Olfactory information processing viewed through mitral and tufted cell-specific channels.

Parallel processing is a fundamental strategy of sensory coding. Through this processing, unique and distinct features of sensations are computed and projected to the central targets. This review proposes that mitral and tufted cells, which are the second-order projection neurons in the olfactory bulb, contribute to parallel processing within the olfactory system. Based on anatomical and functional evidence, I discuss potential features that could be conveyed through the unique channel formed by these neurons.

Neurogenic timing of the inferior olive subdivisions is related to the olivocerebellar projection topography.

The olivocerebellar projection is organized into an intricate topographical connection from the inferior olive (IO) subdivisions to the longitudinally-striped compartments of cerebellar Purkinje Cells (PCs), to play an essential role in cerebellar coordination and learning. However, the central mechanisms for forming topography need to be clarified. IO neurons and PCs are generated during overlapping periods of a few days in embryonic development. Therefore, we examined whether their neurogenic timing is specifically involved in the olivocerebellar topographic projection relationship. First, we mapped neurogenic timing in the entire IO by using the neurogenic-tagging system of neurog2-CreER (G2A) mice and specific labeling of IO neurons with FoxP2. IO subdivisions were classified into three groups depending on their neurogenic timing range. Then, we examined the relationships in the neurogenic-timing gradient between IO neurons and PCs by labeling topographic olivocerebellar projection patterns and PC neurogenic timing. Early, intermediate, and late groups of IO subdivisions projected to late, intermediate, and early groups of the cortical compartments, respectively, except for a few particular areas. The results indicated that the olivocerebellar topographic relationship is essentially arranged according to the reverse neurogenic-timing gradients of the origin and target.

Spontaneous activity in whisker-innervating region of neonatal mouse trigeminal ganglion.

Spontaneous activity during the early postnatal period is thought to be crucial for the establishment of mature neural circuits. It remains unclear if the peripheral structure of the developing somatosensory system exhibits spontaneous activity, similar to that observed in the retina and cochlea of developing mammals. By establishing an ex vivo calcium imaging system, here we found that neurons in the whisker-innervating region of the trigeminal ganglion (TG) of neonatal mice generate spontaneous activity. A small percentage of neurons showed some obvious correlated activity, and these neurons were mostly located close to one another. TG spontaneous activity was majorly exhibited by medium-to-large diameter neurons, a characteristic of mechanosensory neurons, and was blocked by chelation of extracellular calcium. Moreover, this activity was diminished by the adult stage. Spontaneous activity in the TG during the first postnatal week could be a source of spontaneous activity observed in the neonatal mouse barrel cortex.

NeuroGT: A brain atlas of neurogenic tagging CreER drivers for birthdate-based classification and manipulation of mouse neurons.

Neuronal birthdate is one of the major determinants of neuronal phenotypes. However, most birthdating methods are retrospective in nature, allowing very little experimental access to the classified neuronal subsets. Here, we introduce four neurogenic tagging mouse lines, which can assign CreER-loxP recombination to neuron subsets that share the same differentiation timing in living animals and enable various experimental manipulations of the classified subsets. We constructed a brain atlas of the neurogenic tagging mouse lines (NeuroGT), which includes holistic image data of the loxP-recombined neurons and their processes across the entire brain that were tagged on each single day during the neurodevelopmental period. This image database, which is open to the public, offers investigators the opportunity to find specific neurogenic tagging driver lines and the stages of tagging appropriate for their own research purposes.

Striped Distribution Pattern of Purkinje Cells of Different Birthdates in the Mouse Cerebellar Cortex Studied with the Neurog2-CreER Transgenic Line.

Heterogeneity of Purkinje cells (PCs) that are arranged into discrete longitudinally-striped compartments in the cerebellar cortex is related to the timing of PC generation. To understand the cerebellar compartmental organization, we mapped the PC birthdate (or differentiation timing) in the entire cerebellar cortex. We used the birthdate-tagging system of Neurog2-CreER (G2A) mice hybridized with the AldocV strain which visualizes the zebrin (aldolase C) longitudinal striped pattern. The birthdate-specific distribution pattern of PCs was arranged into longitudinally-oriented stripes consistently throughout almost all lobules except for the nodulus, paraflocculus, and flocculus, in which distinct stripes were observed. Boundaries of the birthdate stripes coincided with the boundary of zebrin stripes or located in the middle of a zebrin stripe. Each birthdate stripe contained PCs born in a particular period between embryonic day (E) 10.0 and E 13.5. In the vermis, PCs were chronologically distributed from lateral to medial stripes. In the paravermis, PCs of early birthdates were distributed in the long lateral zebrin-positive stripe (stripe 4+//5+) and the medially neighboring narrow zebrin-negative substripe (3d-//e2-), while PCs of late birthdates were distributed in the rest of all paravermal areas. In the hemisphere, PCs of early and late birthdates were intermingled in the majority of areas. The results indicate that the birthdate of a PC is a partial determinant for the zebrin compartment in which it is located. However, the correlation between the PC birthdate and the zebrin compartmentalization is complex and distinct among the vermis, paravermis, hemisphere, nodulus, and flocculus.

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.

Common Origin of the Cerebellar Dual Somatotopic Areas Revealed by Tracking Embryonic Purkinje Cell Clusters with Birthdate Tagging.

One of the notable characteristics of the functional localization in the cerebellar cortex is the dual representation of the body (somatotopy) on its anterior-posterior axis. This somatotopy is conspicuous in the C1/C3 module, which is demarcated as the multiple zebrin-negative and weekly-positive stripes in dual paravermal areas in anterior and posterior lobules within the cerebellar compartments. In this report, we describe the early formation process of the cerebellar compartmentalization, particularly in the C1/C3 module. As developing PCs guide formation of the module-specific proper neuronal circuits in the cerebellum, we hypothesized that the rearrangement of embryonic Purkinje cell (PC) clusters shapes the adult cerebellar compartmentalization. By identifying PC clusters with immunostaining of marker molecules and genetical birthdate-tagging with Neurog2-CreER (G2A) mice, we clarified the three-dimensional spatial organization of the PC clusters and tracked the lineage relationships among the PC clusters from embryonic day 14.5 (E14.5) till E17.5. The number of recognized clusters increased from 9 at E14.5 to 37 at E17.5. Among E14.5 PC clusters, the c-l (central-lateral) cluster which lacked E10.5-born PCs divided into six c-l lineage clusters. They separately migrated underneath other clusters and positioned far apart mediolaterally as well as rostrocaudally by E17.5. They were eventually transformed mainly into multiple separate zebrin-negative and weakly-positive stripes, which together configured the adult C1/C3 module, in the anterior and posterior paravermal lobules. The results indicate that the spatial rearrangement of embryonic PC clusters is involved in forming the dual somatotopic areas in the adult mouse paravermal cerebellar cortex.

Chemokine receptor CXCR7 non-cell-autonomously controls pontine neuronal migration and nucleus formation.

Long distance tangential migration transports neurons from their birth places to distant destinations to be incorporated into neuronal circuits. How neuronal migration is guided during these long journeys is still not fully understood. We address this issue by studying the migration of pontine nucleus (PN) neurons in the mouse hindbrain. PN neurons migrate from the lower rhombic lip first anteriorly and then turn ventrally near the trigeminal ganglion root towards the anterior ventral hindbrain. Previously we showed that in mouse depleted of chemokine receptor CXCR4 or its ligand CXCL12, PN neurons make their anterior-to-ventral turn at posteriorized positions. However, the mechanism that spatiotemporally controls the anterior-to-ventral turning is still unclear. Furthermore, the role of CXCR7, the atypical receptor of CXCL12, in pontine migration has yet to be examined. Here, we find that the PN is elongated in Cxcr7 knockout due to a broadened anterior-to-ventral turning positions. Cxcr7 is not expressed in migrating PN neurons en route to their destinations, but is strongly expressed in the pial meninges. Neuroepithelium-specific knockout of Cxcr7 does not recapitulate the PN phenotype in Cxcr7 knockout, suggesting that CXCR7 acts non-cell-autonomously possibly from the pial meninges. We show further that CXCR7 regulates pontine migration by modulating CXCL12 protein levels.

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.

A Novel Birthdate-Labeling Method Reveals Segregated Parallel Projections of Mitral and External Tufted Cells in the Main Olfactory System.

A fundamental strategy in sensory coding is parallel processing, whereby unique, distinct features of sensation are computed and projected to the central target in the form of submodal maps. It remains unclear, however, whether such parallel processing strategy is employed in the main olfactory system, which codes the complex hierarchical odor and behavioral scenes. A potential scheme is that distinct subsets of projection neurons in the olfactory bulb (OB) form parallel projections to the targets. Taking advantage of the observation that the distinct projection neurons develop at different times, we developed a Cre-loxP-based method that allows for birthdate-specific labeling of cell bodies and their axon projections in mice. This birthdate tag analysis revealed that the mitral cells (MCs) born in an early developmental stage and the external tufted cells (TCs) born a few days later form segregated parallel projections. Specifically, the latter subset converges the axons onto only two small specific targets, one of which, located at the anterolateral edge of the olfactory tubercle (OT), excludes widespread MC projections. This target is made up of neurons that express dopamine D1 but not D2 receptor and corresponds to the most anterolateral isolation of the CAP compartments (aiCAP) that were defined previously. This finding of segregated projections suggests that olfactory sensing does indeed involve parallel processing of functionally distinct submodalities. Importantly, the birthdate tag method used here may pave the way for deciphering the functional meaning of these individual projection pathways in the future.

Frequent nonrandom shifts in the temporal sequence of developmental landmark events during teleost evolutionary diversification.

Morphological transformations can be generated by evolutionary changes in the sequence of developmental events. In this study, we examined the evolutionary dynamics of the developmental sequence on a macroevolutionary scale in teleosts. Using the information from previous reports describing the development of 31 species, we extracted the developmental sequences of 19 landmark events involving the formation of phylogenetically conserved body parts; we then inferred ancestral developmental sequences by two different parsimony-based methods-event-pairing and continuous analysis. The phylogenetic comparisons of these sequences revealed event-dependent heterogeneity in the frequency of sequence changes. Most of the sequence changes occurred as exchanges of temporally neighboring events. These heterochronic changes in developmental sequences accumulated along evolutionary time, but the precise distribution of the changes over the teleostean phylogeny remains unclear due to technical limitations.

Timing matters: A strategy for neurons to make diverse connections.

Neurogenesis proceeds like a continuous wave, in which each type of neurons is produced over a few days to several days. During this protracted time window, early-born and late-born neurons are sequentially produced with a considerable time lag. Even if they are identical in their genetic and molecular specifications, they could develop different characteristics under the influences of the timing of their birth. In this review, we discuss the potential influences of "timing" as a generic parameter affecting neuronal differentiation, particularly on axon guidance and connections. These ideas have rarely been tested experimentally, but may provide a new strategy by which phenotypic diversity is increased in neurons.

Netrin-1 Derived from the Ventricular Zone, but not the Floor Plate, Directs Hindbrain Commissural Axons to the Ventral Midline.

Netrin-1 (Ntn1) emanating from the ventral midline has been thought to act as a long-range diffusible chemoattractant for commissural axons (CAs). However, CAs still grow towards the midline in the absence of the floor plate (FP), a glial structure occupying the midline. Here, using genetically loss-of-function approaches in mice, we show that Ntn1 derived from the ventricular zone (VZ), but not the FP, is crucial for CA guidance in the mouse hindbrain. During the period of CA growth, Ntn1 is expressed in the ventral two-thirds of the VZ, in addition to the FP. Remarkably, deletion of Ntn1 from the VZ and even from the dorsal VZ highly disrupts CA guidance to the midline, whereas the deletion from the FP has little impact on it. We also show that the severities of CA guidance defects found in the Ntn1 conditional mutants were irrelevant to their FP long-range chemoattractive activities. Our results are incompatible with the prevailing view that Ntn1 is an FP-derived long-range diffusible chemoattractant for CAs, but suggest a novel mechanism that VZ-derived Ntn1 directs CAs to the ventral midline by its local actions.

Expression patterns of Sema3A in developing amniote limbs: With reference to the diversification of peripheral nerve innervation.

Paired limbs were acquired in the ancestor of tetrapods and their morphology has been highly diversified in amniotes in relation to the adaptive radiation to the terrestrial environment. These morphological changes may have been induced by modification of the developmental program of the skeletal or muscular system. To complete limb modification, it is also important to change the neuronal framework, because the functions of the limbs rely on neural circuits that involve coordinated movement. Previous studies have shown that class 3 semaphorins (Sema3 semaphorins), which act as repulsive axonal guidance cues, play a crucial role in the formation of the peripheral nerves in mice. Here, we studied the expression pattern of Sema3A orthologues in embryos of developing amniotes, including mouse, chick, soft-shelled turtle, and ocelot gecko. Sema3A transcripts were expressed in restricted mesenchymal parts of the developing limb primordium in all animals studied, and developing spinal nerves appeared to extend through Sema3A-negative regions. These results suggest that a Sema3A-dependent guidance system plays a key role in neuronal circuit formation in amniote limbs. We also found that Sema3A partially overlapped with the distribution of cartilage precursor cells. Based on these results, we propose a model in which axon guidance and skeletogenesis are linked by Sema3A; such mechanisms may underlie functional neuron rearrangement during limb diversification.

Reallocation of Olfactory Cajal-Retzius Cells Shapes Neocortex Architecture.

The neocortex undergoes extensive developmental growth, but how its architecture adapts to expansion remains largely unknown. Here, we investigated how early born Cajal-Retzius (CR) neurons, which regulate the assembly of cortical circuits, maintain a dense superficial distribution in the growing neocortex. We found that CR cell density is sustained by an activity-dependent importation of olfactory CR cells, which migrate into the neocortex after they have acted as axonal guidepost cells in the olfactory system. Furthermore, using mouse genetics, we showed that CR cell density severely affects the architecture of layer 1, a key site of input integration for neocortical networks, leading to an excitation/inhibition ratio imbalance. Our study reveals that neurons reenter migration several days after their initial positioning, thereby performing sequential developmental roles in olfactory cortex and neocortex. This atypical process is essential to regulate CR cell density during growth, which in turn ensures the correct wiring of neocortical circuitry. VIDEO ABSTRACT.

Telomere Visualization in Tissue Sections using Pyrrole-Imidazole Polyamide Probes.

Pyrrole-Imidazole (PI) polyamides bind to specific DNA sequences in the minor groove with high affinity. Specific DNA labeling by PI polyamides does not require DNA denaturation with harsh treatments of heat and formamide and has the advantages of rapid and less disruptive processing. Previously, we developed tandem hairpin PI polyamide probes (TH59 series), which label telomeres in cultured cell lines more efficiently than conventional methods, such as fluorescence in situ hybridization (FISH). Here, we demonstrate that a TH59 derivative, HPTH59-b, along with immunostaining for specifying cell types in the tissues, visualizes telomeres in mouse and human tissue sections. Quantitative measurements of telomere length with single-cell resolution suggested shorter telomeres in the proliferating cell fractions of tumor than in non-tumor tissues. Thus, PI polyamides are a promising alternative for telomere labeling in clinical research, as well as in cell biology.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance.

The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system.

A common developmental plan for neocortical gene-expressing neurons in the pallium of the domestic chicken Gallus gallus domesticus and the Chinese softshell turtle Pelodiscus sinensis.

The six-layered neocortex is a unique characteristic of mammals and likely provides the neural basis of their sophisticated cognitive abilities. Although all mammalian species share the layered structure of the neocortex, the sauropsids exhibit an entirely different cytoarchitecture of the corresponding pallial region. Our previous gene expression study revealed that the chicken pallium possesses neural subtypes that express orthologs of layer-specific genes of the mammalian neocortex. To understand the evolutionary steps leading toward animal group-specific neuronal arrangements in the pallium in the course of amniote diversification, we examined expression patterns of the same orthologs and a few additional genes in the pallial development of the Chinese softshell turtle Pelodiscus sinensis, and compared these patterns to those of the chicken. Our analyses highlighted similarities in neuronal arrangements between the two species; the mammalian layer 5 marker orthologs are expressed in the medial domain and the layer 2/3 marker orthologs are expressed in the lateral domain in the pallia of both species. We hypothesize that the mediolateral arrangement of the neocortical layer-specific gene-expressing neurons originated in their common ancestor and is conserved among all sauropsid groups, whereas the neuronal arrangement within the pallium could have highly diversified independently in the mammalian lineage.

The synaptic targeting of mGluR1 by its carboxyl-terminal domain is crucial for cerebellar function.

The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca(2+) release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.