Cajal-Retzius Research Articles

Dentate gyrus morphogenesis is regulated by β-catenin function in hem-derived fimbrial glia.

The dentate gyrus, a gateway for input to the hippocampal formation, arises from progenitors in the medial telencephalic neuroepithelium adjacent to the cortical hem. Dentate progenitors navigate a complex migratory path guided by two cell populations that arise from the hem, the fimbrial glia and Cajal-Retzius (CR) cells. As the hem expresses multiple Wnt genes, we examined whether β-catenin, which mediates canonical Wnt signaling and also participates in cell adhesion, is necessary for the development of hem-derived lineages. We report that, in mice, the fimbrial glial scaffold is disorganized and CR cells are mispositioned upon hem-specific disruption of β-catenin. Consequently, the dentate migratory stream is severely affected, and the dentate gyrus fails to form. Using selective Cre drivers, we further determined that β-catenin function is required in the fimbrial glial scaffold, but not in the CR cells, for guiding the dentate migration. Our findings highlight a primary requirement for β-catenin for the organization of the fimbrial scaffold and a secondary role for this factor in dentate gyrus morphogenesis.

Specific contribution of Reelin expressed by Cajal–Retzius cells or GABAergic interneurons to cortical lamination

The extracellular protein Reelin, expressed by Cajal-Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the "inside-out" pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell-derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron-derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell-derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell-derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits.

Tetrodotoxin prevents heat-shock induced granule cell dispersion in hippocampal slice cultures

Granule cell dispersion (GCD) has been associated as a pathological feature of temporal lobe epilepsy (TLE). Early-life epileptiform activity such as febrile seizures has been proposed to have a causal link to developing chronic TLE. During postnatal development, the hippocampus may be particularly vulnerable to hyperexcitability-induced insults since neuronal migration and differentiation are still ongoing in the hippocampus. Further, the extracellular matrix (ECM), here in particular the protein reelin, has been implicated in the pathophysiology of GCD. Thus, loss of reelin-expressing cells, Cajal-Retzius cells and subsets of interneurons, may be related to GCD. To study the possible role of febrile seizures, we previously induced GCD in vitro by subjecting hippocampal slice cultures to a transient heat-shock, which was not accompanied by loss of Cajal-Retzius cells. In order to examine the mechanisms involved in heat-shock induced GCD, the present study aimed to determine whether such dispersion could be prevented by blocking cellular electrical activity. Here we show that the extent of heat-shock induced GCD could be significantly reduced by treatment with the sodium channel blocker tetrodotoxin (TTX), suggesting that electrical activity is an important factor involved in heat-shock induced GCD.

Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development.

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.

Glutamate released by Cajal-Retzius cells impacts specific hippocampal circuits and behaviors.

SUMMARYThe impact of Cajal-Retzius cells on the regulation of hippocampal circuits and related behaviors is unresolved. Here, we directly address this issue by impairing the glutamatergic output of Cajal-Retzius cells with the conditional ablation of vGluT2, which is their main vesicular glutamate transporter. Although two distinct conditional knockout lines do not reveal major alterations in hippocampal-layer organization and dendritic length of principal neurons or GABAergic cells, we find parallel deficits in specific hippocampal-dependent behaviors and in their putative underlying microcircuits. First, conditional knockout animals show increased innate anxiety and decreased feedforward GABAergic inhibition on dentate gyrus granule cells. Second, we observe impaired spatial memory processing, which is associated with decreased spine density and reduced AMPA/NMDA ratio of postsynaptic responses at the perforant- and entorhino-hippocampal pathways. We conclude that glutamate synaptically released by Cajal-Retzius cells is critical for the regulation of hippocampal microcircuits and specific types of behaviors.

Dynamic interplay between thalamic activity and Cajal-Retzius cells regulates the wiring of cortical layer 1.

SummaryCortical wiring relies on guidepost cells and activity-dependent processes that are thought to act sequentially. Here, we show that the construction of layer 1 (L1), a main site of top-down integration, is regulated by crosstalk between transient Cajal-Retzius cells (CRc) and spontaneous activity of the thalamus, a main driver of bottom-up information. While activity was known to regulate CRc migration and elimination, we found that prenatal spontaneous thalamic activity and NMDA receptors selectively control CRc early density, without affecting their demise. CRc density, in turn, regulates the distribution of upper layer interneurons and excitatory synapses, thereby drastically impairing the apical dendrite activity of output pyramidal neurons. In contrast, postnatal sensory-evoked activity had a limited impact on L1 and selectively perturbed basal dendrites synaptogenesis. Collectively, our study highlights a remarkable interplay between thalamic activity and CRc in L1 functional wiring, with major implications for our understanding of cortical development.

Phosphorylation of Focal Adhesion Kinase at Y925: Role in Glia-Dependent and Independent Migration through Regulating Cofilin and N-Cadherin.

The adult neocortex is a six-layered structure, consisting of nearly continuous layers of neurons that are generated in a temporally strictly coordinated order. During development, cortical neurons originating from the ventricular zone migrate toward the Reelin-containing marginal zone in an inside-out arrangement. Focal adhesion kinase (FAK), one tyrosine kinase localizing to focal adhesions, has been shown to be phosphorylated at tyrosine 925 (Y925) by Src, an important downstream molecule of Reelin signaling. Up to date, the precise molecular mechanisms of FAK and its phosphorylation at Y925 during neuronal migration are still unclear. Combining in utero electroporation with immunohistochemistry and live imaging, we examined the function of FAK in regulating neuronal migration. We show that phosphorylated FAK is colocalized with Reelinpositive Cajal-Retzius cells in the developing neocortex and hippocampus. Phosphorylation of FAK at Y925 is significantly reduced in reeler mice. Overexpression and dephosphorylation of FAK impair locomotion and translocation, resulting in migration inhibition and dislocation of both late-born and early-born neurons. These migration defects are highly correlated to the function of FAK in regulating cofilin phosphorylation and N-Cadherin expression, both are involved in Reelin signaling pathway. Thus, fine-tuned phosphorylation of focal adhesion kinase at Y925 is crucial for both glia-dependent and independent neuronal migration.

New Strategies for the Treatment of Neuropsychiatric Disorders Based on Reelin Dysfunction.

Reelin is an extracellular matrix protein that is mainly produced in Cajal-Retzius cells and controls neuronal migration, which is important for the proper formation of cortical layers in the developmental stage of the brain. In the adult brain, Reelin plays a crucial role in the regulation of N-methyl-D-aspartate receptor-dependent synaptic function, and its expression decreases postnatally. Clinical studies showed reductions in Reelin protein and mRNA expression levels in patients with psychiatric disorders; however, the causal relationship remains unclear. Reelin-deficient mice exhibit an abnormal neuronal morphology and behavior, while Reelin supplementation ameliorates learning deficits, synaptic dysfunctions, and spine loss in animal models with Reelin deficiency. These findings suggest that the neuronal deficits and brain dysfunctions associated with the down-regulated expression of Reelin are attenuated by enhancements in its expression and functions in the brain. In this review, we summarize findings on the role of Reelin in neuropsychiatric disorders and discuss potential therapeutic approaches for neuropsychiatric disorders associated with Reelin dysfunctions.

Poly (ADP-Ribose) Polymerase 1 Regulates Cajal-Retzius Cell Development and Neural Precursor Cell Adhesion.

Poly (ADP-ribose) polymerase 1 (PARP1) is a ubiquitously expressed enzyme that regulates DNA damage repair, cell death, inflammation, and transcription. PARP1 functions by adding ADP-ribose polymers (PAR) to proteins including itself, using NAD+ as a donor. This post-translational modification known as PARylation results in changes in the activity of PARP1 and its substrate proteins and has been linked to the pathogenesis of various neurological diseases. PARP1 KO mice display schizophrenia-like behaviors, have impaired memory formation, and have defects in neuronal proliferation and survival, while mutations in genes that affect PARylation have been associated with intellectual disability, psychosis, neurodegeneration, and stroke in humans. Yet, the roles of PARP1 in brain development have not been extensively studied. We now find that loss of PARP1 leads to defects in brain development and increased neuronal density at birth. We further demonstrate that PARP1 loss increases the expression levels of genes associated with neuronal migration and adhesion in the E15.5 cerebral cortex, including Reln. This correlates with an increased number of Cajal–Retzius (CR) cells in vivo and in cultures of embryonic neural progenitor cells (NPCs) derived from the PARP1 KO cortex. Furthermore, PARP1 loss leads to increased NPC adhesion to N-cadherin, like that induced by experimental exposure to Reelin. Taken together, these results uncover a novel role for PARP1 in brain development, i.e., regulation of CR cells, neuronal density, and cell adhesion.

Analysis of the Expression Pattern of Cajal-Retzius Cell Markers in the Xenopus laevis Forebrain

Cajal-Retzius cells are essential for cortical development in mammals, and their involvement in the evolution of this structure has been widely postulated, but very little is known about their progenitor domains in non-mammalian vertebrates. Using in situhybridization and immunofluorescence techniques we analyzed the expression of some of the main Cajal-Retzius cell markers such as Dbx1, Ebf3, ER81, Lhx1, Lhx5, p73, Reelin, Wnt3a, Zic1, and Zic2 in the forebrain of the anuran Xenopus laevis, because amphibians are the only class of anamniote tetrapods and show a tetrapartite evaginated pallium, but no layered or nuclear organization. Our results suggested that the Cajal-Retzius cell progenitor domains were comparable to those previously described in amniotes. Thus, at dorsomedial telencephalic portions a region comparable to the cortical hem was defined in Xenopus based on the expression of Wnt3a, p73, Reelin, Zic1, and Zic2. In the septum, two different domains were observed: a periventricular dorsal septum, at the limit between the pallium and the subpallium, expressing Reelin, Zic1, and Zic2, and a related septal domain, expressing Ebf3, Zic1, and Zic2. In the lateral telencephalon, the ventral pallium next to the pallio-subpallial boundary, the lack of Dbx1 and the unique expression of Reelin during development defined this territory as the most divergent with respect to mammals. Finally, we also analyzed the expression of these markers at the prethalamic eminence region, suggested as Cajal-Retzius progenitor domain in amniotes, observing there Zic1, Zic2, ER81, and Lhx1 expression. Our data show that in anurans there are different subtypes and progenitor domains of Cajal-Retzius cells, which probably contribute to the cortical regional specification and territory-specific properties. This supports the notion that the basic organization of pallial derivatives in vertebrates follows a comparable fundamental arrangement, even in those that do not have a sophisticated stratified cortical structure like the mammalian cerebral cortex.

Reelin restricts dendritic growth of interneurons in the neocortex.

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation.

SMI-32 labeling in Cajal-Retzius cells of feline primary visual cortex

Cajal-Retzius cells are one of the transient elements of the developing cerebral cortex. These cells express some characteristic molecules. One of them, heavy-chain neurofilaments, participating in the construction of the mature cerebral networks, are believed to be a specific feature of the human’s Cajal-Retzius cells. Using histochemical stain for SMI-32 antibody to the non-phosphorylated heavy-chain neurofilaments, large neurons having horizontally oriented soma and bipolar processes were labeled in the molecular layer of the primary visual cortex of cats aged 0–2 postnatal days. Using DiI technique, similar neurons having a well-developed system of parallel vertical branches coming from the two horizontal processes were visualized in these areas. The location and general morphology of these neurons were similar to the Cajal-Retzius cells allowing to suppose for the carnivores to share similar with primates developmental mechanisms of the corticogenesis.

Transitory and Vestigial Structures of the Developing Human Nervous System

As with many body organs, the human central nervous system contains many structures and cavities that may have had functions in embryonic and fetal life but are vestigial or atrophic at maturity. Examples are the septum pellucidum, remnants of the lamina terminalis, Cajal-Retzius neurons, induseum griseum, habenula, and accessory olfactory bulb. Other structures are transitory in fetal or early postnatal life, disappearing from the mature brain. Examples are the neural crest, subpial granular glial layer of Brun over cerebral cortex, radial glial cells, and subplate zone of cerebral cortex. At times persistent fetal structures that do not regress may cause neurological problems or indicate a pathologic condition, such as Blake pouch cyst. Transitory structures thus can become vestigial. Examples are an excessively wide cavum septi pellucidi, suprapineal recess of the third ventricle, trigeminal artery of the posterior fossa circulation, and hyaloid ocular artery. Arrested maturation might be considered another aspect of vestigial structure. An example is the persistent microcolumnar cortical architecture in focal cortical dysplasia type Ia, in cortical zones of chronic fetal ischemia, and in some metabolic/genetic congenital encephalopathies. Some transitory structures in human brain are normal adult structures in lower vertebrates. Recognition of transitory and vestigial structures by fetal or postnatal neuroimaging and neuropathologically enables better understanding of cerebral ontogenesis and avoids misinterpretations.

The multiple facets of Cajal-Retzius neurons

Cajal-Retzius neurons (CRs) are among the first-born neurons in the developing cortex of reptiles, birds and mammals, including humans. The peculiarity of CRs lies in the fact they are initially embedded into the immature neuronal network before being almost completely eliminated by cell death at the end of cortical development. CRs are best known for controlling the migration of glutamatergic neurons and the formation of cortical layers through the secretion of the glycoprotein reelin. However, they have been shown to play numerous additional key roles at many steps of cortical development, spanning from patterning and sizing functional areas to synaptogenesis. The use of genetic lineage tracing has allowed the discovery of their multiple ontogenetic origins, migratory routes, expression of molecular markers and death dynamics. Nowadays, single-cell technologies enable us to appreciate the molecular heterogeneity of CRs with an unprecedented resolution. In this Review, we discuss the morphological, electrophysiological, molecular and genetic criteria allowing the identification of CRs. We further expose the various sources, migration trajectories, developmental functions and death dynamics of CRs. Finally, we demonstrate how the analysis of public transcriptomic datasets allows extraction of the molecular signature of CRs throughout their transient life and consider their heterogeneity within and across species.

Development of the mammalian cortical hem and its derivatives: the choroid plexus, Cajal-Retzius cells and hippocampus.

The dorsal medial region of the developing mammalian telencephalon plays a central role in the patterning of the adjacent brain regions. This review describes the development of this specialized region of the vertebrate brain, called the cortical hem, and the formation of the various cells and structures it gives rise to, including the choroid plexus, Cajal-Retzius cells and the hippocampus. We highlight the ontogenic processes that create these different forebrain derivatives from their shared embryonic origin and discuss the key signalling pathways and molecules that influence the patterning of the cortical hem. These include BMP, Wnt, FGF and Shh signalling pathways acting with Homeobox factors to carve the medial telencephalon into district progenitor regions, which in turn give rise to the choroid plexus, dentate gyrus and hippocampus. We then link the formation of the lateral ventricle choroid plexus with embryonic and postnatal neurogenesis in the hippocampus.

BBS4 protein has basal body/ciliary localization in sensory organs but extra-ciliary localization in oligodendrocytes during human development

Bardet-Biedl syndrome protein 4 (BBS4) localization has been studied in human embryos/fetuses from Carnegie stage 15 to 37 gestational weeks in neurosensory organs and brain, underlying the major clinical signs of BBS. We observed a correlation between the differentiation of the neurosensory cells (hair cells, photoreceptors, olfactory neurons) and the presence of a punctate BBS4 immunostaining in their apical cytoplasm. In the brain, BBS4 was localized in oligodendrocytes and myelinated tracts. In individual myelinated fibers, BBS4 immunolabelling was discontinuous, predominantly at the periphery of the myelin sheath. BBS4 immunolabelling was confirmed in postnatal developing white matter tracts in mouse as well as in mouse oligodendrocytes cultures. In neuroblasts/neurons, BBS4 was only present in reelin-expressing Cajal-Retzius cells. Our results show that BBS4, a protein of the BBSome, has both basal body/ciliary localization in neurosensory organs but extra-ciliary localization in oligodendrocytes. The presence of BBS4 in developing oligodendrocytes and myelin described in the present paper might attribute a new role to this protein, requiring further investigation in the field of myelin formation.

Integrative genomic analysis of early neurogenesis reveals a temporal genetic program for differentiation and specification of preplate and Cajal-Retzius neurons.

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Heat-Shock Induces Granule Cell Dispersion and Microgliosis in Hippocampal Slice Cultures.

Granule cell dispersion (GCD) has been found in the dentate gyrus (dg) of patients with temporal lobe epilepsy (TLE) and a history of febrile seizures but was also recently observed in pediatric patients that did not suffer from epilepsy. This indicates that GCD might not always be disease related, but instead could reflect normal morphological variation. Thus, distribution of newborn granule cells within the hilar region is part of normal dg development at early stages but could be misinterpreted as pathological GCD. In turn, pathological GCD may be caused, for example, by genetic mutations, such as the reeler mutation. GCD in the reeler mutant goes along with an increased susceptibility to epileptiform activity. Pathological GCD in combination with epilepsy is caused by experimental administration of the glutamate receptor agonist kainic acid in rodents. In consequence, the interpretation of GCD and the role of febrile seizures remain controversial. Here, we asked whether febrile temperatures alone might be sufficient to trigger GCD and used hippocampal slice cultures as in vitro model to analyze the effect of a transient temperature increase on the dg morphology. We found that a heat-shock of 41°C for 6 h was sufficient to induce GCD and degeneration of a fraction of granule cells. Both of these factors, broadening of the granule cell layer (gcl) and increased neuronal cell death within the gcl, contributed to the development of a significantly reduced packaging density of granule cells. In contrast, Reelin expressing Cajal–Retzius (CR) cells in the molecular layer were heat-shock resistant. Thus, their number was not reduced, and we did not detect degenerating CR cells after heat-shock, implying that GCD was not caused by the loss of CR cells. Importantly, the heat-shock-induced deterioration of dg morphology was accompanied by a massive microgliosis, reflecting a robust heat-shock-induced immune response. In contrast, in the study that reported on GCD as a non-specific finding in pediatric patients, no microglia reaction was observed. Thus, our findings underpin the importance of microglia as a marker to distinguish pathological GCD from normal morphological variation.

Cell cycle during neuronal migration and neocortical lamination.

In order to understand the relationships between neocortical lamination and cell cycle, various cells, such as neural stem cell, migrating postmitotic neuron, Cajal-Retzius (CR) cell, and mature pyramidal cell in various cell phases were investigated in mouse cortices. With mouse neocortex and hippocampus, the immunofluorescent labeling, BrdU assay, and DiI tracing technique were implemented in the study. (1) During mouse development, the neocortex expressed different proteins, such as FOXP2, CDP, and Nestin, which could be used as the markers for cortical lamination. (2) The neural stem cells were mainly located in the subventricular zone, with the expressions of Nestin, Cyclin A2, Cyclin E1, and CDT1, suggesting that they were in the repeated cell cycle. Furthermore, the migrating neurons in the neocortex were Cyclin D1- (G1 phase-specific marker) positive, suggesting that they were in the G1 phase. However, Pyramidal cells that developed from postmitotic migrating neurons and settled in the cortical plate were Cyclin D1- negative, suggesting that they were in the G0 phase. (3) Reelin positive CR cells appeared in the molecular layer of the neocortex in early embryonic day (E10), which could express Cyclin A2, Cyclin E1, and CDT1 as pyramidal cells, but not Cyclin D1, suggesting that they may have exited the cell cycle and entered the G0 phase. The neural migration, neural proliferation, and cell cycle alterations play an important role during cortical lamination. During the cortical development and lamination, the neural stem cells and migrating postmitotic neurons are in different cell cycle phases, but pyramidal cells and CR cells have exited the cell cycle.

Analysis of Reelin signaling and neurodevelopmental trajectory in primary cultured cortical neurons with RELN deletion identified in schizophrenia

Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca2+/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.