Fujio Murakami

Abstract: 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.

Abstract: Nuclei are anatomical units of the central nervous system (CNS). Their formation sets the structural basis for the functional organization of the brain, a process known as nucleogenesis. In the present study, we investigated the role of the transmembrane immunoglobulin superfamily molecule Neph2 in the nucleogenesis of the pontine nucleus (PN). Neph2 expression is turned on in migrating PN neurons only after they enter the presumptive nuclear region. Neph2 knockdown disrupted the nuclear organization of PN presumably by changing the migratory behavior of PN neurons inside the nuclear region. Moreover, overexpression of the cytoplasmic region of Neph2, which can sequester intracellular signaling of endogenous Neph2, resulted in similar phenotypes. Overall, these results suggest Neph2 is involved in the nucleogenesis of the PN through the control of neuronal migration inside the nucleus.

Abstract: Ptf1a, a basic helix-loop-helix transcription factor, plays an indispensable role for cell fate specification of subsets of neurons in the developing central nervous system. However, downstream molecules induced by Ptf1a during neural development have not been well characterized. In the present study, we identified immunoglobulin superfamily molecules, Nephrin and Neph3, as direct downstream targets of Ptf1a. First, the expression domains of Nephrin and Neph3 closely resembled those of Ptf1a in the developing retina, hypothalamus, cerebellum, hindbrain, and spinal cord. Second, Ptf1a bound directly to a PTF-binding motif in the 5'-flanking region of Nephrin and Neph3 genes. Third, Ptf1a activated transcription driven by the 5'-flanking region of these genes. Finally, the expression of Nephrin and Neph3 was lost in Ptf1a-null mice, whereas ectopic expression of Nephrin and Neph3 was induced by forced expression of Ptf1a. We provided further evidence that Nephrin and Neph3 could interact homophilically and heterophilically, suggesting that Nephrin and Neph3 might regulate certain developmental aspects of Ptf1a-positive neurons as homo- or heterooligomers.

Abstract: Cortical GABAergic interneurons are divided into various subtypes, with each subtype contributing to rich variety and fine details of inhibition. Despite the functional importance of each interneuron subtype, the molecular mechanisms that contribute to sorting them to their appropriate positions within the cortex remain unclear. Here, we show that the chemokine receptor CXCR4 regulates the regional and layer-specific distribution of interneuron subtypes. We removed Cxcr4 specifically in a subset of interneurons at a specific mouse embryonic developmental stage and analyzed the number of interneurons and their laminar distribution in 9 representative cortical regions comprehensively in adults. We found that the number of Cxcr4-deleted calretinin- and that of neuropeptide Y-expressing interneurons were reduced in most caudomedial and lateral cortical regions, respectively, and also in superficial layers. In addition, Cxcr4-deleted somatostatin-expressing interneurons showed a reduction in the number of superficial layers in certain cortical regions but of deep layers in others. These findings suggest that CXCR4 is required for proper regional and laminar distribution in a wider interneuron subpopulation than previously thought and may regulate the establishment of functional cortical circuitry in certain cortical regions and layers.

Abstract: Mature neurons polarize by extending an axon and dendrites. In vitro studies of dissociated neurons have demonstrated that axons are initiated from a nonpolarized stage. Dissociated hippocampal neurons form four to five minor neurites shortly after plating but then one of them starts to elongate rapidly to become the future axon, whereas the rest constitutes the dendrites at later stages. However, neuroepithelial cells as well as migrating neurons in vivo are already polarized, raising the possibility that mature neurons inherit the polarities of immature neurons of neuroepithelial or migrating neurons. Here we show that the axon of interneurons in mouse cortical explant emerges from a morphologically nonpolarized shape. The morphological maturation of cortical interneurons labeled by electroporation at an embryonic stage was analyzed by time-lapse imaging during the perinatal stage. In contrast to earlier stages, most interneurons at this stage show sea urchin-like nonpolarized shapes with alternately extending and retracting short processes. Abruptly, one of these processes extends to give rise to an outstandingly long axon-like process. Given that the interneurons exhibit typical polarized shapes during embryonic development, the present results suggest that axon-dendrite polarity develops from a nonpolarized intermediate stage.

Abstract: The generation of distinct neural subtypes depends on the activities of cell-extrinsic and -intrinsic factors during the development of the vertebrate CNS. Previous studies have provided a molecular basis for how neural progenitors are patterned and generate distinct descendants that are spatially- and temporally-regulated by inductive signals secreted by polarized sources. However, it still remains unknown how the generation of neural descendants by progenitors located at polarized sources of inductive signals is controlled. Sonic hedgehog (Shh), which is expressed at the ventral midline in the forebrain, has been shown to play a critical role for the patterning and specification of distinct neural subtypes in the forebrain. Here, we analyzed the identities and distributions of Shh-descendants generated at discrete time points in the forebrain by using a ShhcreER(T2) mouse driver line in which a tamoxifen-inducible Cre cassette was inserted into the Shh locus together with a Z/EG mouse reporter line. Our results showed that Shh-expressing neural progenitors generated neuronal and glial descendants distributed throughout the telencephalon and diencephalon in a temporally distinct manner. Furthermore, our results showed that Shh-progenitors are located at two spatially distinct sub-domains that can be characterized by their temporally distinct patterns of Shh expression. These results suggest that temporally- and spatially-controlled mechanisms that specify neural subtypes operate in the Shh-expressing progenitor domain, and raise the possibility that the distinct temporal gradient of Shh activity might be responsible for the generation of distinct neural subtypes in the telencephalon. © 2010 Wiley Periodicals, Inc. Develop Neurobiol, 2010.

Abstract: The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.

Abstract: The development of mossy-fibre projecting precerebellar neurons (PCN) presents a classical example of tangential neuronal migration. PCN migrate tangentially along marginal streams beneath the pial surface from the lower rhombic lip to specific locations in the hindbrain, where they form precerebellar nuclei. Among them, the pontine neurons follow a stereotypic anteroventral-directed pathway to form the pontine nuclei in the pons. The guidance mechanisms that determine the marginal migration of PCN and the anterior migration of pontine neurons are poorly understood. Here, we report that a chemokine SDF1 (also known as CXCL12) derived from the meningeal tissue regulates the migratory pathways of PCN. PCN are chemoattracted by the meningeal tissue, an effect that is mimicked by an SDF1 source. Analysis of knockout mice for the Sdf1 receptor Cxcr4 shows that both the marginal migration of PCN and the anterior migration of pontine neurons are disrupted. We provide further evidence that SDF1/CXCR4 signalling regulates these two processes cell-autonomously. As a result of disrupted neuronal migration, pontine nuclei formation was highly abnormal, with the presence of multiple ectopic pontine clusters posteriorly. The ectopic pontine clusters led to ectopic collateral branch formation from the corticospinal tract. Our results together demonstrate crucial roles for SDF1/CXCR4 in multiple aspects of PCN migration and highlight the deleterious consequence of derailed migration on proper nuclei formation. Furthermore, we provide the first in vivo evidence that pontine neurons themselves induce collateral branching from the corticospinal axons.

Abstract: Migrating neurons are thought to travel from their origin near the ventricle to distant territories along stereotypical pathways by detecting environmental cues in the extracellular milieu. Here, we report a novel mode of neuronal migration that challenges this view. We performed long-term, time-lapse imaging of medial ganglionic eminence (MGE)-derived cortical interneurons tangentially migrating in the marginal zone (MZ) in flat-mount cortices. We find that they exhibit a diverse range of behaviors in terms of the rate and direction of migration. Curiously, a predominant population of these neurons repeatedly changes its direction of migration in an unpredictable manner. Trajectories of migration vary from one neuron to another. The migration of individual cells lasts for long periods, sometimes up to 2 d. Theoretical analyses reveal that these behaviors can be modeled by a random walk. Furthermore, MZ cells migrate from the cortical subventricular zone to the cortical plate, transiently accumulating in the MZ. These results suggest that MGE-derived cortical interneurons, once arriving at the MZ, are released from regulation by guidance cues and initiate random walk movement, which potentially contributes to their dispersion throughout the cortex.

Abstract: 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. J. Comp. Neurol. 514:215-225, 2009. (c) 2009 Wiley-Liss, Inc.

Abstract: Accumulating evidence indicates that signaling centers controlling the dorsoventral (DV) polarization of the neural tube, the roof plate and the floor plate, play crucial roles in axon guidance along the DV axis. However, the role of signaling centers regulating the rostrocaudal (RC) polarization of the neural tube in axon guidance along the RC axis remains unknown. Here, we show that a signaling center located at the midbrain-hindbrain boundary (MHB) regulates the rostrally directed growth of axons from midbrain dopaminergic neurons (mDANs). We found that beads soaked with fibroblast growth factor 8 (FGF8), a signaling molecule that mediates patterning activities of the MHB, repelled mDAN axons that extended through the diencephalon. This repulsion may be mediated by semaphorin 3F (sema3F) because (1) FGF8-soaked beads induced an increase in expression of sema3F, (2) sema3F expression in the midbrain was essentially abolished by the application of an FGF receptor tyrosine kinase inhibitor, and (3) mDAN axonal growth was also inhibited by sema3F. Furthermore, mDAN axons expressed a sema3F receptor, neuropilin-2 (nrp2), and the removal of nrp-2 by gene targeting caused caudal growth of mDAN axons. These results indicate that the MHB signaling center regulates the growth polarity of mDAN axons along the RC axis by inducing sema3F.

Abstract: A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal-Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons.

Abstract: During development, neurons migrate from their origin to their final destinations where they form neuronal architectures such as layers and nuclei. While the mechanisms for the formation of laminated structures have been studied extensively, little is known about nucleogenesis. Previously, we analyzed nucleogenesis in neurons from four types of mossy-fiber projecting precerebellar nuclei neurons by gene transfer, and obtained evidence suggesting that the change from tangential to radial migration occurs at the region that will develop into the nucleus (Kawauchi, D., Taniguchi, H., Watanabe, H., Saito, T., Murakami, F., 2006. Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration. Development 133, 1113-1123). Here we analyzed the dynamics of these neurons using mice embryos. We electroporated genes of fluorescent proteins to the lower rhombic lip at embryonic day (E) 12.5 and carried out time-lapse analyses at E14.5, when pontine nuclei begin to be formed. We found that many labeled neurons showed transition from tangential to radial migration in the region that will develop into the nucleus. This transition occurred in two ways. One was initiated by a leading process extending radially while the second was caused by a newly developed radial process from the cell soma. Curiously, we observed that many neurons stopped tangentially migration, paused, and then began radial migration. These findings indicate that a signal to stop and cause the change in tangential to radial migration is critical for nucleogenesis by pontine neurons.

Abstract: Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1(-/-), but not Robo2(-/-), animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1(-/-) mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1(-/-)/Slit2(-/-) mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1(-/-), Robo2(-/-) and Slit1(-/-)/Slit2(-/-), but not in Slit1(-/-) mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.

Abstract: 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.

Abstract: It is well documented that most cortical interneurons originate from the basal forebrain and migrate tangentially to the cortex. However, relatively little is known about their migration after their arrival at the cortex. To elucidate the route and mode of intracortical migration of the interneurons, we performed real-time analysis by utilizing glutamate decarboxylase (GAD)67/green fluorescence protein (GFP) knock-in mice and an electroporation-based gene transfer of DsRed into the ganglionic eminence (GE) of a mouse embryo. Cortical interneurons show a diverse mode of migration. In coronal slices, ventrolateral-to-dorsomedial migration predominantly occurs in the lower-intermediate zone. However, a substantial number of interneurons migrate radially either towards the pial or ventricular surface. There are also quiescent neurons. Observations of the marginal zone or the ventricular zone in flat-mounted cortex from the pial or the ventricular surface, respectively, revealed that the interneurons tangentially migrate in all directions. Medial GE-derived interneurons visualized by DsRed electroporation show similar migratory behaviours. Thus, final settlement of cortical interneurons in their destinations may be a result of successive migratory process of different modes within the cortex.

Abstract: GABAergic neurons are the major inhibitory interneurons that are widely distributed in the central nervous system. It is well established that they originate from a focal region in the embryonic forebrain during development, and then migrate to other regions such as the neocortex. However, the migration of GABAergic neurons remains obscure in other axial levels of the brain. We examined the early development of myelencephalic GABAergic neurons using glutamate decarboxylase 67 / green fluorescent protein (GAD67-GFP) knocking mice. Observation of fixed tissues in coronal sections and flat whole-mount preparations indicated that, while GFP-positive cells are restricted to the subpial region in the ventral aspect of the myelencephalon at an early stage, they spread dorsally and eventually occupy the entire region of the myelencephalon as development proceeds. We developed a flat-mount in vitro preparation in which these patterns of development could be recapitulated. Transplantation of dorsal myelencephalic tissue of a wildtype embryo to a corresponding region of GAD67-GFP mouse embryos clearly demonstrated invasion of dorsally oriented GABAergic neurons from host to donor tissue. These results indicate that ventral-to-dorsal tangential migration of GABAergic neurons takes place in the myelencephalon. Our results extend the observations in the forebrain that inhibitory and excitatory neurons in a specific brain compartment take distinct migratory paths.

Abstract: The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

Abstract: Nuclei are aggregates of neurons distributed in the central nervous system and are fundamental functional units that share anatomical and physiological features. Despite their importance, the cellular basis that leads to nucleogenesis is only poorly understood. Using exo utero electroporation with an enhanced yellow fluorescent protein (EYFP) gene, we show that the precerebellar neurons derived from the lower rhombic lip (lRL) undergo multiple migration steps to form nuclei. After the unilateral transfer of EYFP to the lRL of embryonic day 12.5 mice, EYFP-labelled neurons migrate tangentially from the lRL in two distinct streams, one towards the ventral metencephalon and the other towards the ventral myelencephalon. These neurons cross the ventral midline and then become radially directed. Labelled neurons in the tangential migratory streams form contralateral clusters in the external cuneate nucleus (ECN) and lateral reticular nucleus (LRN) in the myelencephalon, and bilateral clusters in the pontine grey nucleus (PGN) and reticulotegmental nucleus (RTN) in the metencephalon. Before forming the clusters, EYFP-labelled neurons begin to migrate radially towards the ventricle in close apposition to nestin-positive radial fibres, and then they aggregate as they detach from the fibres. Inhibition of cadherin function in ECN and LRN progenitors caused ipsilateral formation of the ECN and LRN, implying that the transition of their migration from tangential to radial involves a cell-intrinsic mechanism. These observations suggest that nucleogenesis of precerebellar neurons is a result of multi-phasic migration, and that ventricle-directed radial glia-guided migration is a key step for nucleogenesis.

Abstract: Most GABAergic interneurons originate from the basal forebrain and migrate tangentially into the cortex. The migratory pathways and mode of interneuron migration within the developing cerebral cortex, however, previously was largely unknown. Time-lapse imaging and in vivo labelling with glutamate decarboxylase (GAD)67-green fluorescence protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)ergic neurons indicated that multidirectional tangential (MDT) migration of interneurons takes place in both the marginal zone (MZ) and the ventricular zone (VZ) of the cortex. Quantitative analysis of migrating interneurons showed that rostrocaudally migrating neurons outnumber those migrating mediolaterally in both of these zones. In vivo labelling with a lipophilic dye showed that the MDT migration in the MZ occurs throughout the cortex over distances of up to 3 mm during a period of a few days. These results indicate that MZ cortical interneurons undergo a second phase of tangential migration in all directions and over long distances, after reaching the cortex by dorsomedial tangential migration. The MDT migration in the MZ may disperse and intermix interneurons within the cortex, resulting in a balanced distribution of interneuron subtypes.

Abstract: In vertebrate embryos, commissural axons extend toward and across the floor plate (FP), an intermediate target at the ventral midline (VM) of the spinal cord. After decussating, many commissural axons turn into the longitudinal plane and elaborate diverse projections. FP contact is thought to alter the responsiveness of these axons so that they can exit the FP and adopt new trajectories. However, a requirement for the FP in shaping contralateral commissural projections has not been established in higher vertebrates. Here we further analyze to what extent FP contact is necessary for the elaboration of decussated commissural projections both in cultured, FP-excised spinal cord preparations and in gli2-deficient mice, which lack a FP. In FP-lacking spinal cords, we observe a large number of appropriately projecting contralateral commissural projections in vivo and in vitro. Surprisingly, even though gli2 mutants lack a FP, slit1-3 mRNA and their receptors (Robo1/2) are expressed in a wild-type-like manner. In addition, blocking Robo-Slit interactions in FP-lacking spinal cord explants prevents commissural axons from leaving the VM and turning longitudinally. Thus, compared to FP contact, Slit-Robo interactions are more critical for driving commissural axons out of the VM and facilitating the elaboration of a subset of contralateral commissural projections.

Abstract: Most post-crossing commissural axons turn into longitudinal paths to make synaptic connections with their targets. Mechanisms that control their rostrocaudal turning polarity are still poorly understood. We used the hindbrain as a model system to investigate the rostral turning of a laterally located commissural tract, identified as the caudal group of contralateral cerebellar-projecting second-order vestibular neurons (cC-VC). We found that the caudal hindbrain possessed a graded non-permissive/repulsive activity for growing cC-VC axons. This non-permissiveness/repulsion was in part mediated by glycosyl-phosphatidylinositol (GPI)-anchored ephrin A. We further demonstrated that ephrin A2 was distributed in a caudal-high/rostral-low gradient in the caudolateral hindbrain and cC-VC axons expressed EphA receptors. Finally, perturbing ephrin A/EphA signalling both in vitro and in vivo led to rostrocaudal pathfinding errors of post-crossing cC-VC axons. These results suggest that ephrin A/EphA interactions play a key role in regulating the polarity of post-crossing cC-VC axons as they turn into the longitudinal axis.

Abstract: Classic cadherins are calcium dependent homophilic cell adhesion molecules that play a key role in developmental processes such as morphogenesis, compartmentalization and maintenance of a tissue. They also play important roles in development and function of the nervous system. Although classic cadherins have been shown to be involved in the migration of non-neuronal cells, little is known about their role in neuronal migration. Here, we show that classic cadherins are essential for the migration of precerebellar neurons. In situ hybridization analysis shows that at least four classic cadherins, cadherin 6 (Cad6), cadherin 8 (Cad8), cadherin11 (Cad11) and N-cadherin (Ncad), are expressed in the migratory streams of lateral reticular nucleus and external cuneate nucleus (LRN/ECN) neurons. Functional analysis performed by electroporation of cadherin constructs into the hindbrain indicates requirement for cadherins in the migration of LRN/ECN neurons both in vitro and in vivo. While overexpression of full-length classic cadherins, NCAD and CAD11, has no effect on LRN/ECN neuron migration, overexpression of two dominant negative (DN) constructs, membrane-bound form and cytoplasmic form, slows it down. Introduction of a DN construct does not alter some characteristics of LRN/ECN cells as indicated by a molecular marker, TAG1, and their responsiveness to chemotropic activity of the floor plate (FP). These results suggest that classic cadherins contribute to contact-dependent mechanisms of precerebellar neuron migration probably via their adhesive property.

Abstract: The mRNA encoding activity-regulated cytoskeleton-associated protein (Arc) is known to be targeted to dendritic regions that have received strong synaptic inputs. However, the cis-acting elements in Arc mRNA that mediate dendritic targeting have not been identified. To identify the dendritic targeting element (DTE) in rat Arc mRNA, we expressed reporter mRNAs containing various regions of Arc in primary hippocampal neurones and analysed their subcellular distribution by in situ hybridization. Here, we report that the 3'-untranslated region of rat Arc mRNA contains a 350-nucleotide DTE with strong dendritic targeting activity and another 370-nucleotide sequence with weaker dendritic targeting activity. The 350-nucleotide DTE does not share any obvious sequence similarity with other known DTEs previously reported.

Abstract: Commissural axons in vertebrates and insects are initially attracted to the nervous system midline, but once they reach this intermediate target they undergo a dramatic switch, becoming responsive to repellent Slit proteins at the midline, which expel them onto the next leg of their trajectory. We have unexpectedly implicated a divergent member of the Robo family, Rig-1 (or Robo3), in preventing premature Slit sensitivity in mammals. Expression of Rig-1 protein by commissural axons is inversely correlated with Slit sensitivity. Removal of Rig-1 results in a total failure of commissural axons to cross. Genetic and in vitro analyses indicate that Rig-1 functions to repress Slit responsiveness similarly to Commissureless (Comm) in Drosophila. Unlike Comm, however, Rig-1 does not produce its effect by downregulating Robo receptors on precrossing commissural axon membranes. These results identify a mechanism for regulating Slit repulsion that helps choreograph the precise switch from attraction to repulsion at a key intermediate axonal target.

Abstract: In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.

Abstract: 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.

Abstract: During development of the central nervous system (CNS), commissural axons grow toward the ventral midline. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. The contacts between the commissural axons and the floor plate cells are involved in this axonal guidance, but their mechanisms or structures have not fully been understood. In this study, we found that nectin-1 and -3, immunoglobulin-like cell-cell adhesion molecules, asymmetrically localized at the contact sites between the commissural axons and the floor plate cells, respectively. In vitro perturbation of the endogenous trans-interaction between nectin-1 and -3 caused abnormal fasciculation of the commissural axons and impairment of the contacts, and resulted in failure in longitudinal turns of the commissural axons at the contralateral sites of the rat hindbrain. These results indicate that the contacts between the commissural axons and the floor plate cells are mediated by the hetero-trans-interaction between nectin-1 and -3 and involved in regulation of the trajectory of the commissural axons.

Abstract: The role of neurotrophins in thalamic axon growth was studied by culturing embryonic rat thalamus on collagen-coated substrate or fixed cortical slices in the presence of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Both BDNF and NT-3 promoted axonal growth, but the axonal growth-promoting activity depended on culture substrates. Axonal growth on collagen-coated membrane was accelerated by BDNF, but not by NT-3. In contrast, axonal outgrowth on fixed cortex was significantly enhanced by NT-3, but not by BDNF. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cultured thalamic cells demonstrated that culture substrates did not alter the expression of their receptors, trkB and trkC. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining further demonstrated that axonal growth promoted by neurotrophins was not due to reduction of cell death. Measurement of the developmental changes in BDNF and NT-3 levels revealed that, in contrast to the rapid elevation of BDNF after the arrival of thalamocortical axons to their target layer, the regulation of NT-3 protein accompanies the phase of their outgrowth in neocortex. These findings suggest that BDNF and NT-3 promote thalamic axon growth in different manners in terms of substrate dependency and developmental stage.

Abstract: Laminar specificity is one of the most striking features of neocortical circuitry. To explore the molecular basis of this specificity, particularly in relation to thalamocortical connectivity, we searched for the genes expressed in the upper cortical layers by constructing a subtraction cDNA library that was enriched for genes expressed in layer 4 of perinatal rat somatosensory cortex. Differential screening, sequence analysis and in situ hybridization demonstrated that a new unc5 family member (unc5h4), deltex-like gene, stem cell factor (SCF) and myocyte-specific enhancer factor-2C (MEF-2C) were specifically expressed in layer 4 or layers 2/3-4 at postnatal day 7, by when laminar organization and fundamental cortical circuitries have been established. In terms of regional specificity, unc5h4 and SCF signals were stronger in sensory cortices, whereas MEF-2C and deltex-like gene were expressed rather uniformly in all neocortical regions. Analysis during development demonstrated that expression of these genes was pronounced between late embryonic and early postnatal developmental stages, except for MEF-2C expression, which continued in later stages. These results demonstrate that certain types of molecules including transcription factors, receptor and ligand molecules, are expressed specifically in the upper layers of the developing neocortex, suggesting a role in laminar specification of cortical cells and circuitry.

Abstract: Regulation of axonal fasciculation plays an important role in the precise patterning of neural circuits. Selective fasciculation contributes to the sorting of different types of axons and prevents the misrouting of axons. However, axons must defasciculate once they reach the target area. To study the regulation of fasciculation, we focused on the primary vestibulo-cerebellar afferents (PVAs), which show a dramatic change from fasciculated axon bundles to defasciculated individual axons at their target region, the cerebellar primordium. To understand how fasciculation and defasciculation are regulated in this system, we investigated the roles of murine SC1-related protein (MuSC), a molecule belonging to the immunoglobulin superfamily. We show: (i) by comparing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) labelling and anti-MuSC immunohistochemistry, that downregulation of MuSC in PVAs during development is concomitant with the defasciculation of PVA axons; (ii) in a binding assay with cells expressing MuSC, that MuSC has cell-adhesive activity via a homophilic binding mechanism, and this activity is increased by multimerization; and (iii) that MuSC also displays neurite outgrowth-promoting activity in vestibular ganglion cultures. These findings suggest that MuSC is involved in axonal fasciculation and its downregulation may help to initiate the defasciculation of PVAs.

Abstract: Channel density is a fundamental factor in determining neuronal firing and is primarily regulated during development through transcriptional and translational regulation. In adult rats, striatal cholinergic interneurons have a prominent A-type current and co-express Kv4.1 and Kv4.2 mRNAs. There is evidence that Kv4.2 plays a primary role in producing the current in adult neurons. The contribution of Kv4.2 and Kv4.1 to the A-type current in cholinergic interneurons during development, however, is not known. Here, using patch-clamp recording and semi-quantitative single-cell reverse transcription-polymerase chain reaction (RT-PCR) techniques, we have examined the postnatal development of A-type current and the expression of Kv4.2 and Kv4.1 in rat striatal cholinergic interneurons. A-type current was detectable at birth, and its amplitude was up-regulated with age, reaching a plateau at about 3 wk after birth. At all ages, the current inactivated with two time constants: one ranging from 15 to 27 ms and the other ranging from 99 to 142 ms. Kv4.2 mRNA was detectable at birth, and the expression level increased exponentially with age, reaching a plateau by 3 wk postnatal. In contrast, Kv4.1 mRNA was not detectable during the first week after birth, and the expression level did not show a clear tendency with age. Taken together, our results suggest that Kv4.2 plays an essential role in producing the A-type current in striatal cholinergic interneurons during the entire course of postnatal development.

Abstract: Axons of the trigeminal ganglion convey sensory information from mechanoreceptors, thermoreceptors, and nociceptors in the face and nasal mucosa, then terminate on several groups of neurons including the principal sensory nucleus and the nuclei of the spinal trigeminal tract. To understand guidance mechanisms during the development of trigeminal sensory axons (TA) in the embryonic brain, we first investigated the growth pattern of TA in relation to organization in the hindbrain using flat whole-mount preparation from rat. We found that the primary TA from the trigeminal ganglion entered the brainstem and grew longitudinally within the hindbrain. Whereas descending axons ran just medial to the primary vestibular axons to innervate the spinal nucleus, ascending axons stayed near the entry point. In flat whole-mount culture, the TA extended both ascending and descending branches as they do in vivo. Rostral hindbrain was found to be a less permissive substrate for the TA compared to caudal hindbrain. In addition, the nonpermissive property of the ventral hindbrain substrate restricted the invasion of TA along the entire length of the hindbrain. Thus, cooperation of absolute and relative permissiveness of the substrate plays important roles in the guidance of TA to their targets.

Abstract: Many ionic currents undergo significant rundown during whole-cell recording. Although rundown is an artifact associated with the recording method, studying the mechanism of rundown may lead to understanding mechanisms regulating channel functions in physiological conditions. The mechanisms for rundown, however, remain obscure for many channels. Here we have studied the mechanism for rundown of an A-type K(+) current in mouse striatal cholinergic interneurons. The interneuron expressed a prominent component of A-type current which exhibited significant rundown during whole-cell recording. When the current was assessed with a highly hyperpolarized prepotential (-140 mV), however, the rundown was virtually fully suppressed, suggesting its being dependent on voltage. Estimation of channel voltage dependence revealed that both activation and inactivation curves shifted towards hyperpolarized potentials during rundown. The shift was suppressed by intracellular ATP, but was affected neither by phosphatase inhibitors nor by antioxidative reagents. The gradual shift of inactivation curve towards negative potentials would make the holding potential progressively inactivate the channel, resulting in apparent loss of activity of the channels. Our results thus provide a biophysical explanation for rundown of A-type current. .

Abstract: Neuronal migration is crucial for the construction of neuronal architecture such as layers and nuclei. Most inhibitory interneurons in the neocortex derive from the basal forebrain and migrate tangentially; however, little is known about the mode of migration of these neurons in the cortex. We used glutamate decarboxylase (Gad)67-green fluorescent protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)-ergic neurons and performed time-lapse analysis. In coronal slices, many GFP-positive neurons in the lower intermediate zone (IZ) and subventricular zone (SVZ) showed robust tangential migration from lateral to medial cortex, while others showed radial and non-radial migration mostly towards the pial surface. In flat-mount preparations, GFP-positive neurons of the marginal zone (MZ) showed multidirectional tangential migration. Some of these neurons descended toward the cortical plate (CP). Intracortical migration of these neurons was largely unaffected by a treatment that cleaves glycosylphosphatidylinositol (GPI) anchors. These findings suggest that tangential migration of cortical interneurons from lateral to medial cortex predominantly occurs in the IZ/SVZ and raise the possibility that a part of the pial surface-directed neurons in the IZ/SVZ reach the MZ, whereby they spread into the whole area of the cortex. At least a part of these neurons may descend toward the CP. Our results also suggest that intracortical migration of GABAergic neurons occurs independent of GPI-anchored proteins.

Abstract: To understand the range of competence of embryonic stem (ES) cell-derived neural precursors, we have examined in vitro differentiation of mouse and primate ES cells into the dorsal- (neural crest) and ventralmost (floor plate) cells of the neural axis. Stromal cell-derived inducing activity (SDIA; accumulated on PA6 stromal cells) induces cocultured ES cells to differentiate into rostral CNS tissues containing both ventral and dorsal cells. Although early exposure of SDIA-treated ES cells to bone morphogenetic protein (BMP)4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the fourth day of coculture causes differentiation of neural crest cells and dorsalmost CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons. Notably, high concentrations of Shh efficiently promote differentiation of HNF3beta(+) floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naive precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals.

Abstract: Neuronal migration is required for the establishment of specific neural structures, such as layers and nuclei. Neurons migrate along specific migratory routes toward their final destinations, sometimes across long distances. However, the cellular and molecular interactions that control neuronal migration are largely unknown. Here, we examined the mechanism underlying the transmedian migration of precerebellar neurons using a flat whole-mount preparation of the rat embryo. These neurons were initially attracted by the floor plate (FP) at the ventral midline. However, after crossing the midline, they lost their responsiveness to the FP and became attracted by the alar plate (AP). Although the loss of responsiveness to FP cues was caused by an encounter of migrating cells with the FP, the gain of responsiveness to AP cues occurred irrespective of their encounter with the FP. These results identify a crucial change in the response of migrating cells to attractive guidance cues during the transmedian migration of precerebellar neurons.

Abstract: During development of the central nervous system, growth cones navigate along specific pathways, recognize their targets and then form synaptic connections by elaborating terminal arbors. To date, a number of developmental and in vitro studies have characterized the nature of the guidance cues that underlie various types of axonal behavior, from initial outgrowth to synapse formation, including pathway selection, polarized growth, orientated growth, termination and branching. New approaches in molecular biology have identified several types of guidance cues, most of which are likely to act as local cues. Moreover, recent studies have indicated that axonal responsiveness to guidance cues changes dynamically, which appears to be elicited by environmental factors encountered by the navigating growth cones. This article addresses what molecular cues are responsible for guidance mechanisms including axonal responsiveness, focusing on axonal behavior in the developmental stages.

Abstract: The role was studied of ephrin-B3, a ligand of the Eph family of tyrosine kinase receptors, in the formation of cortical connectivity. In situ hybridization and immunohistochemistry showed that EphA4, a receptor of ephrin-B3, was expressed in the lateral thalamus (visual and somaotosensory thalamus) of the developing rat brain, but not in the medial thalamic nuclei which project to the limbic cortex. Correspondingly, ephrin-B3 was expressed strongly in the developing limbic cortex including amygdala, entorhinal cortex and hippocampus. To examine the action of ephrin-B3 on thalamic axons, either lateral or medial thalamic explants were cultured on membranes obtained from ephrin-B3-expressing COS cells. Axonal growth was inhibited for cells from the lateral thalamus but not from the medial thalamus. These results suggest that ephrin-B3 contributes to regional specificity by suppressing axonal growth of lateral thalamic neurons.

Abstract: 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.

Abstract: 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.

Abstract: Information that originates from peripheral sensory organs is conveyed by axons of cephalic sensory cranial ganglia connecting the sensory organs to appropriate central targets in the brain. Thus, the establishment of correct axonal projections by sensory afferents is one of the most important issues in neural development. Previously, we examined the development of the vestibular nerve that originates from the VIIIth ganglion using a flat whole-mount preparation of the rat hindbrain and developed an in vitro, culture preparation that can recapitulate vestibular nerve development (Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M., Heizmann, C. W. and Murakami, F. (2000) J. Comp. Neurol. 417, 491-500). Both in vivo and in vitro, the ascending branch of the VIIIth ganglion projecting to the cerebellum reaches the base of the cerebellar primordium and starts to splay out towards the rhombic lip, apparently avoiding the ventral metencephalon. We now examine the nature of cues that guide vestibulocerebellar axons by applying various manipulations to the flat whole-mount in vitro preparation. Our observations suggest that local nonpermissive cues and oriented cues play a pivotal role in the guidance of vestibular axons to their central target.

Abstract: To visualize and isolate live dopamine (DA)-producing neurons in the embryonic ventral mesencephalon, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene promoter. In the transgenic mice, GFP expression was observed in the developing DA neurons containing tyrosine hydroxylase. The outgrowth and cue-dependent guidance of GFP-labeled axons was monitored in vitro with brain culture systems. To isolate DA neurons expressing GFP from brain tissue, cells with GFP fluorescence were sorted by fluorescence-activated cell sorting. More than 60% of the sorted GFP(+) cells were positive for tyrosine hydroxylase, confirming that the population had been successfully enriched with DA neurons. The sorted GFP(+) cells were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a recovery from Parkinsonian behavioral defects. This strategy for isolating an enriched population of DA neurons should be useful for cellular and molecular studies of these neurons and for clinical applications in the treatment of Parkinson's disease.

Abstract: Spine-like dendritic protrusions (SLDPs) emanating from developing dendrites have been proposed to play an important role in early synaptogenesis. We previously analyzed synaptic termination sites on soma-dendritic membrane of newborn cats and found that corticorubral (CR) axons form synapses preferentially on SLDPs (Saito et al., 1997). In the present study, we examined CR synapses in adult cats to elucidate the maturation process of CR synapses in relation to SLDPs. Electron microscopic observation of serial thin sections of Phaseolus vulgaris-leucoagglutinin-labeled axons revealed that approximately 60% of CR terminals in adult cats formed synapses on dendritic spines. We also found that CR axons terminate on dendritic spines originating from the intermediate or distal dendrites of rubrospinal cells (more than 200 microm apart from the soma), in contrast to kittens in which CR fibers terminate on SLDPs originating from the proximal dendrites (less than 100 microm apart from the soma) of rubrospinal cells (Saito et al. [1997] J. Neurosci. 17:8792-8803). These results suggest that CR synapses undergo remarkable remodeling after initial termination on SLDP during postnatal development.

Abstract: During development of the vertebrate CNS, commissural axons initially grow circumferentially toward the ventral midline floor plate. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. Although recent studies have unraveled the mechanisms that control navigation of these axons along the circumferential axis, those that result in the transition from circumferential to longitudinal trajectory remain unknown. Here, we examined whether an interaction with the floor plate is a prerequisite for the initiation of trajectory transition of commissural axons, using in vitro preparations of the rat metencephalon. We found that commissural axons in the metencephalon, once having crossed the floor plate, turned sharply to grow longitudinally. In contrast, axons extending in floor plate-deleted preparations, continued to grow circumferentially, ignoring the hypothetical turning point. These results suggest that a prior interaction of commissural axons with floor plate cells is a key step for these axons to activate a navigation program required for their change in axonal trajectory from the circumferential to the longitudinal axis.

Abstract: The subthalamic nucleus (STN) directly innervates the output structures of the basal ganglia, playing a key role in basal ganglia function. It is therefore important to understand the regulatory mechanisms for the activity of STN neurons. In the present study, we aimed to investigate how the intrinsic membrane properties of STN neurons interact with their synaptic inputs, focusing on their generation and the properties of the long-lasting, plateau potential. Whole cell recordings were obtained from STN neurons in slices prepared from postnatal day 14 (P14) to P20 rats. We found that activation of glutamate receptor-mediated excitatory synaptic potentials (EPSPs) evoked a plateau potential in a subpopulation of STN neurons (n = 13/22), in a voltage-dependent manner. Plateau potentials could be induced only when the cell was hyperpolarized to more negative than about -75 mV. Plateau potentials, evoked with a depolarizing current pulse, again only from a hyperpolarized state, were observed in about half of STN neurons tested (n = 162/327). Only in neurons in which a plateau potential could be evoked by current injection did EPSPs evoke plateau potentials. L-type Ca(2+) channels, Ca(2+)-dependent K(+) channels, and TEA-sensitive K(+) channels were found to be involved in the generation of the potential. The stability of the plateau potential, tested by the injection of a negative pulse current during the plateau phase, was found to be robust at the early phase of the potential, but decreased toward the end. As a result the early part of the plateau potential was resistant to membrane potential perturbations and would be able to support a train of action potentials. We conclude that excitatory postsynaptic potentials, evoked in a subpopulation of STN neurons at a hyperpolarized state, activate L-type Ca(2+) and other channels, leading to the generation of a plateau potential. Thus about half of STN neurons can transform short-lasting synaptic excitation into a long train of output spikes by voltage-dependent generation of a plateau potential.

Abstract: During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.

Abstract: During development, thalamocortical axons form arbors primarily in layer 4 of the neocortex. This lamina-specific branch formation was studied in cultures of rat thalamic explants grown next to chemically fixed cortical slices. After a week in vitro, thalamic axons formed branches specifically in the target layer of fixed cortical slices, regardless of the orientation of the ingrowth. This in vitro system permits a direct assessment of contributions of membrane-associated molecules to thalamic axon branch formation. To this end, the present study uses three enzymatic perturbations: chondroitinase, phosphatidylinositol phospholipase C, or the polysialic acid (PSA)-specific endoneuraminidase (endo N). With endo N pretreatment of cortex, the number of branch points was increased significantly, whereas branch tip length was decreased. In addition, the localization of branch points to the target layer was weakened considerably. These features of branch formation were not altered by the other two enzymatic treatments, except that branch tips were shortened by chondroitinase treatment to the same extent as in endo N treatment. These results suggest that membrane-bound components are involved in lamina-specific branch formation of thalamocortical axons, and in particular that PSA moieties contribute to laminar specificity by inhibiting branch emergence in inappropriate layers.

Abstract: In vertebrates, sensory neurons interconnect a variety of peripheral tissues and central targets, conveying sensory information from different types of sensory receptors to appropriate second-order neurons in the central nervous system (CNS). To explore the possibility that the different rhombomere environments where sensory neurons enter into the hindbrain affect the pathfinding capability of growth cones, we studied the development of the VIIIth ganglion afferent both in vivo and in vitro. We focused on the vestibular nerve because it is the only cranial nerve projecting to the cerebellum, allowing for ready identification from its pattern of projection. Embryonic rat brain was cut along the dorsal midline and, with the VIIIth and Vth ganglia still attached, flat mounted and visualized with antibodies specific for sensory ganglia. Axons reached the cerebellar primordium at embryonic day (E) 13, then splayed out towards the edges of the rhombic lip of rostral hindbrain. In vitro, the VIIIth ganglion showed development similar to that in vivo and innervated the cerebellum, an appropriate target, indicating that mechanisms for axon guidance and target recognition are preserved in vitro. When the VIIIth ganglion was transplanted to the position of the Vth ganglion, axons from the transplanted ganglion entered the cerebellar primordium with a trajectory characteristic of the VIIIth nerve. These results indicate that the central projection pattern of the VIIIth nerve is not affected by the environment of nerve entry into the brainstem, suggesting that axons of sensory cranial ganglion intrinsically possess the capacity to find their target correctly.

Abstract: Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development.

Abstract: The subthalamic nucleus (STN) plays a key role in motor control. Although previous studies have suggested that Ca(2+) conductances may be involved in regulating the activity of STN neurons, Ca(2+) channels in this region have not yet been characterized. We have therefore investigated the subtypes and functional characteristics of Ca(2+) conductances in STN neurons, in both acutely isolated and slice preparations. Acutely isolated STN cells were identified by retrograde filling with the fluorescent dye, Fluoro-Gold. In acutely isolated STN neurons, Cd(2+)-sensitive, depolarization-activated Ba(2+) currents were observed in all cells studied. The current-voltage relationship and current kinetics were characteristic of high-voltage-activated Ca(2+) channels. The steady-state voltage-dependent activation curves and inactivation curves could both be fitted with a single Boltzmann function. Currents evoked with a prolonged pulse, however, inactivated with multiple time constants, suggesting either the presence of more than one Ca(2+) channel subtype or multiple inactivation processes with a single channel type in STN neurons. Experiments using organic Ca(2+) channel blockers revealed that on average, 21% of the current was nifedipine sensitive, 52% was sensitive to omega-conotoxin GVIA, 16% was blocked by a high concentration of omega-agatoxin IVA (200 nM), and the remainder of the current (9%) was resistant to the co-application of all blockers. These currents had similar voltage dependencies, but the nifedipine-sensitive current and the resistant current activated at slightly lower voltages. omega-Agatoxin IVA at 20 nM was ineffective in blocking the current. Together, the above results suggest that acutely isolated STN neurons have all subtypes of high-voltage-activated Ca(2+) channels except for P-type, but have no low-voltage-activated channels. Although acutely isolated neurons provide a good preparation for whole cell voltage-clamp study, dendritic processes are lost during dissociation. To gain information on Ca(2+) channels in dendrites, we thus studied Ca(2+) channels of STN neurons in a slice preparation, focusing on low-voltage-activated channels. In current-clamp recordings, a slow spike was always observed following termination of an injected hyperpolarizing current. The slow spike occurred at resting membrane potentials and was sensitive to micromolar concentrations of Ni(2+), suggesting that it is a low-threshold Ca(2+) spike. Together, our results suggest that STN neurons express low-voltage-activated Ca(2+) channels and several high-voltage-activated subtypes. Our results also suggest the possibility that the low-voltage-activated channels have a preferential distribution to the dendritic processes.

Abstract: The vertebrate CNS is composed of a variety of longitudinal axonal tracts extending rostrally and caudally. Although recent studies have demonstrated that chemoattraction and chemorepulsion play key roles in axon guidance along the circumferential axis in the neural tube of the vertebrate, mechanisms of axonal elongation along the longitudinal axis, and most importantly, what determines rostrocaudal polarity of axonal growth, remains unknown. Here, we examined the mechanism that guides midbrain dopaminergic axons rostrally, using flat whole-mount preparations of embryonic rat brain both in vivo and in vitro. At embryonic day 11 (E11) and early stage E12, dopaminergic neurons in the ventral midbrain extended short axons dorsally. By middle stage E12, these axons had increased in number, some deflecting rostrally and others caudally. At E13, almost all axons showed rostrally oriented growth heading toward the forebrain targets. In in vitro whole-mount preparations prepared from an E12 embryo and cultured for 24 hr, these axons showed rostrally oriented growth, but when they were forced to grow on substratum of reversed rostrocaudal polarity, they turned abruptly and grew following the polarity of the reversed midbrain substratum. These results suggest that local directional cues in the midbrain guide these axons rostrally and support the idea that substratum-associated polarized cues play an important role in axon guidance along the longitudinal axis.

Abstract: Developing axons reach their final targets as a result of a series of axonal projections to successive intermediate targets. Long-range chemoattraction by intermediate targets plays a key role in this process. Growing axons, however, do not stall at the intermediate targets, where the chemoattractant concentration is expected to be maximal. Commissural axons in the metencephalon, initially attracted by a chemoattractant released from the floor plate, were shown to lose responsiveness to the chemoattractant when they crossed the floor plate in vitro. Such changes in axon responsiveness to chemoattractants may enable developing axons to continue to navigate toward their final destinations.

Abstract: In mammals, topographic maps emerge from initially diffuse projections during development. To gain insight into the mechanisms governing the transition from a diffuse projection to a topographic map, we studied topographic specificity of functional connections during development, using the cat corticorubral system as a model. In the adult cat, rubrospinal neurons in the dorsomedial part of the red nucleus (RN) receive input primarily from the forelimb area of the sensorimotor cortex, whereas those in the ventrolateral part receive input primarily from the hindlimb area. During development, axons from the sensorimotor cortex arrive in the RN at embryonic day 50 (E50) (Song et al., 1995a) and are diffusely distributed in the RN until postnatal day 13 (P13) (Higashi et al., 1990). Here, we studied the development of the pattern of functional cortical inputs to individual rubrospinal neurons, using synaptic potentials recorded in vivo. The functional topography in each rubrospinal neuron in developing cats was examined and classified either as adult-like or nonadult-like by comparison with the adult pattern. In preterm kittens from E61 to E65, only about half of the recorded neurons (41%; n = 22) showed adult-like functional topography. This percentage, however, increased to 82% (n = 56) in P1-P8 kittens and to 93% (n = 42) in P13-P28 kittens. These results, in conjunction with the above mentioned anatomical observations, suggest that corticorubral axons make functional synapses nonselectively with rubrospinal neurons before birth. Furthermore, the functional topographic map developed earlier than the anatomical map (<P8 vs >P13), suggesting that there is a developmental step of selective promotion of synapse formation and/or selective enhancement of synaptic efficacy in topographically appropriate regions in the RN, before the emergence of the mature anatomical map.

Abstract: We describe the recent progress of studies on the mechanisms of early neural patterning, focusing on the guidance of circumferentially migrating axons in the vertebrate brain. We also refer to the underlying molecular mechanisms revealed by recent studies.

Abstract: The formation of synaptic contacts is a crucial event during neural development and is thought to be achieved by complex interactions between incoming axons and the neurons in the target. We have focused on spine-like dendritic protrusions (SLDPs), which are transient pleomorphic protrusive structures seen in developing brains. Although the functional significance of SLDPs remains unknown, accumulating in vitro evidence suggests that the SLDP plays an important role in synaptogenetic interactions with axons. As a test of this idea, the present study was performed to examine whether the SLDPs are the preferential sites of synapse formation in vivo. The ultrastructure of biocytin-labeled corticorubral (CR) terminals was examined in serial thin sections during the period of synaptogenesis in newborn cats. We found that a major proportion (86%) of the CR synapses was formed on SLDPs. The presynaptic terminals were often invaginated by fine processes extending from the tips of SLDPs. Synaptic structures presumably of cortical origin were also found on SLDPs of HRP-labeled rubrospinal cells, suggesting that SLDPs postsynaptic to labeled CR terminals originate at least in part from rubrospinal cells. Taken together, these results indicate that SLDPs may represent preferred sites of synapse formation and support the notion that SLDPs play a role in synaptogenic interactions during brain development.

Abstract: Netrin-1, a diffusible signal secreted by floor plate cells at the ventral midline of the vertebrate CNS, can attract ventrally migrating axons and repel a subset of dorsally migrating axons in the spinal cord and rostral hindbrain in vitro. Whether netrin-1 can act as a global cue to guide all circumferentially migrating axons is, however, unknown. Here, we show that netrin-1 can attract alar plate axons that cross the floor plate along its entire rostrocaudal axis. Dorsally directed axons forming the posterior commissure are, however, repelled by the floor plate by a netrin-independent mechanism. These results suggest that netrin-1 functions as a global guidance cue for attraction to the midline. Moreover, floor plate-mediated chemorepulsion may also operate generally to direct dorsal migrations, but its molecular basis may involve both netrin-dependent and -independent mechanisms.

Abstract: Ordered neuronal connections in mature brains are thought to be sculpted from initially diffuse projections by elimination of inappropriate projections and strengthening of appropriate ones. Although evidence suggests that neuronal activity plays a role in these processes, the mechanism behind the modification of neuronal connections remains obscure. To gain insight into the mechanisms of axonal elimination and projection strengthening, we examined the morphology of individual axons that were to be eliminated as well as the consequences of partial denervation. While corticorubral projections in adult cats are thought to be uncrossed, early in postnatal development and after early unilateral lesions to the sensorimotor cortex, however, a significant amount of crossed corticorubral projections occurs. We examined the morphology of individual corticorubral axons in fetal cats and kittens from embryonic day 59 to postnatal day 48 and those that had received early unilateral lesions to the cortex, by serial reconstruction of Phaseolus-vulgaris-leucoagglutinin- or biocytin-labeled axons. For about 2 weeks during pre- and postnatal development, crossed axons remained simple in morphology, with few branches. Thereafter, they showed an increase in branch number, but then began to show fewer branches again. Axons and their collaterals were found in nonrestricted areas of the red nucleus (RN) throughout the period of observation, indicating that axons can sit at an inappropriate target for weeks but fail to ramify. In contrast, crossed corticorubral axons in kittens with cortical lesions showed terminal-arbor-like structures in the RN region that are in mirror symmetry to topographically appropriate areas in the ipsilateral RN, although some showed simple morphology without arbors. These complicated forms of morphology of individual axons during development and after partial denervation may not be explained by a simple activity-dependent mechanism.

Abstract: To elucidate guidance mechanisms of brain commissural axons, we examined the navigation of cerebellofugal axons. Axons were labeled by implantation of the fluorescent tracer Dil into the cerebellar plate (CP) of fixed, flat whole-mount embryonic rat brain. Axons initially grew straight toward the ventral midline floor plate (FP) in the rostral hindbrain and then, after crossing it, made a right-angled turn to grow either caudally or rostrally along the longitudinal axis. In collagen gel culture, CP axons showed directed growth toward both FP explants and heterologous cells expressing netrin-1, a FP-derived chemoattractant for spinal commissural axons. These results suggest that CP axons are guided to the midline by FP-derived chemoattractant(s) and then reoriented, possibly by another guidance cue, for longitudinal extension. Considering that the basic structures of the neural tube, including the FP, extend up to the caudal diencephalon, these results suggest that common guidance mechanisms operate for ventrally decussating commissural axons in both the brain and spinal cord.

Abstract: 1. The development of action potential conduction was studied by intracellular recording of antidromic spikes in cat rubrospinal cells. 2. The distance between the C1 and L1 spinal segments increased linearly from 5.6 cm at embryonic day (E) 59 to 9.8 cm at postnatal day (P) 30. 3. The conduction time from the C1 segment to the rubrospinal neuron soma, estimated from antidromic spike latency evoked by stimulation of the C1 segment, decreased rapidly prior to birth and then slowly thereafter. This coincided with a reduction in conduction time variation between cells. 4. The conduction time from the red nucleus to the L1 segment followed a similar time course during development. The conduction time reached the adult value by P30, at which time the spinal cord is only half the adult length. 5. The conduction velocity between the C1 and L1 segments increased monotonically between E59 and P30, from a low of 1 m s-1 to a maximum of 34 m s-1. 6. The rise time of rubrospinal neuron somadendritic spikes followed a developmental time course similar to that for conduction time. 7. Myelination of rubrospinal axons, as judged by the presence of myelinated segment spikes, began to occur prior to E59. 8. These findings suggest that development of action potential propagation in rubrospinal cells can be divided into an early and a late stage: conduction time reaches the adult value during the early stage, i.e. by the first postnatal month, and is maintained during the late stage. We propose that myelination, axon diameter increase and maturation of membrane properties act to reduce conduction time to adult values during the early stage, while a proportional increase in fibre diameter with axonal length results in a constant conduction time during the late stage.

Abstract: Cerebrorubral (CR) and cerebellorubral (CBR) afferents are localized to distal dendrites and somata of red nucleus (RN) neurons, respectively, in adult cat. To test if this synaptic site segregation is established by a sequential arrival of the two afferents, the development of CR and CBR projections was studied. CR axons arrived in the RN at embryonic day (E) 50 or 51, while CBR axons entered the RN before E35. These results suggest the possibility that arrival of CBR axons before CR axons leads to the segregation of synaptic sites.

Abstract: In order to discover molecules involved in axonal outgrowth during development of the central nervous system (CNS), monoclonal antibodies (MAbs) were raised against homogenates of the early postnatal rat cerebral cortex. A novel MAb, 5H, was obtained which recognizes a developmentally regulated antigen in the CNS. In the cerebral cortex of postnatal (PN) day 0-6 rats, 5H-immunoreactive punctate or fiber-like processes were observed. In the PN day 12 cortex, 5H immunostaining was mostly present as puncta. The expression of the 5H antigens decreased in the second PN week and completely disappeared from the cortex of rats older than PN day 18. In the hippocampal region, 5H immunoreactivity exhibited similar developmental changes, but with a more prolonged period of expression of the antigen (up to 5 weeks postnatally). They were also absent from the hippocampus of the adult rat. Similar developmental changes were also observed in the other CNS regions, but the only region in the adult CNS that showed 5H immunoreactivity was the olfactory bulb, in which synaptic turnover normally occurs even in the adult. Immunoelectron microscopy revealed that 5H immunoreactivity was localized to the cytoplasm of neuronal processes, including synaptic boutons. The expression of the 5H antigen in neurons cultured from rat cerebral cortex obtained from embryonic day 16 to PN day 6 was mostly restricted to neurite tips, including growth cones. These results suggest that the MAb 5H antigen is associated with axonal outgrowth during early development in the CNS.

Abstract: Axons in the central nervous system of mature mammals generally fail to regenerate following injury. Although the reason for this regenerative failure remains unknown, several lines of evidence suggest that it is due to nonpermissiveness of oligodendrocytes for axonal elongation. However, most of the in vitro experiments carried out so far used neural-crest-derived peripheral neurons to test the permissiveness of oligodendrocytes, although studying the interactions between central neurons and oligodendrocytes is crucially important for elucidating their roles in vivo. In this study we cultured retinas and dorsal root ganglia of the chick embryo with oligodendrocytes obtained from postnatal rat spinal cord and performed time-lapse analysis. Oligodendrocytes were identified with galactocerebroside antibody. Retinal growth cones readily grew over oligodendrocytes, while growth cones of the dorsal root ganglion collapsed and grew away on contacting the oligodendrocytes. Correspondingly, neurite-free areas centered by oligodendrocytes were formed behind growth cones in DRG-oligodendrocyte but not in retina-oligodendrocyte co-culture. These observations suggest the possibility that responsiveness of growth cones to oligodendrocytes is dependent on neuronal type.

Abstract: In the bilaterally symmetrical vertebrate CNS, all developing axons must choose between remaining on the same side of the midline or growing across it. The mechanism underlying this axonal pathfinding is, however, poorly understood. Here we demonstrate that the ventral midline floor plate (FP) chemorepels two types of ipsilaterally projecting axons, one from the alar plate and another from the basal plate in the mesencephalon. We further demonstrate that the FP chemoattracts contralaterally projecting myelencephalic as well as metencephalic axons. The FP at all axial levels displayed both chemoattractive and chemorepellent activities, suggesting that FP chemoattraction and chemorepulsion may be at work throughout the neuraxis. Chemotropic guidance by the FP may therefore play a key role in the establishment of neuronal projection laterality.

Abstract: Accumulating evidence suggests that an inhibitory influence of the environment on growth cones plays a crucial role in development and regeneration of neuronal projections. Oligodendrocyte-associated neurite growth inhibiting substance is one of the most extensively studied molecules. Molecular biological studies, however, remain slow in progress. Although finding clonal cells that express such factors would facilitate the analysis of inhibitory influences on neurite growth, few cell lines have been reported to express neurite growth inhibitor. We therefore investigated the possibility of a clonal glial cell line to differentiate and express inhibitory or non-permissive features for neurite outgrowth in culture. We chose the C6 glioblastoma cell line and examined neurite extension from chick dorsal root ganglion (DRG) explants. Neurites from embryonic day 9 DRG extensively grew on C6 cells that were cultured at low cell density, while they failed to grow on C6 cells cultured at high density, even in the presence of nerve growth factor in high concentrations. Membrane extract from high density C6 cells, when used as culture substratum, was less permissive for neurite outgrowth compared to extract from low density cells. Treatment of the membrane extract derived from high density C6 cells with trypsin made it less non-permissive for neurite growth. These results suggest that C6 cells are induced to express a non-permissive property for neurite outgrowth by culturing them at high density.

Abstract: The corticorubral projections in adult cats are primarily uncrossed. However, early in development and after early unilateral lesions of the sensorimotor cortex, crossed corticorubral projections are also observed. The present study was performed to disclose (1) whether the crossed projections originate from neuronal subpopulations different from those producing uncrossed ones and (2) how the neurons that give rise to the crossed projections in the lesioned animals are related to those occurring in normal development. We injected fluorescent latex microspheres into the red nucleus of two groups of animals: (1) intact kittens at postnatal week 3 and (2) kittens that had received unilateral ablation of the cerebral cortex at this stage and were then allowed to survive for at least 4 weeks. Red fluorescing microspheres were injected on one side and green ones on the other. In both normal and lesioned kittens, a number of cells in the cortex were labeled as a result of the contralateral as well as the ipsilateral injections, and no difference in size or distribution was found between the cells labeled from contralateral and ipsilateral injections. More than half of the cells labeled from contralateral injections were double-labeled in both groups of animals. These results indicate that individual corticorubral cells project bilaterally in normal development as well as following unilateral lesions of the cortex. With respect to the cells producing crossed projections, they were similar in both laminar and regional distributions between the intact and lesioned animal, suggesting that the crossed projections arise from the same neuronal subpopulation before and after cortical lesions. This view was supported by sequential injections of the tracers, which indicated that cells normally projecting contralaterally maintained the crossed projection after the lesions. Taking into account our previous observations that growth and proliferation of crossed corticorubral axons took place in the red nucleus (Murakami et al. 1991a), it is likely that growth and proliferation of the axons in denervated targets play a major role in lesion-induced establishment of aberrant projections.

Abstract: We examined whether transient projections in the developing central nervous system of Mammalia form functional synapses on their target neurons, using transient ipsilateral interpositorubral (iIR) projection in kittens as a model system. Intracellular recordings were made from red nucleus (RN) neurons in 26 kittens aged 6-26 postnatal days (PD6-26). RN neurons were identified by monosynaptic excitatory postsynaptic potentials (EPSPs) evoked by stimulation of contralateral nucleus interpositus (IN), and additionally by intracellular staining in a few cells. Sixty-nine out of 362 RN neurons responded to stimulation of the ipsilateral IN. Of the 69 cells, 25 showed depolarizing responses with relatively short latency (2.1-6.7 ms) in kittens up to PD20. Such responses were not observed in older animals. Varying stimulus strength revealed that the potentials were unitary. Paired-pulse facilitation of the potential was observed, suggesting that the depolarizations are EPSPs. Several lines of evidence were obtained suggesting that the EPSPs are evoked monosynaptically. They followed high-frequency stimulation up to 50 Hz, and their latencies remained constant with varying stimulus strength. The latencies of ipsilaterally induced EPSPs were always longer than those of contralateral ones, evidence consistent with the longer course of ipsilaterally projecting axons than that of contralateral ones (Song and Murakami 1990). The age of disappearance of the monosynaptic EPSPs, i.e., PD20, also corresponds roughly with that of the anatomically demonstrable iIR fibers (PD15-PD25; Song and Murakami 1990). It is thus concluded that the transient iIR fibers in kittens form functional synapses on RN neurons.

Abstract: Subcellular localization and in vitro expression of a newly raised monoclonal antibody 374 antigen, which outlines a subset of neurons in various regions of the rat CNS, were studied. Electron microscopy revealed that the immunoreactivity is associated with the outer surfaces of neuronal subsets, the surfaces of glial cell processes facing such neurons and less frequently, with the Golgi apparatus and inner cell surfaces. The monoclonal antibody also stained part of the cytoplasm of a population of dendrites. In order to test whether the antigen originates from neurons, we examined the expression of the antigen in cultured cortical cells. Double immunofluorescence staining with neuron-specific enolase and glial fibrillary acidic protein revealed that the antigen is expressed on a subset of neuronal cells and to a lesser extent on glial cells. The antigen is considered to be expressed on the outer surface because the cells were stained by the monoclonal antibody irrespective of whether they were alive or fixed. The monoclonal antibody 374 antigen may be a novel neuronal surface molecule which plays a role in neuron-neuron or glial-neuron interactions in the CNS.

Abstract: Cerebrorubral and cerebellorubral inputs are localized to distal dendrites and somata of red nucleus neurons in adult cats, respectively. To examine if this segregation is established early in development, we performed intracellular recording from rubrospinal neurons of fetal cats aged from embryonic day 58 to 65. Stimulation of the contralateral cerebellar nuclei evoked excitatory postsynaptic potentials (EPSPs). EPSPs were also induced by stimulation of the ipsilateral pericruciate cortext but they were much slower in time course and smaller in amplitude compared to cerebellar ones. We suggest that cerebrorubral and cerebellorubral synapses are segregated on soma-dendritic membrane of rubrospinal neurons early in development.

Abstract: Developing neurons transiently grow numerous spine- or filopodium-like dendritic protrusions (SLDPs). Electron microscopy on identified input and intracellular staining of postsynaptic cells were performed to gain insight into their significance. Newborn kitten-corticorubral axons, labelled with biocytin, commonly made synapses on SLDP, often multiply invaginated by the SLDPs. Correspondingly, intracellularly labelled kitten rubrospinal cells had numerous SLDPs. Taking into account that corticorubral synapses are largely formed on dendritic shafts in adult cats, it is likely that the SLDPs play some important role in the development of corticorubral synapses. We hypothesize that rubrospinal cells elongate SLDPs searching for corticorubral axons to form synapses.

Abstract: Although mature nervous systems show substantial malleability following various surgical or environmental manipulations, developing brains show far more prominent plasticity, particularly in terms of morphological features. Neuronal circuits, for example, can be dramatically rewired following neonatal but not adult brain lesions. It remains unknown why neuronal circuits in developing brains show such remarkable plasticity. A number of anatomical and physiological studies suggest that there are transient projections in developing brains and they are eliminated by cell death and/or collateral elimination as development proceeds. This raises a possibility that aberrant projections observed following various surgical or environmental manipulations such as partial denervation, results from retention or stabilization of transient projections. However, evidence suggests that cell death does not play an important role in developmental fine-tuning of neuronal projections. Furthermore, although the elimination of axon collaterals takes place, individual neurons appear to elaborate axonal arbors in appropriate target areas, resulting in a net increase in the size of axonal arbor emerging from individual neurons. In accord with these observations, the number of synapses appear to increase during the period when axonal elimination proceeds. Taken together, reinforcement of appropriate projections rather than elimination of excessive connections plays a major role in developmental specification of neuronal connections. Appearance of aberrant projections after partial denervation may not be a consequence of disordered axonal growth, since they form topographic maps which precisely mirrors those for normal projections. They may be induced due to reinforcement of pre-existing neuronal connections rather than to construction of novel pathways. Observations of axonal morphology in denervated areas indicate that lesion-induced enlargement of projections is due to transformation of axonal morphology, from simple and poorly branched to multiply branched. Perhaps such simple and poorly branched axons in inappropriate target areas may represent ones in the course of elimination but they may serve as a source of sprouting when denervated. In other words, after total elimination of axons any surgical or environmental manipulation cannot induce enlargement of projections. The mechanisms underlying such modifiability of neuronal connections remains unclarified but possible participation of an activity-dependent competitive mechanism is discussed.

Abstract: The corticorubral projections in newborn kittens are bilateral, while the projections are unilateral in adults. We addressed the question whether or not the crossed corticorubral projection in kitten forms synaptic contacts in the red nucleus. The neurons in the sensorimotor cortex of the kitten were labeled by Phaseolus vulgaris-leucoagglutinin or biocytin. Electron microscopic observations revealed that corticofugal axons form synapses both in the contralateral and the ipsilateral red nucleus; most of them were on small postsynaptic profiles, possibly dendritic spines or distal dendrites.

Abstract: The aberrant crossed corticorubral projection of the cat, which is very weak compared to the uncrossed one at about 1 month postnatal, becomes pronounced following unilateral lesions of the sensorimotor cortex. In order to determine whether or not terminal proliferation of pre-existing axons underlie this enlargement, the morphological changes of the crossed axons were examined, using the anterograde tracer Phaseolus vulgar- is leukoagglutinin (PHA-L). The crossed corticorubral axons in normal kittens were mostly simple in morphology with infrequent branching and did not often exhibit growth-cone-like axonal endings at 1 month postnatal. Two to 5 days after unilateral lesions of the sensorimotor cortex placed at this age, the axons were as simple as those in normal animals but ended in growth cones more frequently. Seven to 10 days post-lesion, the axons often bore side-branches which ended in growth cones. Two to 3 weeks post-lesion axons with sprays of finger-like fine sprouts occurred throughout the projection zone. There was no clear topography for the crossed projection in normal animals, but at 1-2 weeks post-lesion the axons started to show a certain amount of localization in the regions of the red nucleus which corresponded to the densely innervated region on the ipsilateral side. The topography of the crossed projections roughly mirrors that of the ipsilateral projection at about 1 month post-lesion. Thus, the lesions of the sensorimotor cortex induce substantial growth and proliferation of the crossed corticorubral axons. The post-lesion changes in axonal morphology and topographic refinement are reminiscent of developmental events. It is likely that the lesions permit the crossed axons, which normally fail to develop, to develop like the uncrossed ones.

Abstract: Localization of a telencephalon-specific glycoprotein, telencephalin (TCLN), in the olfactory bulb of the rabbit was studied with an electron microscope. Anti-TCLN antisera appeared to stain plasma membrane, Golgi apparatus and multivesicular bodies of granule cells which are local circuit interneurons in the bulb. Principal neurons, mitral and tufted cells, were not immunoreactive. No glial cells showed immunoreactivity. Thus, expression of telencephalin is specific not only to the telencephalic segment of the brain, but also to the neuronal types.

Abstract: Hemicerebellectomy has been reported to induce aberrant ipsilateral projection from the nucleus interpositus (IN) of the remaining hemisphere to the red nucleus (RN) in the kitten but not in adult cats. The cellular mechanisms for this age-at-lesion effect were investigated. The experiments were designed to compare interpositorubral (IN-RN) projections in normal kittens and in kittens hemicerebellectomized at corresponding ages. A sparse ipsilateral IN-RN projection was found in normal kittens aged 7-16 postnatal days (PND) with the PHA-L method, but was not found in those aged 26-43 PND. Among the hemicerebellectomized kittens, ipsilateral IN-RN projections were observed only in those which received the lesion on PND 17 or earlier. Such temporal coincidence between the two events strongly suggests that the exuberant ipsilateral IN-RN projection in normal kittens is related to the establishment of the aberrant ipsilateral IN-RN projection observed after hemicerebellectomy. This notion is strengthened by other lines of evidence; the fibers projecting to the ipsilateral RNs appeared to enter the nucleus from the contralateral one by recrossing the midline between them in both the normal and the lesioned animals. In kittens in which hemicerebellectomy induced the aberrant ipsilateral projection, labeling of fibers within the ipsilateral RNs was much more extensive than that in the normal kittens, while labeling on the pathway of the projection was comparable to that in the normal animals. These observations support the interpretation for aberrant projections observed after a neonatal lesion as being terminal proliferations of pre-existing exuberant projections.

Abstract: Chick neocortical cells were cultured on cryostat tissue sections of the brain. Cells preferentially attached to the gray matter of adult rat central nervous system (CNS) tissues. In contrast, they attached to any part of the brain when cultured on developing rat or mature frog brain tissues. Transection of fiber bundles at the superior cerebellar peduncle decussation of adult rat, which reportedly causes regeneration of cerebellofugal axons, made nearby white matter permissive to cell attachment. Superimposition of the gray matter of one section onto the white matter of another, converted the former into a nonpermissive substrate for cell attachment, evidence suggesting that preferential cell attachment to the gray matter of adult rat CNS is due to inhibitory factor(s) localized in the white matter. This inhibitory factor appears to be absent in frog brain and developing rat brain. These results taken together suggest possible involvement of this factor in the regulation of axonal elongation in vivo.

Abstract: Cellular mechanisms for decision of laterality of projections are discussed based on the observation of corticorubral and interpositorubral projections in the kitten. In both systems unilateral projections in adults appear to emerge from bilateral projections which occur early in development. Early unilateral lesions of the cerebral cortex or the interpositus nucleus cause persistent bilateral projections. Thus the neurons which eventually make a unilateral projection have the capability of forming synaptic connections with their target cells on both sides of the brain. Aberrant crossed (or uncrossed) projections in neonatal animals retract without forming complex axonal arbors, while those in lesioned animals form complex axonal arbors. Thus, success or failure in forming axonal arbors may be crucial for the maintenance of the aberrant crossed (or uncrossed) projections.

Abstract: Morphological changes in individual corticorubral fibers and the pattern of crossed and uncrossed corticorubral projections were studied during the postnatal development of cats in order to understand cellular mechanisms for restriction of corticorubral projections with development. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into restricted areas of the pericruciate cortex in kittens and PHA-L-labeled axons in the red nucleus were examined at postnatal days (PND) 7-73. In accordance with our previous study (Murakami and Higashi, Brain Res. 1988; 447:98-108), a crossed corticorubral projection was observed in addition to the uncrossed one in every experimental animal. During the early period of development (PND 7-8), swellings of irregular shape were observed along the entire course of the axons and they were often interconnected with extremely fine axonal segments. These axons bifurcated only infrequently and often ended as growth cones. These features were common to both uncrossed and crossed corticorubral axons. At later stages of development (PND 28 or later), the total number of swellings decreased and axonal swellings with smooth contours became dominant. A quantitative examination of axonal branches indicated that axons on the ipsilateral side branch occurred more frequently at later stages of development. However, there was no substantial change in branching frequency for the crossed corticorubral fibers during development. In parallel with morphological changes in individual axons, the crossed projection that was initially relatively abundant was reduced during development. Since a PHA-L injection can be confined to a small region of cortex, topographic projections can easily be detected. At PND 7-8 there was no well-defined topographic order in the ipsilateral corticorubral projection. Adult-like topography was first discernible at PND 13. These observations suggest that the unilateral uncrossed corticorubral projection in the adult cat is achieved at least in part by the formation of axonal arbors in the uncrossed projection. This was accompanied by the failure of crossed fibers to form complex arbors. It is possible that a similar mechanism also operates in the formation of topographic maps.

Abstract: Among a panel of monoclonal antibodies generated against monkey brain tissue, a class of antibodies was found to produce perineuronal staining of small subsets of mammalian central neurons. Three antibodies (MAbs 473, 376, 528) we report here define two different, though partially overlapping, neuronal subsets in the monkey neocortex. All 3 antibodies stain in addition certain chondrocytes. The neural immunoreactivities were lost, and the chondral immunoreactivities either lost or enhanced, after treatment of the sections with chondroitinase ABC. Independently, 3 other antibodies (MAbs 1B5, 9A2, 3B3) with established specificity to glycosaminoglycan epitopes also produced perineuronal staining of a related subset of central neurons. Immunoblot experiments with two of the antibodies revealed bands of high molecular weight. These findings indicate that certain glycosaminoglycans occur surrounding mammalian central neurons, and suggest that different neuronal subsets are associated with different combinations of proteoglycan epitopes.

Abstract: A monoclonal antibody was obtained by immunizing mice with an extract of monkey brain. The monoclonal antibody 473 stained a small subpopulation of neurons in various regions of monkey and rat central nervous system. The perimeters of neuronal somata and the proximal parts of dendrites bound the antibody. Electron microscopic analysis showed that the immunoreactivity was associated with the outer surface of the cell. The immunoreactivity in the rat cerebral cortex appeared gradually during the second four weeks after birth. The antibody stained fetal cartilages but otherwise was specific to the nervous system. Experiments on the stability of the immunoreactivity to enzymatic and chemical treatments of the sections suggest that the antigen molecule is of proteoglycan nature.

Abstract: Dissociated chick neocortical neurons were cultured on cryostat sections of the rat central nervous system. The neurons adhered to and grew on the gray matter of the tissue derived from various parts of the central nervous system (CNS), but were not seen on the white matter. However, cell attachment was seen on sciatic nerve. This preferred adhesion to and growth on the gray matter of the CNS was abolished by irradiation of ultraviolet light which is supposed to denaturate proteins without disturbing tissue architecture. These observations suggest that differential cell adhesion to the gray and white matter could be ascribable to localization of some adhesive molecule(s) in the gray matter or to localization of nonpermissive molecule(s) in the CNS gray matter.

Abstract: After injection of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the interposed nucleus of the cat, labelled fibers were found in the granular layer as well as in the molecular layer of the cerebellar cortex. Those in the molecular layer were judged from their location and morphology as climbing fibers. Such labelling of climbing fibers which have not been labelled by autoradiography could be attributed to higher sensitivity and resolution of PHA-L method. In addition, Purkinje cell soma and its dendrites were labelled in a narrow stripe in the vermal and paravermal cortex. The pattern of labelling suggests that they were labelled by retrograde transport of the lectin.

Abstract: Brain lesions made during early developmental stages produce more prominent remodeling of synaptic organization than those made in adults. This difference in the extent of neuronal or synaptic plasticity between immature and mature animals may be due to difference in the capacity for axonal elongation. Alternatively, it could be due to the prevention of retraction of exuberant projections present only in the early developmental stages. Aberrant crossed corticorubral projections seen after neonatal hemispherectomy have been ascribed to collateral sprouting. To determine whether these results from the prevention of retraction of crossed fibers, we studied the corticorubral pathway in normal kittens and compared it with that observed after unilateral cortical lesion, using the plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L). One to two weeks after injection of PHA-L, many immunocytochemically labelled fibers were observed in the red nucleus (RN) ipsilateral to the cortical injection. Although very few, labelled fibers were also seen in the RN contralateral to the injection in normal kittens. By contrast, many labelled fibers were seen in the RN contralateral to the injection in lesioned animals. Many growth-cone like axonal endings were also observed. The abundant crossed corticorubral fibers seen in lesioned animals may be ascribed to the increase in the number of fibers crossing the midline towards the contralateral RN or they could be due to increased branching of pre-existing crossed fibers.

Abstract: Pairing of the stimulus to the cerebral peduncle (CP) with that to the forearm skin leads cats to flex their forearms within a 10-day training period in response to stimulus to CP, which was initially ineffective. Behavioral study and extracellular unit analysis suggested that the cellular mechanism for this conditioning lies at the corticorubral (CR) synapses. Since formation of new CR synapses occurs in parallel with the recovery from behavioral deficits after brain damage and peripheral nerve cross-innervation, we explored the possibility that the formation of new CR synapses underlies conditioning. We investigated the time course of the CR excitatory postsynaptic potentials (EPSPs) as well as the distribution of the CR synapses on the somadendritic membrane of the red nucleus neurons and compared them with those observed in control animals. In conditioned animals, the times-to-peak of the CR EPSPs were significantly shorter than those in control animals. Electron microscopic studies demonstrated that more CR synapses make contact with large, i.e. proximal, dendrites and somata of red nucleus neurons in conditioned cats than in control ones. These results support the view that the formation of new synapses on the proximal dendrites and soma underlies classical conditioning in the cat.

Abstract: Giant neurons of the red nucleus of the cat were stained intracellularly with horseradish peroxidase and examined using light microscopy, electron microscopy of thin sections, and high voltage electron microscopy of thick sections (2-5 microns). Special attention was paid to the arrangement of dendritic spines and other appendages relative to the distribution of synaptic contacts from known sources. In the region of the neuron known to receive synaptic contacts from the nucleus interpositus of the cerebellum (soma and proximal 200-300 microns of dendrites), the dendrites were relatively unbranched, and free of long spines or complex appendages. The surface of the neurons in this region was covered with a dense layer of short thin appendages that invaginated or penetrated between the synaptic terminals that cover this part of the cells. The small spines received synapses of the types associated both with the cerebellar afferent fibers and with the local inhibitory interneurons. These same terminals made synaptic contacts directly onto the surface of the neurons and onto the lateral surfaces of the spines, suggesting that the spines may serve primarily to increase the available synaptic surface area. The more distal portion of the dendritic field, where cerebellar afferents do not make synaptic contacts, exhibited a dramatically different appearance. The dendrites were much more branched, and exhibited many and varied dendritic appendages. The appendages were of three general types. One was a large protrusion with a cup-shaped head that formed the principal postsynaptic component of a glomerular arrangement also involving an axon terminal and usually a presynaptic dendrite. A second was a long thin filiform process that usually occurred around the glomeruli. This appendage was occasionally postsynaptic. The third was a spherical appendage containing many lysosomal organelles resembling residual bodies. The glomerular dendritic protrusions were very common in the distal portion of the dendritic field, numbering at least 1000 per cell. At least some of the glomeruli are specialized for receipt of synaptic input from the corticorubral pathway, since lesions of sensorimotor cortex resulted in degeneration of the central synaptic terminal in some glomeruli on horseradish peroxidase-injected rubrospinal neurons. These specializations of dendritic structure may contribute to the differences in excitatory postsynaptic potential wave shape between cortical and cerebellar inputs, and they may play a role in the changes in the cortical excitatory postsynaptic potential that develop after lesions of cerebellar inputs.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: An electron microscopic quantitative study of corticorubral synapses was performed in the cat which acquired classical conditioning. Conditioned stimulus was applied to the cerebral peduncle and the unconditioned stimulus was an electrical shock to the forelimb skin. The proportion of corticorubral synapses contacting with somata and proximal dendrite was increased after conditioning. It was suggested that collateral sprouting and the formation of new synapses underlie classical conditioning.

Abstract: An immunocytochemical study using anti-GAD serum was performed to examine the plastic changes of GABAergic inhibitory synapses in the red nucleus (RN) after lesions of the nucleus interpositus (IP) of the cat. Light-microscopic analyses revealed that 20-175 d after the unilateral lesion of the IP, somatic profiles of large neurons in the magnocellular RN contralateral to the lesion were more densely covered with GAD-immunoreactive puncta than those in the ipsilateral RN. Electron-microscopic analyses demonstrated that the GAD-immunoreactive puncta observed with the light microscope were synaptic terminals and that the number of GAD-immunoreactive synaptic terminals per unit length of somatic membrane of RN neurons was increased on the deafferented side. The GAD-immunoreactive terminals on somata of RN neurons made symmetric synaptic contacts with somatic membranes on both the deafferented and control sides. The number of immunoreactive synapses on somata of RN neurons was markedly increased on the deafferented side following IP lesion, whereas that of the unlabeled asymmetric synapses was decreased. These observations indicate that new GABAergic synapses were formed on somata of RN neurons after deafferentation from the IP.

Abstract: A new iterative single-electrode voltage clamp method was applied to the measurement of synaptic currents in the red nucleus (RN) neuron of the cat. Voltage clamp was attained within 10 repetitions with great stability and the new algorithm was demonstrated to be superior to the original algorithm of iterative voltage clamp. With a conventional microelectrode, it was possible to measure the synaptic current with the time resolution of 50 microseconds. The synaptic currents evoked by stimulation of the contralateral interpositus nucleus (IP) had time-to-peak ranging from 200 to 540 microseconds and fitted well to alpha functions. Corticorubral (CR) synaptic current was also measured by making use of synaptic plasticity. The stimulation of the ipsilateral cerebral peduncle in cats with chronic lesion of the contralateral IP evoked fast rising EPSPs, as reported previously. The CR-EPSPs with times-to-peak less than 1 ms were subjected to voltage clamp. The CR synaptic currents had times-to-peak ranging from 350 to 880 microseconds. Since most of the interpositorubral (IR) synapses and a part of the CR synapses in IP-lesioned cats are situated on the somatic membrane of RN neurons and some of the CR synaptic currents were as rapid as the IR synaptic currents, the observed synaptic currents evoked by stimulation of the IP and those of the fast-rising CR-EPSPs were taken to originate from the synaptic membrane under space-clamp, i.e. soma. The present study provided additional evidence for the sprouting of the CR fibers as well as the time course of the synaptic current at the dendritic synapses remote from the soma, for the first time.

Abstract: The appearance of crossed corticorubral projections following ablations of the ipsilateral cortex is shown to result from the formation of new connections and is not due to the preservation of pre-existing bilateral connections. At least some of these crossed projections are collaterals of the pyramidal tract. Post-tetanic potentiation can be demonstrated both intra- and extracellularly following ipsilateral cerebral peduncle stimulation whereas no changes in excitatory postsynaptic potential amplitude are observed following contralateral cerebral peduncle stimulation.

Abstract: The presence of glutamic acid decarboxylase (GAD), the enzyme synthesizing gamma-aminobutyric acid (GABA), was investigated in the red nucleus by an immunocytochemical method. The ipsilateral sensorimotor cortex was ablated prior to the immunocytochemical procedures to examine whether cortical neurons make synaptic contacts with GAD-immunoreactive neurons. Small GAD-immunoreactive neurons with a major diameter of 16.1 +/- 3.2 micron (mean +/- S.D.) were observed in the red nucleus under both light and electron microscopy. They were uniformly distributed throughout the nucleus. Degenerating axon terminals were found making synaptic contact with GAD-immunoreactive neurons in the red nucleus, which suggests that there is an input from the ipsilateral sensorimotor cortex to these neurons. This observation, along with our previous findings that GABAergic axon terminals make synaptic contact with the rubrospinal neurons, provides anatomical evidence for the presence of intrinsic GABAergic interneurons which mediate cortical inhibition in cat rubrospinal neurons.

Abstract: A quantitative electron microscopic study of corticorubral synapses was performed in the red nucleus (RN) of adult cats to determine the morphological correlates for the changes in time course of corticorubral excitatory post-synaptic potentials, which occur following cross-innervation of forelimb extensor and flexor nerves. Corticorubral synaptic endings were identified by anterograde degeneration after lesions of the ipsilateral sensorimotor cortex. Rubrospinal neurons innervating upper spinal segments were electrophysiologically identified and filled with horseradish peroxidase (HRP). These cells were mainly situated in the dorsomedial part of RN. Electron micrographs of the degenerating corticorubral synaptic endings were taken in the region surrounding HRP-filled neurons and the diameter of the dendrites contacted by such terminals was measured. In the cross-innervated animals many degenerating terminals were found to synapse on dendrites with large diameter and the somata of neurons in RN. This is in contrast to the previous observations in normal cats, in which very few corticorubral synapses were found to synapse on proximal dendrites and somata of RN neurons. The diameter of HRP-filled neurons in cats which were cross-innervated was slightly smaller than those observed in normal animals. These results indicate that new corticorubral synapses were formed on proximal dendrites and somata of RN neurons as a consequence of cross-innervation.

Abstract: Several suggestions have been made with regard to the functional significance of dendritic spines in connection with synaptic plasticity. We have shown that for a constant synaptic current, when the synaptic resistance is large compared to the spine-stem resistance, a morphological change in the spine does not produce a marked change in the postsynaptic potential (PSP). When the synaptic resistance is comparable to the spine-stem impedance a morphological change in the spine can induce changes in the synaptic current and the PSP due to the so-called nonlinear effect to the synapse (Kawato and Tsukahara, 1983, 1984). Consequently, in a study of the electrical properties of dendritic spines the input impedance of the parent dendrite, the spinestalk conductance and the conductance change associated with synaptic activity must be considered. We quantitatively estimated all three factors. By comparing electrophysiological data with morphological data, we estimated the synaptic conductance which causes corticorubral EPSP. Its maximum amplitude was 43 nS with a time-to-peak value of 0.3 ms. With this value, the effects of the spine were examined using an improved algorithm based on that of Butz and Cowan (1974). It uses a three-dimensional morphology of the rubrospinal (RS) neurons, which was reconstructed from serial sections containing HRP-filled RS cells. As the spine shortens, the amplitude of the EPSP becomes considerably larger, but its time-to-peak value does not markedly change. Moreover, if unitary EPSP in the RS cell is produced by the activation of several synaptic terminals a morphological change of the spine has a smaller effect on the EPSPs.

Abstract: Afterhyperpolarization (AHP) following single or short trains of spikes in rubrospinal neurones (RN neurones) of the cat has been studied with intracellular recording techniques. The AHP amplitude was potential dependent; it increased with depolarization and decreased with hyperpolarization and had an extrapolated reversal potential about 20 mV below resting membrane potential. The AHP was associated with an increase in the membrane conductance and it was concluded that the AHP is primarily caused by an increase in membrane conductance to potassium ions. The time course of the conductance change underlying the AHP was measured with short current pulses and calculated from the AHP voltage. The AHP following a single spike was conditioned at different interspike intervals by a preceding spike (or several spikes). In many RN neurones the AHP (conductance) following a spike added approximately linear to that generated by a preceding spike. In most cells, however, the AHP following a spike was instead depressed by a preceding spike. The summation of AHPs increased progressively, while the depression appeared to be already maximal with one preceding spike. The depression was then approximately constant for interspike intervals less than the AHP duration. It will be shown in a following paper that these properties of the AHP are reflected in the behaviour of the repetitive discharge evoked by constant current pulses in the same neurones.

Abstract: Synaptic inputs of rubrospinal (RN) neurons from the cerebral cortex, pretectal area (PRT), and medial lemniscus (ML) were investigated electrophysiologically in the cat. Stimulation of the ipsilateral parietal association cortex (PASC) and secondary sensory area (SII) produced slow-rising about 3 msec rise time monosynaptic EPSPs which were, in some cases, followed by hyperpolarizations, similar to the sensorimotor cortex (SM)-induced PSPs previously observed. Stimulation of the contralateral cerebral cortex never produced detectable PSPs. Topographical arrangement of PASC-rubral projection was found. Stimulation of the lateral part of PASC induced EPSPs predominantly in RN cells innervating the cervicothoracic spinal segments, while stimulation of the medial part of PASC produced EPSPs predominantly in RN cells innervating the lumbosacral cord. Furthermore, PASC-induced EPSPs were more frequently recorded at the rostral half of RN than at the caudal half. Monosynaptic EPSPs and multisynaptic IPSPs were induced by stimulation of the ipsilateral PRT and ML. PRT- and ML-induced EPSPs had times-to-peak of 1.0 +/- 0.4 msec (mean +/- S.D.) and 1.6 +/- 0.5 msec, respectively, which were intermediate to those of the cerebral peduncle (CP)- and nucleus interpositus of the cerebellum (IP)-induced EPSPs. Furthermore, sensitivity of amplitudes of PRT-induced EPSPs to membrane hyperpolarization was intermediate to those of CP- and IP-EPSPs, and that of ML-induced EPSPs was lower than that of IP-EPSPs. Therefore, it is likely that synapses of PRT and ML fibers are formed between the distal dendrites where CP-rubral synapses terminate and soma where IP-rubral synapses terminate. PASC-induced EPSPs after chronic IP and SM lesions had a new fast-rising component and the effectiveness of ML stimulation to induce the unit spike of RN cells was clearly increased in IP and SM lesioned cats. It was suggested that PASC-rubral fibers sprouted and formed new synapses at the proximal portions of soma-dendritic membranes of RN cells after IP and SM destructions. Collateral fibers to RN cells of the pyramidal tract were also shown to sprout new synapses following IP and ML lesions.

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Abstract: GABAergic synapses on rubrospinal neurons were demonstrated with immunocytochemistry combined with intracellular injection of horseradish peroxidase. Sections containing red nucleus neurons were processed for glutamic acid decarboxylase (GAD) immunohistochemistry. GAD-immunoreactive synaptic endings formed synaptic contacts with somata and dendrites of red nucleus neurons and identified rubrospinal neurons. Our observation provides further evidence that GABA acts as an inhibitory transmitter mediating cortically evoked inhibitory postsynaptic potentials in red nucleus neurons.

Abstract: Intracellular recordings were made from neurons of the red nucleus (RN) in cats where the cerebellar cortical effects were removed by chronic ablation of the intermediate part of the anterior lobe of the cerebellum. A prolonged depolarization could be elicited by stimulating the nucleus interpositus (IP) of the cerebellum, nucleus reticularis tegmenti pontis (NRTP) and the nucleus reticularis paramedianus (PMRN). This prolonged depolarization was abolished after cooling the inferior and middle cerebellar peduncles and persisted after ablation of the cerebral sensorimotor cortex. The prolonged depolarization was also recorded intracellularly from IP neurons. It was concluded that the prolonged depolarization set up in RN neurons is due to the repetitive discharges of IP neurons which produces tonic bombardment onto RN cells. The mechanisms of the repetitive discharges of IP neurons are considered to be due to impulse reverberation via the IP. The dynamic properties of the reverberating circuits were characterized by regenerative behavior. Above and below threshold, there were two states, the excited state where many constituent neurons were active, and the resting state where all neurons were inactive. It was found that cats with chronically stimulated cerebral peduncle (CP), and tested in an acute experiment, showed sometimes effective for inducing the prolonged depolarization and repetitive discharges of RN neurons by stimulation of IP. The prolonged depolarization thus produced could be reversibly abolished by cooling the middle and inferior cerebellar peduncles. The possible constituent neurons of the reverberating circuits were investigated in light of previous physiological investigations of stimulating the NRTP, PMRN, nucleus reticularis lateralis (LRN), nucleus olivaris inferior (IO) and recording EPSPs in RN cells. The RN cells receive axon reflex activation from NRTP and PMRN, and disynaptic excitation from NRTP, PMRN, LRN and IO. Based on these and other available data, the components of the cerebello-precerebellar reverberating circuits are discussed.

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular alpha-bungarotoxin (alpha BT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9S intracellular alpha BT binding component, with a pI value close to that of the extrajunctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.

Abstract: A quantitative electron microscopic analysis of the corticorubral projection was performed in the red nucleus (RN) of adult cats to determine morphological correlates of synaptic reorganization that occur following a lesion of the interpositus nucleus (IP). Corticorubral synaptic endings were identified by lesioning the sensorimotor cortex 2-6 days before electrophysiological experiments. Horseradish peroxidase (HRP) was injected into electrophysiologically identified RN neurons. Sagittal sections 100 micrometers thick were cut and reacted by diaminobenzidine. Sections containing HRP-positive neurons were selected and embedded in Epon. In normal cats, degenerating corticorubral terminals in the RN region frequently made contact with dendritic profiles, having small cross-sections, while a few made contact with somatic profiles. Similar results were obtained when degenerating terminals making contact with HRP-filled dendrites were analyzed. In the experimental animals, the cortical lesion was performed more than 8 weeks after lesion of the IP. In these animals, degenerating corticorubral terminals were frequently found on proximal dendrites and somata in RN region and HRP-positive neurons in contrast to the findings in normal cats. The results indicate that new corticorubral synapses were formed on proximal dendrites and somata of RN neurons as a consequence of IP lesions.

Abstract: Reciprocal excitatory connections which give rise to reverberatory activity were investigated by intracellular recording and retrograde horseradish peroxidase (HRP) transport. Neurons in the interpositus nucleus (IP) were activated antidromically from the nucleus reticularis tegmenti pontis (NRTP) and paramedian reticular nucleus (PMRN). Stimulation of these nuclei and lateral reticular nucleus (LRN) elicited monsynaptic EPSPs in IP neurons. PMRN neurons could be activated antidromically as well as orthodromically from IP. HRP-positive neurons were found in NRTP, PMRN and LRN following injection of HRP into IP. Neuronal connections of IP-NRTP-IP, IP-PMRN-IP and IP-RN-LRN-IP were suggested as possible components of reverberating circuits.

Abstract: 1. Properties of synaptic transmission during and after repetitive activation of the newly formed cortico-rubral were examined in the red nucleus neurons (RN) of cats after lesions of the nucleus interpositus of the cerebellum (chronic cats) as well as in normal ones. 2. A prominent facilitation of the amplitude of cortico-rubral unitary EPSPs was observed in both normal and chronic cats when a stimulus to the cerebral peduncle (CP) was preceded by another stimulus by 2-50 msec. 3. Time course of the facilitation shows that it attains maximum at the interval of about 3 msec and decays approximately exponentially lasting for 50 msec or more. 4. When three successive stimuli of identical intensity were applied to CP, the degree of facilitation was more prominent than that for double shock. 5. There was a positive correlation between the time to peak of the cortico-rubral EPSPs and their maximum value of facilitation. 6. The posttetanic potentiation of the cortico-rubral EPSPs was observed after tetanic stimulation to CP in chronic and normal cats. It lasts for a few minutes in both cases.

Abstract: 1. Unitary EPSPs were recorded intracellularly from neurons of the red nucleus (RN) by stimulating the cortico-rubral fibers in normal cats as well as those with chronic lesion of interpositus nucleus (IP) of the cerebellum. 2. Two groups of unitary EPSPs were recorded in cats with IP lesion. One consisted of cortico-rubral unitary EPSPs with a shorter time to peak and larger amplitude than those in normal cats. The other consisted of unitary EPSPs of the normal range. 3. The appearance of the fast rising group of cortico-rubral EPSPs caused by IP lesions was theoretically predictable based on Rall's compartmental model by assuming that new synapses were formed close to the soma of the RN cells. 4. The amplitude of the fast rising EPSPs increased more prominently by hyperpolarizing the membrane potential than that of the slowly rising ones. 5. It was concluded that the fast rising group of unitary EPSPs which appeared following IP lesion was due to the formation of new synapses on the proximal portion of the soma-dendritic membrane of RN cell.

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Abstract: Membrane electrical constants have been studied in neurons of the red nucleus (RN) of the cat which were identified antidromically from the spinal cord. For each cell, the input resistance was determined from the membrane potential changes to current steps and was found to be 2.5+/-0.9 Momega in twenty five RN cells studied. In addition, linear summation of the membrane responses induced by two current pulses was demonstrated. 2. From the membrane voltage transients to current steps, the first membrane time constant, tau0, and second time constant, tau1, were determined as 5.6+/-1.0 msec and 0.6+/-0.2 msec, respectively. The ratio of the amplitudes of two exponential functions, E1/E0, was 0.18+/-0.05. A linear relation was found between the ratio of these amplitudes of exponential functions and that of the two time constants. 3. The cable parameter (electrotonic length, L) of the combined soma and dendrites of the RN neutrons was estimated as 1.1 from membrane transient data using the relation developed by Rall (1969). 4. By using this parameter, an attempt was made to estimate the location of the two excitatory inputs on the soma-dendritic membrane of RN cells.

Abstract: Intracellular recording was made from cat's red nucleus after chronic lesion of the nucleus interpositus (IP) of the cerebellum and the properties of the corticorubral EPSPs were examined. 2. It was found that the time to peak of the corticorubral ESPSs induced from cerebral peduncle (CP EPSP) of chronic cats was much faster than that of normal cats. There was no simple decay following the rapid rise and early summit; instead there was another peak as if the slow normal CP EPSPs were superimposed on the fast ones. 3. By stimulating two loci, sensorimotor cortex and the cerebral peduncle, the conduction velocities of the fibers responsible for the newly appeared fast-rising component of the corticorubral EPSPs were measured. They were almost the same as those of normal corticorubral EPSPs.