has been known to have endocrine-disrupting activity to induce reproductive and behavioral abnormalities in offspring of laboratory animal species. However, morphological basis of this abnormality during brain development is largely unknown. Cerebral cortex plays a crucial role in higher brain function, and its precisely laminated structure is formed by neuronal migration. In the present study, transfecting a plasmid (pCAG-mCherry) by in utero electroporation (IUE), we visualized developing neurons and investigated the possible effects of in utero BPA exposure on neuronal migration. Pregnant mice were exposed to BPA by osmotic pump at estimated daily doses of 0, 40 (BPA-40), or 400 (BPA-400) µg/kg from embryonic day 14.5 (E14.5) to E18.5. IUE was performed at E14.5 and neuronal migration was analyzed at E18.5. Compared with the control group, neuronal migration in the cortical plate was significantly decreased in the BPA-40 group; however, there was no significant difference in the BPA-400 group. Among several neuronal migration-related genes and cortical layer-specific genes, TrkB in the BPA-400 group was found significantly upregulated. In conclusion, in utero exposure to low BPA dose was found to disrupt neuronal migration in the cerebral cortex in a dose-specific manner.

Resilience in schizophrenia: A comparative study between a remote island and an urban area in Japan.

The resilience levels between patients with schizophrenia residing in a rural island and a metropolitan area in Tokyo, Japan, was compared and the factors associated with resilience were explored. The Resilience Scale (RS) and EuroQol were assessed, together with biological markers and multiple demographic variables. No significant difference was found in the RS scores between the two groups (40 subjects each). However, longer duration of illness and higher EuroQol score were significantly associated with a greater RS score, which indicates that potentially successful adaptation and subjective perspectives appear more pertinent than the degree of urbanicity in determining resilience levels.

Developmental origin of abnormal dendritic growth in the mouse brain induced by in utero disruption of aryl hydrocarbon receptor signaling.

Increased prevalence of mental disorders cannot be solely attributed to genetic factors and is considered at least partly attributable to chemical exposure. Among various environmental chemicals, in utero and lactational dioxin exposure has been extensively studied and is known to induce higher brain function abnormalities in both humans and laboratory animals. However, how the perinatal dioxin exposure affects neuromorphological alterations has remained largely unknown. Therefore, in this study, we initially studied whether and how the over-expression of aryl hydrocarbon receptor (AhR), a dioxin receptor, would affect the dendritic growth in the hippocampus of the developing brain. Transfecting a constitutively active AhR plasmid into the hippocampus via in utero electroporation on gestational day (GD) 14 induced abnormal dendritic branch growth. Further, we observed that 14-day-old mice born to dams administered with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dose: 0, 0.6, or 3.0 µg/kg) on GD 12.5 exhibited disrupted dendritic branch growth in both the hippocampus and amygdala. Finally, we observed that 16-month-old mice born to dams exposed to perinatal TCDD as described above exhibited significantly reduced spine densities. These results indicated that abnormal micromorphology observed in the developing brain may persist until adulthood and may induce abnormal higher brain function later in life.

Neuronal Heterotopias Affect the Activities of Distant Brain Areas and Lead to Behavioral Deficits.

Neuronal heterotopia refers to brain malformations resulting from deficits of neuronal migration. Individuals with heterotopias show a high incidence of neurological deficits, such as epilepsy. More recently, it has come to be recognized that focal heterotopias may also show a range of psychiatric problems, including cognitive and behavioral impairments. However, because focal heterotopias are not always located in the brain areas responsible for the symptoms, the causal relationship between the symptoms and heterotopias remains elusive. In this study, we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited spatial working memory deficit and low competitive dominance behavior, which have been shown to be closely associated with the activity of the medial prefrontal cortex (mPFC) in rodents. Analysis of the mPFC activity revealed that the immediate-early gene expression was decreased and the local field potentials of the mPFC were altered in the mice with heterotopias compared with the control mice. Moreover, activation of these ectopic and overlying sister neurons using the DREADD (designer receptor exclusively activated by designer drug) system improved the working memory deficits. These findings suggest that cortical regions containing focal heterotopias can affect distant brain regions and give rise to behavioral abnormalities. Significance statement: Recent studies reported that patients with heterotopias have a variety of clinical symptoms, such as cognitive disturbance, psychiatric symptoms, and autistic behavior. However, the causal relationship between the symptoms and heterotopias remains elusive. Here we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited behavioral deficits that have been shown to be associated with the mPFC activity in rodents. The existence of heterotopias indeed altered the neural activities of the mPFC, and direct manipulation of the neural activity of the ectopic neurons and their sister neurons in the overlying cortex improved the behavioral deficit. Thus, our results indicate that focal heterotopias could affect the activities of distant brain areas and cause behavioral abnormalities.