Cortical Divergent Projections in Mice Originate from Two Sequentially Generated, Distinct Populations of Excitatory Cortical Neurons with Different Initial Axonal Outgrowth Characteristics.

Excitatory cortical neurons project to various subcortical and intracortical regions, and exhibit diversity in their axonal connections. Although this diversity may develop from primary axons, how many types of axons initially occur remains unknown. Using a sparse-labeling in utero electroporation method, we investigated the axonal outgrowth of these neurons in mice and correlated the data with axonal projections in adults. Examination of lateral cortex neurons labeled during the main period of cortical neurogenesis (E11.5-E15.5) indicated that axonal outgrowth commonly occurs in the intermediate zone. Conversely, the axonal direction varied; neurons labeled before E12.5 and the earliest cortical plate neurons labeled at E12.5 projected laterally, whereas neurons labeled thereafter projected medially. The expression of Ctip2 and Satb2 and the layer destinations of these neurons support the view that lateral and medial projection neurons are groups of prospective subcortical and callosal projection neurons, respectively. Consistently, birthdating experiments demonstrated that presumptive lateral projection neurons were generated earlier than medial projection neurons, even within the same layer. These results suggest that the divergent axonal connections of excitatory cortical neurons begin from two types of primary axons, which originate from two sequentially generated distinct subpopulations: early-born lateral (subcortical) and later-born medial (callosal) projection neuron groups.

From migration to settlement: the pathways, migration modes and dynamics of neurons in the developing brain.

Neuronal migration is crucial for the construction of the nervous system. To reach their correct destination, migrating neurons choose pathways using physical substrates and chemical cues of either diffusible or non-diffusible nature. Migrating neurons extend a leading and a trailing process. The leading process, which extends in the direction of migration, determines navigation, in particular when a neuron changes its direction of migration. While most neurons simply migrate radially, certain neurons switch their mode of migration between radial and tangential, with the latter allowing migration to destinations far from the neurons' site of generation. Consequently, neurons with distinct origins are intermingled, which results in intricate neuronal architectures and connectivities and provides an important basis for higher brain function. The trailing process, in contrast, contributes to the late stage of development by turning into the axon, thus contributing to the formation of neuronal circuits.

Excitatory cortical neurons with multipolar shape establish neuronal polarity by forming a tangentially oriented axon in the intermediate zone.

The formation of axon-dendrite polarity is crucial for neuron to make the proper information flow within the brain. Although the processes of neuronal polarity formation have been extensively studied using neurons in dissociated culture, the corresponding developmental processes in vivo are still unclear. Here, we illuminate the initial steps of morphological polarization of excitatory cortical neurons in situ, by sparsely labeling their neuroepithelial progenitors using in utero electroporation and then examining their neuronal progeny in brain sections and in slice cultures. Morphological analysis showed that an axon-like long tangential process formed in progeny cells in the intermediate zone (IZ). Time-lapse imaging analysis using slice culture revealed that progeny cells with multipolar shape, after alternately extending and retracting their short processes for several hours, suddenly elongated a long process tangentially. These cells then transformed into a bipolar shape, extending a pia-directed leading process, and migrated radially leaving the tangential process behind, which gave rise to an "L-shaped" axon. Our findings suggest that neuronal polarity in these cells is established de novo from a nonpolarized stage in vivo and indicate that excitatory cortical neurons with multipolar shape in the IZ initiate axon outgrowth before radial migration into the cortical plate.

Formation of axon-dendrite polarity in situ: initiation of axons from polarized and non-polarized cells.

Neurons are polarized cells that extend a single axon and several dendrites. Historically, how neurons establish their axon-dendrite polarity has been extensively studied using dissociated hippocampal cells in culture. Although such studies have identified the cellular and molecular mechanisms underlying axon-dendrite polarization, the conclusions have been limited to in vitro conditions. Recent progress using live imaging has enabled us to directly observe axon formation in situ, revealing distinct cellular mechanisms that regulate axon-dendrite polarization in vivo. In this review, we compare the cellular events during axon formation studied in various systems both in vivo and in vitro and discuss possible common mechanisms underlying the axon-dendrite polarization.

Filamin A-interacting protein (FILIP) regulates cortical cell migration out of the ventricular zone.

Precisely regulated radial migration out of the ventricular zone is essential for corticogenesis. Here, we identify a mechanism that can tether ventricular zone cells in situ. FILIP interacts with Filamin A, an indispensable actin-binding protein that is required for cell motility, and induces its degradation in COS-7 cells. Degradation of Filamin A is identified in the cortical ventricular zone, where filip mRNA is localized. Furthermore, most ventricular zone cells that overexpress FILIP fail to migrate in explants. These results demonstrate that FILIP functions through a Filamin A F-actin axis to control the start of neocortical cell migration from the ventricular zone.

How Do Cortical Excitatory Neurons Terminate Their Migration at the Right Place? Critical Roles of Environmental Elements.

Interactions between neurons and their environment are crucial for proper termination of neuronal migration during brain development. In this review, we first introduce the migration behavior of cortical excitatory neurons from neurogenesis to migration termination, focusing on morphological and behavioral changes. We then describe possible requirements for environmental elements, including extracellular matrix proteins and Cajal-Retzius cells in the marginal zone, radial glial cells, and neighboring neurons, to ensure proper migration termination of these neurons at their final destinations. The requirements appear to be highly linked to sequential and/or concurrent changes in adhesiveness of migrating neurons and their surroundings, which allow the neurons to reach their final positions, detach from substrates, and establish stable laminar structures.

Role of RhoA in activity-dependent cortical axon branching.

During development, axon branching is influenced by sensory-evoked and spontaneous neural activity. We studied the molecular mechanism that underlies activity-dependent branch formation at horizontally elongating axons (horizontal axons) in the upper cortical layers, focusing on Rho family small GTPases. Axonal labeling with enhanced yellow fluorescent protein showed that horizontal axons formed several branches in organotypic slice cultures. This branch formation was considerably increased by introducing constitutively active RhoA and was slightly inhibited by dominant-negative RhoA. Activators and inhibitors of endogenous RhoA signaling also promoted and inhibited branching, respectively. Daily imaging of horizontal axon growth further demonstrated that constitutively active RhoA increased the dynamic addition and loss of branches. Moreover, the amount of active RhoA relative to the total amount of RhoA was examined by a pull-down assay in cortical slices treated with sodium channel or glutamate receptor blockers to reduce neural activity. Activity blockade significantly decreased active RhoA compared with normal culture conditions, in which spontaneous firing is prominent. These findings suggest that RhoA signaling acts as a positive regulator for activity-dependent axon branching in cortical neurons.

Semaphorin 6A-Plexin A2/A4 Interactions with Radial Glia Regulate Migration Termination of Superficial Layer Cortical Neurons.

Precise regulation of neuronal migration termination is crucial for the establishment of brain cytoarchitectures. However, little is known about how neurons terminate migration. Here we focused on interactions between migrating cortical neurons and their substrates, radial glial (RG) cells, and analyzed the role of Plexin A2 and A4 (PlxnA2/A4) receptors and their repulsive ligand, Semaphorin 6A (Sema6A), for this process. In both PlxnA2/A4 double-knockout and Sema6A mutant mice, the outermost cortical plate neurons ectopically invade layer 1 at a stage when they should reach their destinations. PlxnA2/A4 proteins are abundantly expressed on their leading processes, whereas Sema6A mRNA is enriched in RG cell somata. Cell-targeted gene expression and conditional knockouts indicate critical roles for these molecules. We hypothesize that the timely appearance of repulsive signaling mediated by Sema6A-PlxnA2/A4 weakens migrating neuron-RG cell interactions, leading to migration termination.

One-chip sensing device (biomedical photonic LSI) enabled to assess hippocampal steep and gradual up-regulated proteolytic activities.

We developed an implantable one-chip biofluoroimaging device (termed biomedical photonic LSI; BpLSI) which enabled real-time molecular imaging with conventional electrophysiology in vivo in deep brain areas. The multimodal LSI enabled long-term sequential imaging of the fluorescence emitted by proteolysis-linked fluorogenic substrate. Using the BpLSI, we observed a process of stimulation-dependent modulation at synapse with multi-site (16 x 19 pixel) in widespread area and a high-speed video rate, and found that the gradual up-regulated proteolytic activity in a wide range of hippocampal CA1 area and the steep activity in local area, indicating that the proteolysis system is a basis for the fixation of long-term potentiation in post-excited synapses in the hippocampus. Mathematical data analysis confirmed the direct involvement of functional proteolysis for neural plasticity.

Neuronal polarization in the developing cerebral cortex.

Cortical neurons consist of excitatory projection neurons and inhibitory GABAergic interneurons, whose connections construct highly organized neuronal circuits that control higher order information processing. Recent progress in live imaging has allowed us to examine how these neurons differentiate during development in vivo or in in vivo-like conditions. These analyses have revealed how the initial steps of polarization, in which neurons establish an axon, occur. Interestingly, both excitatory and inhibitory cortical neurons establish neuronal polarity de novo by undergoing a multipolar stage reminiscent of the manner in which polarity formation occurs in hippocampal neurons in dissociated culture. In this review, we focus on polarity formation in cortical neurons and describe their typical morphology and dynamic behavior during the polarization period. We also discuss cellular and molecular mechanisms underlying polarization, with reference to polarity formation in dissociated hippocampal neurons in vitro.

In vitro analysis of the origin, migratory behavior, and maturation of cortical pyramidal cells.

During development neurons migrate from their site of origin to their final destinations under a variety of mechanisms. Although evidence has been accumulating that the cells from cortical ventricular zone (VZ) migrate radially and produce pyramidal cells, evidence that directly links the origin and the terminal phenotype of radially migrating cells has been limited. Further, the relation between the migratory behavior of these cells and their mature morphology remains obscure. To address these issues, we developed an in vitro preparation that enables visualization of cells derived from the cortical VZ. VZ cells of a rat cortex at embryonic days 18 to 19 were labeled by injecting green fluorescent protein (GFP)-encoding plasmid into the lateral ventricle, followed by electroporation. The cortex was then sliced and cultured organotypically. After 1 day, GFP(+) cells exhibited neural progenitor and radial glial cell natures. Over the next few days, many GFP(+) cells migrated toward the pial surface, extending leading processes toward the pial surface and leaving a thin trailing process that almost reached the VZ. The leading processes of these neurons were positive for microtubule-associated protein 2, and some transformed into dendritic arbor-like structures by day 5 or 6, and their trailing processes exhibited morphologic features indicative of prospective axons. Time-lapse analysis confirmed extension of the trailing processes. Expression of molecular markers and morphologic analysis demonstrated that the vast majority of the migrated GFP(+) cells differentiated into excitatory neurons with pyramidal cell-like morphology. These results strongly suggested that cells derived from the cortical VZ generate neurons that migrate radially. These neurons appeared to extend prospective dendrites in front and leave prospective axons behind, subsequently differentiating into pyramidal cells.

Distinct migratory behavior of early- and late-born neurons derived from the cortical ventricular zone.

Time-lapse studies indicate that ventricular zone (VZ)-derived cells show two migratory modes in the cerebral cortex at different stages of mammalian embryogenesis: somal translocation and locomotion. We carried out a systematic analysis to examine whether the migratory behavior of cortical neurons derived from the cortical VZ is stage-dependent. We labeled VZ cells of mouse embryos with green fluorescent protein (gfp) -encoding plasmids by in utero electroporation and evaluated the labeled cells after appropriate survival periods. After electroporation at either embryonic day (E) 12.5 or E15.5, GFP+ VZ cells were initially spindle-shaped and radially oriented. After leaving the VZ, they transformed into round or horizontally oriented fusiform neurons with many short processes. They then seemed to gradually change into radially oriented bipolar cells as they moved upward. Whereas the earliest emigrants from the VZ labeled at E12.5 (early-born neurons) reached the top of the cortical plate (CP) after these changes, VZ cells labeled at E15.5 (late-born neurons) further migrated along the length of radial fibers to reach the top of the CP. A dominant negative form of the gene for cyclin-dependent kinase 5 (Cdk5DN) was then introduced into VZ cells. Transfection of E12.5 VZ with cdk5dn did not disrupt the migration of the early-born neurons. However, this caused a failure in migration of the late-born neurons, although they transformed into bipolar shapes in the intermediate zone. Thus, there appear to be at least two distinct migratory phases of cortical neurons: one common to the early- and late-born neurons, and the other specific to late-born neurons and Cdk5-dependent.

Distinct roles of neuropilin 1 signaling for radial and tangential extension of callosal axons.

Cortical excitatory neurons migrate from their origin in the ventricular zone (VZ) toward the pial surface. During migration, these neurons exhibit a stellate shape in the intermediate zone (IZ), transform into bipolar cells, and then initiate radial migration, extending a trailing process, which may lead to an axon. Here we examined the role of neuropilin 1 (NRP1) in these developmental events. Both NRP1 mRNA and protein were highly expressed in the IZ, where stellate-shaped cells were located. DiI labeling experiments showed that neuronal migration occurred normally in Nrp1 mutant mice up to embryonic day (E) 14.5, the latest day to which the mutant survives, with only subtle axonal defasciculation. However, interference with Nrp1 signaling at a later stage caused pathfinding errors: when a dominant negative form of Nrp1 was electroporated into the cortical VZ cells at E12.5 or E15.5 and examined perinatally, guidance errors were found in tangential axonal extension toward the midline. In contrast, no significant effect was noted on the migration of cortical excitatory neurons. These findings indicate that NRP1 plays an important role in the guidance of callosal axons originating from cortical excitatory neurons but does not support a role in their migration. Moreover, insofar as radial axonal extension within the cortical plate was unaffected, the present findings imply that molecular mechanisms for the axonal extension of excitatory neurons within the cortical plate are distinct from those in the white matter.

A multimodal sensing device for fluorescence imaging and electrical potential measurement of neural activities in a mouse deep brain.

We have developed a multimodal CMOS sensing device to detect fluorescence image and electrical potential for neural activities in a mouse deep brain. The device consists of CMOS image sensor with on-chip electrodes and excitation light sources, all of which are integrated on a polyimide substrate. The novel feature of this device is its embedded on-chip electrodes which are partially transmit incident light so that the whole image can be acquired by the sensor. We have demonstrated the CMOS sensor device successfully operates in hippocampus area of an anesthetized mouse.

Crucial roles of Robo proteins in midline crossing of cerebellofugal axons and lack of their up-regulation after midline crossing.

BACKGROUND: Robo1, Robo2 and Rig-1 (Robo3), members of the Robo protein family, are candidate receptors for the chemorepellents Slit and are known to play a crucial role in commissural axon guidance in the spinal cord. However, their roles at other axial levels remain unknown. Here we examine expression of Robo proteins by cerebellofugal (CF) commissural axons in the rostral hindbrain and investigate their roles in CF axon pathfinding by analysing Robo knockout mice. RESULTS: We analysed the expression of Robo proteins by CF axons originating from deep cerebellar neurons in rodent embryos, focusing on developmental stages of their midline crossing and post-crossing navigation. At the stage of CF axon midline crossing, mRNAs of Robo1 and Robo2 are expressed in the nuclear transitory zone of the cerebellum, where the primordium of the deep cerebellar nuclei are located, supporting the notion that CF axons express Robo1 and Robo2. Indeed, immunohistochemical analysis of CF axons labelled by electroporation to deep cerebellar nuclei neurons indicates that Robo1 protein, and possibly also Robo2 protein, is expressed by CF axons crossing the midline. However, weak or no expression of these proteins is found on the longitudinal portion of CF axons. In Robo1/2 double knockout mice, many CF axons reach the midline but fail to exit it. We find that CF axons express Rig-1 (Robo3) before they reach the midline but not after the longitudinal turn. Consistent with this in vivo observation, axons elicited from a cerebellar explant in co-culture with a floor plate explant express Rig-1. In Rig-1 deficient mouse embryos, CF axons appear to project ipsilaterally without reaching the midline. CONCLUSION: These results indicate that Robo1, Robo2 or both are required for midline exit of CF axons. In contrast, Rig-1 is required for their approach to the midline. However, post-crossing up-regulation of these proteins, which plays an important role in spinal commissural axon guidance, does not appear to be required for the longitudinal navigation of CF axons after midline crossing. Our results illustrate that although common mechanisms operate for midline crossing at different axial levels, significant variation exists in post-crossing navigation.