Kazunori Toida

Molecular Identity of Periglomerular and Short Axon Cells

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic–GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65+ GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic–GABAergic cells have extensive interglomerular projections. DAergic–GABAergic SA cells comprise two subgroups. One subpopulation contacts 5–12 glomeruli and is referred to as “oligoglomerular.” Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON→ET→SA circuit. The second population of DAergic–GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive “polyglomerular” network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

Regulation of interkinetic nuclear migration by cell cycle‐coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical–basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1‐phase and apically during G2‐phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle‐dependent linkage of cell‐autonomous and non‐autonomous mechanisms. During S to G2 progression, the microtubule‐associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2‐phase by altering microtubule organization. Thus, Tpx2 links cell‐cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S‐phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1‐phase nuclei depends on a displacement effect by G2‐phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.

Cytokinesis of neuroepithelial cells can divide their basal process before anaphase

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time‐lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal‐to‐apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F‐actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP–anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal‐to‐apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M‐phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.

Molecular Characterization of Heat Shock-Like Factor Encoded on the Human Y Chromosome, and Implications for Male Infertility

Abstract Azoospermia and oligospermia are major causes of male infertility. Some genes located on the Y chromosome are suggested as candidates. Recently, HSFY, which is similar to the HSF (heat shock transcription factor) family, has been mapped on the human Y chromosome as multicopies. However, newly available sequence data deposited at NCBI shows that only the HSFY gene located on Yq has a long open reading frame containing a HSF-type DNA-binding domain. HSFY is similar to LW-1 on the human X chromosome and a murine HSFY-like sequence (mHSFYL), 4933413G11Rik, on the mouse chromosome 1. LW-1 and mHSFYL have 53% and 70% homology to HSFY for amino acid sequences of their presumed DNA-binding domains, respectively. Comparison of the presumed DNA-binding domains unveiled that the three HSF-like factors, HSFY, LW-1, and mHSFYL, belong to a different class than conventional HSFs. When we screened for deletions on the Yq of males suffering from infertility, we found that HSFY was involved in interstitial deletions on the Y chromosomes for two azoospermic males who had DBY, USP9Y, and DAZ but did not have RBMY located on the AZFb. Expression analysis of HSFY, LW-1, and mHSFYL unveiled that they are expressed predominantly in testis. Furthermore, immunhistochemistry of HSFY in testis showed that its expression is restricted to both Sertoli cells and spermatogenic cells and that it exhibits a stage-dependent translocation from the cytoplasm to the nucleus in spermatogenetic cells during spermatogenesis. These results may suggest that deletion of HSFY is involved in azoospermia or oligospermia.

The expression of prostate stem cell antigen in human clear cell renal cell carcinoma: a quantitative reverse transcriptase‐polymerase chain reaction analysis

To analyse the gene expression level of prostate stem cell antigen (PSCA) in human clear cell renal cell carcinoma (CC‐RCC) and its relationship with conventional clinicopathological manifestations, to evaluate its prognostic value for patient outcome, and to determine the effect of PSCA on the progression of CC‐RCC.

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain

Accumulating evidence implicates the significance of the physical properties of the niche in influencing the behavior, growth and differentiation of stem cells. Among the physical properties, extracellular stiffness has been shown to have direct effects on fate determination in several cell types in vitro. However, little evidence exists concerning whether shifts in stiffness occur in vivo during tissue development. To address this question, we present a systematic strategy to evaluate the shift in stiffness in a developing tissue using the mouse embryonic cerebral cortex as an experimental model. We combined atomic force microscopy measurements of tissue and cellular stiffness with immunostaining of specific markers of neural differentiation to correlate the value of stiffness with the characteristic features of tissues and cells in the developing brain. We found that the stiffness of the ventricular and subventricular zones increases gradually during development. Furthermore, a peak in tissue stiffness appeared in the intermediate zone at E16.5. The stiffness of the cortical plate showed an initial increase but decreased at E18.5, although the cellular stiffness of neurons monotonically increased in association with the maturation of the microtubule cytoskeleton. These results indicate that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. Taken together, our method profiles the stiffness of living tissue and cells with defined characteristics and can therefore be utilized to further understand the role of stiffness as a physical factor that determines cell fate during the formation of the cerebral cortex and other tissues.

Prostate stem cell antigen predicts tumour recurrence in superficial transitional cell carcinoma of the urinary bladder

To evaluate the relationship between prostate stem cell antigen (PSCA) expression level in transitional cell carcinoma (TCC) of the urinary bladder and various clinicopathological features, including stage and grade; and to determine whether PSCA mRNA expression predicts disease recurrence in superficial (not muscle‐invasive) TCC of the bladder.

Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory processing is well known to be regulated by centrifugal afferents from other brain regions, such as noradrenergic, acetylcholinergic, and serotonergic neurons. Serotonergic neurons widely innervate and regulate the functions of various brain regions. In the present study, we focused on serotonergic regulation of the olfactory bulb (OB), one of the most structurally and functionally well‐defined brain regions. Visualization of a single neuron among abundant and dense fibers is essential to characterize and understand neuronal circuits. We accomplished this visualization by successfully labeling and reconstructing serotonin (5‐hydroxytryptamine: 5‐HT) neurons by infection with sindbis and adeno‐associated virus into dorsal raphe nuclei (DRN) of mice. 5‐HT synapses were analyzed by correlative confocal laser microscopy and serial‐electron microscopy (EM) study. To further characterize 5‐HT neuronal and network function, we analyzed whether glutamate was released from 5‐HT synaptic terminals using immuno‐EM. Our results are the first visualizations of complete 5‐HT neurons and fibers projecting from DRN to the OB with bifurcations. We found that a single 5‐HT axon can form synaptic contacts to both type 1 and 2 periglomerular cells within a single glomerulus. Through immunolabeling, we also identified vesicular glutamate transporter 3 in 5‐HT neurons terminals, indicating possible glutamatergic transmission. Our present study strongly implicates the involvement of brain regions such as the DRN in regulation of the elaborate mechanisms of olfactory processing. We further provide a structure basis of the network for coordinating or linking olfactory encoding with other neural systems, with special attention to serotonergic regulation. J. Comp. Neurol. 523:262–280, 2015.

Localization of 5α‐reductase in the rat main olfactory bulb

The enzyme steroid 5α‐reductase catalyzes the production of dihydroprogesterone and dihydrotestosterone, which were recently recognized as neurosteroids in the brain with variably potential neuroactivity. The present study reports for the first time detailed localization of 5α‐reductase type 1 in the rat main olfactory bulb. The occurrence of 5α‐reductase in the olfactory bulb was detected by reverse transcription‐polymerase chain reaction and Western blotting analyses. In addition, the enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed that 5α‐reductase immunoreactive cells of variable intensity were present in all layers of the olfactory bulb. Multiple immunolabeling revealed that 5α‐reductase was mainly localized in glial cells, namely, in S‐100β‐ and glial fibrillary acidic protein‐immunoreactive astrocytes, 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase)‐immunoreactive oligodendrocytes, and in S‐100β‐ and neuropeptide‐Y‐immunoreactive olfactory ensheathing cells, whereas the bulbar neurons exhibited little immunoreactivity. Quantitative analysis revealed that the number of 5α‐reductase‐immunoreactive cells was greatest in the olfactory nerve layer. The most intense 5α‐reductase‐immunoreactivity was found in the olfactory ensheathing cells, and next in the CNPase‐immunoreactive cells. The 5α‐reductase in the olfactory bulb was expressed constantly throughout different ages and sexes and in neutered and hypophysectomized rats. Thus, 5α‐reductase may contribute via 5α‐reduced metabolites to the formation and maintenance of olfactory inputs and outputs, which were closely associated with the olfactory ensheathing cells and the oligodendrocytes, respectively. J. Comp. Neurol. 493:381–395, 2005.

Identification of viruses in patients with postviral olfactory dysfunction.

Objective: Causative viruses of postviral olfactory dysfunction (PVOD) have not yet been identified. The aim of this study was to investigate causative viruses in patients with PVOD.