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Biomedical subjects

Yoshihiro Yoshihara

Publications and source records attributed to Yoshihiro Yoshihara.

At least 19 recordsLinked to original sources
PubMed2006

Odorant receptor map in the mouse olfactory bulb: in vivo sensitivity and specificity of receptor-defined glomeruli.

Odorant identity is represented in the olfactory bulb (OB) by the glomerular activity pattern, which reflects a combination of activated odorant receptors (ORs) in the olfactory epithelium. To elucidate this neuronal circuit at the molecular level, we established a functional OR identification strategy based on glomerular activity by combining in vivo Ca(2+) imaging, retrograde dye labeling, and single-cell RT-PCR. Spatial and functional mapping of OR-defined glomeruli revealed that the glomerular positional relationship varied considerably between individual animals, resulting in different OR maps in the OB. Notably, OR-defined glomeruli exhibited different ligand spectra and far higher sensitivity compared to the in vitro pharmacological properties of corresponding ORs. Moreover, we found that the olfactory mucus was an important factor in the regulation of in vivo odorant responsiveness. Our results provide a methodology to examine in vivo glomerular responses at the receptor level and further help address the long-standing issues of olfactory sensitivity and specificity under physiological conditions.

Animals↗
PubMed2006

A transcriptional enhancer that directs telencephalon-specific transgene expression in mouse brain.

Telencephalin (TLCN, intercellular adhesion molecule-5 [ICAM-5]) is a cell adhesion molecule belonging to the immunoglobulin superfamily, which plays important roles in dendritic morphogenesis and synaptic plasticity. The expression of TLCN is restricted to neurons in the most rostral brain segment, telencephalon. Here, we examined a transcriptional regulatory mechanism underlying the telencephalon-specific expression of TLCN. TLCN gene is located in the ICAM gene cluster containing ICAM-1, ICAM-4, and TLCN (ICAM-5) within 30 kb on the mouse chromosome 9. The nucleotide sequence of the 5'-flanking region of mouse TLCN gene is highly homologous to that of human and dog orthologs, suggesting the presence of important regulatory elements for its transcription. To determine the telencephalon-specific enhancer region, we generated several lines of transgenic mice that harbor transgenes consisting of different length of the 5'-flanking region of mouse TLCN gene (3.9, 1.5, 1.1, and 0.2 kb) fused to humanized renilla green fluorescent protein cDNA as a fluorescent reporter. Consequently, we identified a crucial region between 1.1 and 0.2 kb upstream of the transcription start site that directs the telencephalon-specific expression. This enhancer was applied to the Cre/loxP-mediated conditional expression system to generate several transgenic lines with different patterns of recombination in the telencephalon and will be further used as a powerful tool for genetic manipulation in the telencephalic neurons.

5' Flanking Region↗
PubMed2006

Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously.

Fez is a zinc-finger gene encoding a transcriptional repressor that is expressed in the olfactory epithelium, hypothalamus, ventrolateral pallium and prethalamus at mid-gestation. To reveal its function, we generated Fez-deficient mice. The Fez-deficient mice showed several abnormalities in the olfactory system: (1) impaired axonal projection of the olfactory sensory neurons; (2) reduced size of the olfactory bulb; (3) abnormal layer formation in the olfactory bulb; and (4) aberrant rostral migration of the interneuron progenitors. Fez was not expressed in the projection neurons, interneurons or interneuron progenitors. Transgene-mediated expression of Fez in olfactory sensory neurons significantly rescued the abnormalities in olfactory axon projection and in the morphogenesis of the olfactory bulb in Fez-knockout mice. Thus, Fez is cell-autonomously required for the axon termination of olfactory sensory neurons, and Fez non-cell-autonomously controls layer formation and interneuron development in the olfactory bulb. These findings suggest that signals from olfactory sensory neurons contribute to the proper formation of the olfactory bulb.

Animals↗
PubMed2006

Telencephalin slows spine maturation.

Dendritic filopodia are highly dynamic structures, and morphological maturation from dendritic filopodia to spines is intimately associated with the stabilization and strengthening of synapses during development. Here, we report that telencephalin (TLCN), a cell adhesion molecule belonging to the Ig superfamily, is a negative regulator of spine maturation. Using cultured hippocampal neurons, we examined detailed localization and functions of TLCN in spine development and synaptogenesis. At early stages of synaptogenesis, TLCN immunoreactivity gradually increased and was present in dendritic shafts and filopodia. At later stages, TLCN tended to be excluded from mature spine synapses in which PSD-95 (postsynaptic density-95) clusters were apposed to presynaptic synaptophysin clusters. To elucidate the function of TLCN in spine maturation, we analyzed the dendrite morphology of TLCN-overexpressing and TLCN-deficient neurons. Overexpression of TLCN caused a dramatic increase in the density of dendritic filopodia and a concomitant decrease in the density of spines. Conversely, TLCN-deficient mice showed a decreased density of filopodia and an acceleration of spine maturation in vitro as well as in vivo. These results demonstrate that TLCN normally slows spine maturation by promoting the filopodia formation and negatively regulating the filopodia-to-spine transition. In addition, we found that spine heads of mature neurons were wider in TLCN-deficient mice compared with wild-type mice. Thus, the preservation of immature synapses by TLCN may be an essential step for refinement of functional neural circuits in the telencephalon, that take charge of higher brain functions such as learning, memory, and emotion.

Animals↗
PubMed2005

Mutually exclusive glomerular innervation by two distinct types of olfactory sensory neurons revealed in transgenic zebrafish.

The olfactory epithelium of fish contains two major types of olfactory sensory neurons (OSNs) that are distinct morphologically (ciliated vs microvillous) and possibly functionally. Here, we found that these OSNs express different sets of signal transduction machineries: the ciliated OSNs express OR-type odorant receptors, cyclic nucleotide-gated channel A2 subunit, and olfactory marker protein (OMP), whereas the microvillous OSNs express V2R-type receptors and transient receptor potential channel C2 (TRPC2). To visualize patterns of axonal projection from the two types of OSNs to the olfactory bulb (OB), we generated transgenic zebrafish in which spectrally distinct fluorescent proteins are expressed in the ciliated and microvillous OSNs under the control of OMP and TRPC2 gene promoters, respectively. An observation of whole-mount OB in adult double-transgenic zebrafish revealed that the ciliated OSNs project axons mostly to the dorsal and medial regions of the OB, whereas the microvillous OSNs project axons to the lateral region of the OB. A careful histological examination of OB sections clarified that the axons from the two distinct types of OSNs target different glomeruli in a mutually exclusive manner. This segregation is already established at very early developmental stages in zebrafish embryos. These findings clearly demonstrate the relationships among cell morphology, molecular signatures, and axonal terminations of the two distinct types of OSNs and suggest that the two segregated neural pathways are responsible for coding and processing of different types of odor information in the zebrafish olfactory system.

Animals↗
PubMed2005

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system.

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory, consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the ast phenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.

Animals↗
PubMed2005

A novel phenylalanine-based targeting signal directs telencephalin to neuronal dendrites.

Neurons sort out a variety of functional molecules to appropriate subcellular destinations. Telencephalin (TLCN; intercellular adhesion molecule-5) is a cell adhesion molecule specifically localized to somatodendritic membranes in the telencephalic neurons. Here, we established a new in vivo strategy to analyze neuronal sorting mechanisms by ectopic expression of molecules of interest in the cerebellar Purkinje cells of transgenic mice. By using this system, we identified a novel dendritic targeting determinant in the cytoplasmic tail region of TLCN. A full-length TLCN ectopically expressed in the Purkinje cells was localized exclusively to dendrites but not to axons. In contrast, a deletion of cytoplasmic C-terminal 12 amino acids (residues 901-912) or a point mutation of Phe905 to Ala abrogated the dendrite-specific targeting with appearance of the truncated and point-mutated TLCN in both axons and dendrites. Furthermore, an addition of the C-terminal 17 amino acids (residues 896-912) of TLCN to an unrelated molecule (CD8) was sufficient for its specific targeting to dendrites in several types of neurons. Because the C-terminal region of TLCN does not contain any canonical dendritic targeting sequences such as the tyrosine-based motif or the dileucine motif, this study suggests a novel mechanism of protein trafficking to the dendritic compartment of neurons.

Amino Acid Motifs↗
PubMed2005

Arx homeobox gene is essential for development of mouse olfactory system.

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. We report here crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein was expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice showed severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb, (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors, (3) loss of tyrosine hydroxylase-positive periglomerular cells, (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons.

Animals↗
PubMed2004

Laminar organization of the developing lateral olfactory tract revealed by differential expression of cell recognition molecules.

The projection neurons in the olfactory bulb (mitral and tufted cells) send axons through the lateral olfactory tract (LOT) onto several structures of the olfactory cortex. However, little is known of the molecular and cellular mechanisms underlying establishment of functional connectivity from the bulb to the cortex. Here, we investigated the developmental process of LOT formation by observing expression patterns of cell recognition molecules in embryonic mice. We immunohistochemically identified a dozen molecules expressed in the developing LOT and some of them were localized to subsets of mitral cell axons. Combinatorial immunostaining for these molecules revealed that the developing LOT consists of three laminas: superficial, middle, and deep. Detailed immunohistochemical, in situ hybridization, and 5-bromodeoxyuridine labeling analyses suggested that the laminar organization reflects: 1) the segregated pathways from the accessory and main olfactory bulbs, and 2) the different maturity of mitral cell axons. Mitral cell axons of the accessory olfactory bulb were localized to the deep lamina, segregated from those of the main olfactory bulb. In the main olfactory pathway, axons of mature mitral cells, whose somata is located in the apical sublayer of the mitral cell layer, were localized to the middle lamina within LOT, while those of immature mitral cells that located in the basal sublayer were complementarily localized to the superficial lamina. These results suggest that newly generated immature axons are added to the most superficial lamina of LOT successively, leading to the formation of piled laminas with different maturational stages of the mitral cell axons.

Animals↗
PubMed2004

Mitral and tufted cells differ in the decoding manner of odor maps in the rat olfactory bulb.

Mitral and tufted cells in the mammalian olfactory bulb are principal neurons, each type having distinct projection pattern of their dendrites and axons. The morphological difference suggests that mitral and tufted cells are functionally distinct and may process different aspects of olfactory information. To examine this possibility, we recorded odorant-evoked spike responses from mitral and middle tufted cells in the aliphatic acid- and aldehyde-responsive cluster at the dorsomedial part of the rat olfactory bulb. Homologous series of aliphatic acids and aldehydes were used for odorant stimulation. In response to adequate odorants, mitral cells showed spike responses with relatively low firing rates, whereas middle tufted cells responded with higher firing rates. Examination of the molecular receptive range (MRR) indicated that most mitral cells exhibited a robust inhibitory MRR, whereas a majority of middle tufted cells showed no or only a weak inhibitory MRR. In addition, structurally different odorants that activated neighboring clusters inhibited the spike activity of mitral cells, whereas they caused no or only a weak inhibition in the middle tufted cells. Furthermore, responses of mitral cells to an adequate excitatory odorant were greatly inhibited by mixing the odorant with other odorants that activated neighboring glomeruli. In contrast, odorants that activated neighboring glomeruli did not significantly inhibit the responses of middle tufted cells to the adequate excitatory odorant. These results indicate a clear difference between mitral and middle tufted cells in the manner of decoding the glomerular odor maps.

Action Potentials↗
PubMed2004

Pax6 and engrailed 1 regulate two distinct aspects of renshaw cell development.

Many of the interneuron cell types present in the adult spinal cord contribute to the circuits that control locomotion and posture. Little is known, however, about the embryonic origin of these cell types or the molecular mechanisms that control their differentiation. Here we provide evidence that V1 interneurons (INs), an embryonic class of interneurons that transiently express the En1 transcription factor, differentiate as local circuit inhibitory interneurons and form synapses with motor neurons. Furthermore, we show that a subset of V1 INs differentiates as Renshaw cells, the interneuronal cell type that mediates recurrent inhibition of motor neurons. We analyze the role that two V1 IN-related transcription factor genes play in Renshaw cell development. Pax6 (paired box gene 6) is necessary for an early step in Renshaw cell development, whereas Engrailed 1 (En1), which is genetically downstream of Pax6, regulates the formation of inhibitory synapses between Renshaw cells and motor neurons. Together, these results show that Pax6 and En1 have essential roles in establishing the recurrent inhibitory circuit between motor neurons and Renshaw cells.

Animals↗
PubMed2004

Aberrant transcription of unrearranged T-cell receptor beta gene in mouse brain.

The nervous system and the immune system share several functional molecules involved in various cell-cell interaction events. In this study, we used in situ hybridization to identify immune molecules that are expressed by a restricted population of neurons in the mouse brain and found that mRNA for the beta subunit of T-cell receptor (TCRbeta) was predominantly and strongly localized to neurons in deep layers of the cerebral neocortex and weakly expressed in the thalamus. Developmentally, TCRbeta mRNA expression started at embryonic day 15 in the thalamic nuclei and at postnatal day 1 in the cerebral neocortex. The level of TCRbeta mRNA in the neocortex subsequently increased until postnatal day 21, and it remained high in the adult. Detailed analysis revealed that only the Cbeta2 segment of TCRbeta, not the Cbeta1 or Vbeta segments, was expressed by the brain neurons. By the 5' rapid amplification of cDNA ends method, we determined a brain-specific transcription start site in the Jbeta2 region locus, not in the Vbeta region locus. Furthermore, we confirmed that the aberrant transcription around the Jbeta2 region took place only in neurons and lymphocytes in transgenic mice. These results demonstrate that the transcriptional machinery for unrearranged TCRbeta expression is shared by the nervous and immune systems and raise a possibility of gene rearrangement in neurons under certain circumstances.

Alternative Splicing↗
PubMed2004

Activated natural killer cells adhere to cultured hippocampal neurons and affect the dendritic morphology.

To examine the manner of interactions between immune cells and central nervous system (CNS) neurons, mouse hippocampal neurons were co-cultured with lymphokine (IL-2)-activated killer (LAK) cells. Immunocytochemical and time-lapse observations indicated that LAK cells migrated along neuronal processes and made adhesive contacts with them. In addition to the direct physical effects, LAK cells released glutamate, induced the formation of beads-like structure in the dendrites of about 14% of hippocampal neurons and caused the reduction of dendritic protrusions. These results suggest that infiltrating immune cells can form direct adhesive connections with CNS neurons and affect their dendritic morphology.

Animals↗
PubMed2004

Improved parameter estimation for variance-stabilizing transformation of gene-expression microarray data.

A gene-expression microarray datum is modeled as an exponential expression signal (log-normal distribution) and additive noise. Variance-stabilizing transformation based on this model is useful for improving the uniformity of variance, which is often assumed for conventional statistical analysis methods. However, the existing method of estimating transformation parameters may not be perfect because of poor management of outliers. By employing an information normalization technique, we have developed an improved parameter estimation method, which enables statistically more straightforward outlier exclusion and works well even in the case of small sample size. Validation of this method with experimental data has suggested that it is superior to the conventional method.

Algorithms↗
PubMed2003

Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse.

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner. Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development. Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster. Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed. We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor-one neuron rule.

Alleles↗
PubMed2003

Inverse expression of olfactory cell adhesion molecule in a subset of olfactory axons and a subset of mitral/tufted cells in the developing rat main olfactory bulb.

The projection of olfactory sensory neuron (OSN) axons from the olfactory epithelium (OE) to the olfactory bulb (OB) is highly organized but topographically complex. Evidence suggests that odorant receptor expression zones in the OE map to the OB about orthogonal axes. One candidate molecule for the formation of zone-specific targeting of OSN axon synapses onto the OB is the olfactory cell adhesion molecule (OCAM). OCAM(+) OSNs are restricted to three of the four zones in the OE and project their axons to the ventral OB where they form synapses with mitral/tufted (M/T) cells. To determine when this zonal connection is established, we have examined OCAM expression in rat olfactory system, during seminal periods of glomerular formation. OCAM(+) axons sort out in the ventral olfactory nerve layer of the OB before glomerular formation. Surprisingly, OCAM was also expressed transiently by subsets of M/T cell dendrites located in the dorsal OB. The expression of OCAM by OSN axons and M/T dendrites was asymmetrical; in the dorsal OB, OCAM(-) OSN axons synapsed on OCAM(+) M/T dendrites, whereas in the ventral OB, OCAM(+) OSN axons synapsed on OCAM(-) M/T dendrites. The restricted spatial map of OCAM(+) M/T cells appeared earlier in development than the zonal segregation of OCAM(+) OSN axons. Thus, OCAM on M/T cell dendrites may act in a spatiotemporal window to specify regions of the developing rat OB, thereby establishing a foundation for mapping of the OE zonal organization onto the OB.

Animals↗
PubMed2003

Developmental study of dendritic bundles in layer 1 of the rat granular retrosplenial cortex with special reference to a cell adhesion molecule, OCAM.

In the granular retrosplenial cortex (GRS) of adult rats, callosally projecting pyramidal neurons in layer 2 form dendritic bundles, 30-100 micro m wide, in layer 1. The distinctness of these bundles makes the GRS an attractive model system for investigating the developmental, microcircuitry, and basic organizational features related to dendritic modularity. In this report, we investigate the developmental time course of the dendritic bundles, visualized by immunohistochemistry for microtubule-associated protein 2 (MAP2) and glutamate receptor subunits 2/3 (GluR2/3). Bundles in layer 1 are apparent as early as postnatal day 5, first with GluR2/3 and then, from postnatal day 14, with MAP2. As a step toward understanding the mechanisms of dendritic aggregation, we further investigated the ontogeny of expression of the cell adhesion molecule OCAM. OCAM exhibits a patchy distribution in layer 1 from postnatal day 3 to adult, and the regions of weak OCAM immunoreactivity selectively correspond to the dendritic bundles (in both GluR2/3 and MAP2). The periodic geometry of OCAM-immunoreactive regions, the time course of their appearance and the distinct localization complementary to the bundles support the possibility that this molecule is one contributor to the establishment and maintenance of dendritic modules. The interdigitating relationship between regions of high OCAM immunoreactivity and the dendritic bundles in layer 1 suggests that OCAM may have a repellent influence on the formation of these bundles.

Aging↗