Miwa Kawasaki

OCAM: A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons

Zone-to-zone projection of olfactory and vomeronasal sensory axons underlies the topographic and functional mapping of chemoreceptor expression zones of the sensory epithelia onto zonally arranged glomeruli in the main and accessory olfactory bulbs. Here we identified OCAM (R4B12 antigen), an axonal surface glycoprotein expressed by subsets of both olfactory and vomeronasal axons in a zone-specific manner. OCAM is a novel homophilic adhesion molecule belonging to the immunoglobulin superfamily with striking structural homology to neural cell adhesion molecule. In both the main and accessory olfactory systems, OCAM mRNA is expressed by sensory neurons in restricted chemoreceptor expression zones, and OCAM protein-expressing axons project to the glomeruli in the corresponding zones of the main and accessory bulbs. OCAM protein is expressed on subsets of growing sensory axons in explant cultures even in the absence of the target bulb. These results demonstrate a precisely coordinated zonal expression of chemoreceptors and OCAM and suggest that OCAM may play important roles in selective fasciculation and zone-to-zone projection of the primary olfactory axons.

A Genetic Approach to Visualization of Multisynaptic Neural Pathways Using Plant Lectin Transgene

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.

BIG-1: A new TAG-1/F3-related member of the immunoglobulin superfamily with neurite outgrowth-promoting activity

We have cloned a rat cDNA for a novel brain-derived immunoglobulin (Ig) superfamily molecule, BIG-1, by using PCR based on the amino acid sequences of the two closely related and well-known Ig superfamily members, rat TAG-1 and mouse F3. BIG-1 is a glycosylphosphatidylinositol-anchored membrane protein with six Ig-like domains and four fibronectin type III repeats, belonging to the TAG-1/F3 subgroup. The expression of BIG-1 mRNA is developmentally regulated with the highest level in the adult brain. It is restricted to subsets of neurons such as Purkinje cells of the cerebellum, granule cells of the dentate gyrus, and neurons in the superficial layers of the cerebral cortex. Recombinant BIG-1 protein has a neurite outgrowth-promoting activity when used as a substrate for neurons in vitro. These results suggest that BIG-1 may be involved in the formation and maintenance of neuron type-specific networks in the brain.

Interaction between Telencephalin and ERM Family Proteins Mediates Dendritic Filopodia Formation

Dendritic filopodia are long, thin, actin-rich, and dynamic protrusions that are thought to play a critical role as a precursor of spines during neural development. We reported previously that a telencephalon-specific cell adhesion molecule, telencephalin (TLCN) [intercellular adhesion molecule-5 (ICAM-5)], is highly expressed in dendritic filopodia, facilitates the filopodia formation, and slows spine maturation. Here we demonstrate that TLCN cytoplasmic region binds ERM (ezrin/radixin/moesin) family proteins that link membrane proteins to actin cytoskeleton. In cultured hippocampal neurons, phosphorylated active forms of ERM proteins are colocalized with TLCN in dendritic filopodia, whereas α-actinin, another binding partner of TLCN, is colocalized with TLCN at surface membranes of soma and dendritic shafts. Expression of constitutively active ezrin induces dendritic filopodia formation, whereas small interference RNA-mediated knockdown of ERM proteins decreases filopodia density and accelerates spine maturation. These results indicate the important role of TLCN–ERM interaction in the formation of dendritic filopodia, which leads to subsequent synaptogenesis and establishment of functional neural circuitry in the developing brain.

Expression of prostaglandin endoperoxide synthase in rat brain.

Developmental and regional expression of prostaglandin endoperoxide synthase (PES) transcript was examined in the rat brain and in primary mixed cultures of neurons and glial cells from neonatal brain. Although the PES mRNA level in the brain was much lower than that in peripheral rat tissues such as lung, liver, spleen and kidney, a significant 3.0 kb band was detected in brain samples by Northern blot analysis. During development, PES mRNA was first detectable at postnatal day 7, and increased thereafter toward adulthood. The highest level of 3.0 kb PES mRNA was observed in the olfactory bulb, midbrain, and hypothalamus; and the lowest level in the hippocampus. In primary cultures of neonatal brain cells, the level of 3:0 kb transcript of PES transiently and dramatically increased about 30-fold on the third day after plating. Simultaneously, two cross-hybridizing signals were detected at 4.0 and 7.0 kb. This increase in PES mRNAs was completely inhibited by addition of cytosine-1-beta-D-arabinofuranoside. The induction of PES mRNA was in parallel with the increase in PES protein, as assessed by Western blot analysis. Immunostaining of cultured cells with anti-PES monoclonal antibody revealed that PES protein was induced mainly in neurons but not in glial cells. These results suggest that PES is expressed in the central nervous system at a low concentration under normal conditions, and that the neuronal cells possess an ability to express high levels of PES mRNA and protein.

Ontogeny of cytosolic phospholipase A2 activity in rat brain

We investigated developmental changes in the activity of cytosolic phospholipase A2 (cPLA2) in the rat brain. When the cytosolic fractions from rat brain of various ages were examined by gel filtration chromatography, cPLA2 activity was detected at about 100 kDa in all developmental stages. However, the magnitude of cPLA2 activity differed significantly. The cPLA2 activity was highest in the brain of day-12 embryo, gradually decreased toward birth, and retained a constant level into adulthood. This result suggests that cPLA2 plays an important role in the early development of the nervous system.

Vitronectin binds telencephalin and regulates dendritic spine morphogenesis

tic vesicle marker, in the axon terminal of olfactory sensory neurons (OSNs) during the period of synaptogenesis. In contrast, overexpression of PTP in OSNs rescued the phenotype of PTP morphants. Moreover, expression of dominant-negative form of PTP (PTP C1556S) in OSNs also increased VAMP2-EGFP punctate area and punctum number in the axon terminals. Electron microscopic analyses of transgenic zebrafish stably carrying OSN specific promoter-driven PTP C1556S revealed the little effect on the ultrastructure of OSN-mitral cell synapses. On the other hand, we observed a significant increase in the density of OSN-mitral cell synapses. These results suggest that PTP regulates synapse number during development of olfactory systems.

Dendritic filopodia formation is mediated by the interaction between telencephalin and ERM proteins

Repetitive activation of parallel fibers, biforked axons of granule cells (GCs), at short intervals elicits a well-known short-term presynaptic plasticity, a paired-pulse facilitation of the amplitude of excitatory postsynaptic currents (EPSCs) recorded from Purkinje cells. GCs innervate not only onto Purkinje cells but also to GABAergic interneurons. We here report that repetitive activation of GC axons at short intervals (30–100 ms) caused a novel presynaptic plasticity, a paired-pulse facilitation of the decay-time constant (PPFdecay) of the EPSCs recorded from the interneurons. The PPFdecay was significantly suppressed by the low-affinity competitive antagonist of AMPA receptors, but unchanged if postsynaptic AMPA receptors were prevented from the saturation by pharmacological reduction of the glutamate contents in the presynaptic vesicles. The result suggests that the PPFdecay is due to increase in the sequential mobilization of the multiple vesicles upon repetitive activation of the GC axon terminal.

Visualization of functional neural pathways using WGA transgene-2: Olfactory pathway

Recently, there has been a great progress in the understanding of the olfactory system by employing electrophisiological and molecular biological techniques. In particular, the findings of two basic principles in projection patterns of primary olfactory axons (glomerular convergence and zone-to-zone projection) and molecular receptive ranges of the olfactory bulb neurons have shed light on molecular mechanisms of the information processing in the primary olfactory system. However, the functional and molecular analyses of the central connections from the olfactory bulb to the olfactory cortex remain to elucidated. In this study, we applied our WGA transgene technology to the olfactory system. WGA mRNA and protein were robustly expressed in the olfactory sensory neurons under the control of the OMP (olfactory marker protein) promoter elements in transgenic mice. WGA protein was anterogradely transported from the olfactory epithelium to the olfactory bulb, and trans-synaptically transffered in glomeruli to the dendrites of mitral/tufted cells (the second-order neurons). Furthermore, WGA protein was detected in target regions of mitral/tufted cells such as anterior olfactory nucleus, the olfactory tubercle,and the piriform cortex. Thus, this technique for visualization of specific multi-synaptic neural pathways will provide extremely valuable tool for the studies of formation, refinement, maintenance, and remodelling of neural networks in the brain.

1013 Primary structure of OCAM, a novel cell adhesion molecule of the immunoglobulin superfamily

Takashi Kitsukawa’ , Takeshi Yagi*, Makoto Sanbo’, Hajime Fujisawa3 Neuropilin is a type I membrane protein which is expressed on the cell surface of elongating axons of particular types of neurons in Xenopus tadpoles, and in chick and mouse embyros, and has been suggested to be involved in axon fasciculation and guidance. To assess the role of neuropilin in the development of nervous system in vivo directly, we disrupted neuropilin gene in mice by homologous recombination. In the neuropilin-deficient mouse embryos, patterned pathways and projections of either the cranial and spinal nerve fibers which normally express neuropilin are impaired. In the homozygous mutant embryos the trigeminal nerves were overshot, and the spinal nerves were defasciculated and ectopically sprouted. These results emphasize the central role of neuropilin in precise guidance of peripheral nerve fibers.