Maturation of Complex Synaptic Connections of Layer 5 Cortical Axons in the Posterior Thalamic Nucleus Requires SNAP25.

Synapses are able to form in the absence of neuronal activity, but how is their subsequent maturation affected in the absence of regulated vesicular release? We explored this question using 3D electron microscopy and immunoelectron microscopy analyses in the large, complex synapses formed between cortical sensory efferent axons and dendrites in the posterior thalamic nucleus. Using a Synaptosome-associated protein 25 conditional knockout (Snap25 cKO), we found that during the first 2 postnatal weeks the axonal boutons emerge and increase in the size similar to the control animals. However, by P18, when an adult-like architecture should normally be established, axons were significantly smaller with 3D reconstructions, showing that each Snap25 cKO bouton only forms a single synapse with the connecting dendritic shaft. No excrescences from the dendrites were formed, and none of the normally large glomerular axon endings were seen. These results show that activity mediated through regulated vesicular release from the presynaptic terminal is not necessary for the formation of synapses, but it is required for the maturation of the specialized synaptic structures between layer 5 corticothalamic projections in the posterior thalamic nucleus.

Lysophosphatidic acid in medicinal herbs enhances prostaglandin E2 and protects against indomethacin-induced gastric cell damage in vivo and in vitro.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that induces diverse biological responses. Recently, we found that LPA ameliorates NSAIDs-induced gastric ulcer in mice. Here, we quantified LPA in 21 medicinal herbs used for treatment of gastrointestinal (GI) disorders. We found that half of them contained LPA at relatively high levels (40-240 µg/g) compared to soybean seed powder (4.6 µg/g), which we previously identified as an LPA-rich food. The LPA in peony (Paeonia lactiflora) root powder is highly concentrated in the lipid fraction that ameliorates indomethacin-induced gastric ulcer in mice. Synthetic 18:1 LPA, peony root LPA and peony root lipid enhanced prostaglandin E2 production in a gastric cancer cell line, MKN74 cells that express LPA2 abundantly. These materials also prevented indomethacin-induced cell death and stimulated the proliferation of MKN74 cells. We found that LPA was present in stomach fluids at 2.4 µM, which is an effective LPA concentration for inducing a cellular response in vitro. These results indicated that LPA is one of the active components of medicinal herbs for the treatment of GI disorder and that orally administered LPA-rich herbs may augment the protective actions of endogenous LPA on gastric mucosa.

Subsecond Regulation of Synaptically Released Dopamine by COMT in the Olfactory Bulb.

UNLABELLED: The efficacy of neurotransmission depends on multiple factors, including presynaptic vesicular release of transmitter, postsynaptic receptor populations and clearance/inactivation of the transmitter. In the olfactory bulb (OB), short axon cells (SACs) form an interglomerular circuit that uses GABA and dopamine (DA) as cotransmitters. Selective optical activation of SACs causes GABA and DA co-release, resulting in a fast, postsynaptic GABA inhibitory response and a slower G-protein-coupled DA rebound excitation. In most systems, vesicular release of DA is cleared by the dopamine transporter (DAT). However, in the OB, high levels of specific DA metabolites suggest that enzymatic catalysis by catechol-O-methyl-transferase (COMT) predominates over DAT re-uptake. To assess this possibility we measured the amount of the DA breakdown enzyme, COMT, present in the OB. Compared with the striatum, the brain structure richest in DA terminals, the OB contains 50% more COMT per unit of tissue. Furthermore, the OB has dramatically less DAT compared with striatum, supporting the idea that COMT enzymatic breakdown, rather than DAT recycling, is the predominant mechanism for DA clearance. To functionally assess COMT inactivation of vesicular release of DA we used fast-scan cyclic voltammetry and pharmacological blockade of COMT. In mice expressing ChR2 in tyrosine hydroxylase-containing neurons, optical activation of SACs evoked robust DA release in the glomerular layer. The COMT inhibitor, tolcapone, increased the DA signal ∼2-fold, whereas the DAT inhibitor GBR12909 had no effect. Together, these data indicate that the OB preferentially employs COMT enzymatic inactivation of vesicular release of DA. SIGNIFICANCE STATEMENT: In the olfactory bulb (OB), odors are encoded by glomerular activation patterns. Dopaminergic short axon neurons (SACs) form an extensive network of lateral connections that mediate cross talk among glomeruli, releasing GABA and DA onto sensory nerve terminals and postsynaptic neurons. DA neurons are ∼10-fold more numerous in OB than in ventral tegmental areas that innervate the striatum. We show that OB has abundant expression of the DA catalytic enzyme catechol-O-methyl-transferase (COMT), but negligible expression of the dopamine transporter. Using optogenetics and fast-scan cyclic voltammetry, we show that inhibition of COMT increases DA signals ∼2-fold. Thus, in contrast to the striatum, which has the brain's highest proportion of DAergic synapses, the DA catalytic pathway involving COMT predominates over re-uptake in OB.

Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice.

Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes.

Hepatitis C virus core protein suppresses mitophagy by interacting with parkin in the context of mitochondrial depolarization.

Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). We investigated whether HCV affects mitophagy in terms of mitochondrial quality control. The effect of HCV on mitophagy was examined using HCV-Japanese fulminant hepatitis-1-infected cells and the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone as a mitophagy inducer. In addition, liver cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice were examined for mitophagy. Translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without a reduction of pentaerythritol tetranitrate-induced kinase 1 activity in the presence of HCV infection both in vitro and in vivo. Coimmunoprecipitation assays revealed that Parkin associated with the HCV core protein. Furthermore, a Yeast Two-Hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment. Silencing Parkin suppressed HCV core protein expression, suggesting a functional role for the interaction between the HCV core protein and Parkin in HCV propagation. The suppressed Parkin translocation to the mitochondria inhibited mitochondrial ubiquitination, decreased the number of mitochondria sequestered in isolation membranes, and reduced autophagic degradation activity. Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting Parkin translocation to the mitochondria. This inhibition may amplify and sustain HCV-induced mitochondrial injury.

Effects of estradiol on dopaminergic synapse formation in the mouse olfactory bulb.

Olfactory glomeruli are the sites of initial synaptic integration in olfactory information processing. They are surrounded by juxtaglomerular (JG) cells, which include periglomerular, superficial short axon, and external tufted cells. A subpopulation of JG cells expresses the dopamine synthetic enzymes, tyrosine hydroxylase (TH), and aromatic l-amino acid decarboxylase (AADC). TH cells corelease γ-aminobutyric acid (GABA) and their processes extend to multiple glomeruli forming intra- and interglomerular circuits. It is well established that 17ß-estradiol (E2) exerts wide ranging effects in the central nervous system. However, participation of E2 in the modulation of neurotransmission and synaptic plasticity of TH cells in olfactory glomeruli is unknown. To address this, we subcutaneously implanted a 60-day release pellet of E2 or placebo into intact male mice and compared glomerular TH, AADC, and vesicular γ-aminobutyric acid transporter (VGAT) immunoreactivity between them. High-voltage electron microscopy (HVEM) and ultra-HVEM using immunogold revealed significantly increased immunoreactive intensity at the cellular level for TH and AADC after E2 treatment and for VGAT in TH cells. These results indicate that E2 may affect the interplay between dopaminergic and GABAergic systems. Moreover, random-section electron microscopy analysis showed a significant increase in the number of symmetrical synapses from TH cell to mitral/tufted cell dendrites after E2 treatment. This result was supported by quantitative immunofluorescence staining with synapse markers. Together, these data indicate that E2 may regulate inhibition between TH cells and olfactory bulb neurons within the glomerulus via interaction between dopaminergic and GABAergic systems, thereby contributing to neuromodulation of odor information processing.

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 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.

Dopamine D1 receptor signaling system regulates ryanodine receptor expression after intermittent exposure to methamphetamine in primary cultures of midbrain and cerebral cortical neurons.

Regulatory mechanisms of ryanodine receptor (RyR) expression are not well known, although methamphetamine (METH) has been reported to up-regulate RyRs in mouse brain. This study investigate regulatory mechanisms of RyR expression by dopaminergic system using the midbrain and cerebral cortical neurons in primary culture intermittently exposed to METH and dopamine receptor (DR) agonists (1 h/day, for 3 days). Intermittent METH (10 µM) exposure enhanced RyR-1 and -2 proteins and their mRNA, but not RyR-3 expression in the both types of the neurons. These METH-induced increases of RyR proteins and their mRNA were dose-dependently blocked by SCH23390 (a selective D(1) DR antagonist), but not a D(2)DR antagonist sulpiride, suggesting a regulatory role of D(1)DRs in RyR expression by METH in these neurons. In cerebral cortical neurons, intermittent SKF82958 (a selective D(1)DR agonist) exposure increased RyR-1 and -2 proteins and their mRNA, whereas quinpirole (a selective D(2)DR agonist) showed no effects. KT5720, a protein kinase A inhibitor, dose-dependently attenuated the METH-stimulated RyR-1 and -2 expressions in cerebral cortical neurons. METH significantly increased phosphorylation of cAMP-response element-binding protein, which was completely suppressed by SCH23390. These results indicate that RyR-1 and -2 expressions are regulated by D(1)DRs via the signal transduction linked to D(1)DRs.

Cocaine increases ryanodine receptors via dopamine D1 receptors.

For review there are little available data on regulatory mechanisms of ryanodine receptor (RyR) expression with cocaine treatment, though methamphetamine was reported to up-regulate RyRs in mouse brain. This study attempted to investigate regulatory mechanisms of RyR expression using the cerebral cortical neurons in primary culture intermittently exposed to a psychostimulant, cocaine. Intermittent exposure to cocaine (10 µM) significantly enhanced RyR 1 and 2 proteins and their mRNA, but not RyR 3 expression in the neurons. These cocaine-induced increases of RyR proteins and their mRNA were dose-dependently blocked by a dopamine D1 receptor antagonist (SCH23390), but not by a dopamine D2 receptor antagonist (sulpiride). These results indicate a regulatory role of dopamine D1 receptors in RyR expression bycocaine.

Involvement of NMDA receptors in ryanodine receptor expression in dopaminergic neurons in the ventral tegmental area of mice with intermittent methamphetamine treatment.

Excitatory synapses on dopaminergic neurons of the ventral tegmental area (VTA) represent an important role in psychostimulant-induced rewarding effect. This study investigated the regulation of ryanodine receptor (RyR) and N-methyl-D-aspartate (NMDA) receptor expression in mice under intermittent methamphetamine (METH) treatment using a place preference procedure. RyR-1 and -2 significantly increased in the VTA of mice with METH-induced place preference, whereas RyR-3 showed no changes. In addition, the levels of NR1, NR2A, and NR2B subunits were increased in the VTA. The METH-induced place preference was inhibited by intracerebroventricular pretreatment with MK-801, a noncompetitive NMDA receptor antagonist, and ifenprodil, a selective NR2B subunit-containing NMDA receptor antagonist, in a dose-dependent manner. Under these conditions, the increase of RyR-1 and -2 in the VTA was significantly blocked by ifenprodil. The immunohistochemical analysis revealed the colocalization of RyR-1 and -2 with NR2B subunits in dopaminergic neurons in the mouse VTA. These findings suggest that RyRs could be involved in the development of METH-induced place preference and that NR2B subunit-containing NMDA receptors in mice showing METH-induced place preference play an important role in expression of RyRs.

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

Olfactory input is processed in the glomerulus of the main olfactory bulb (OB) and relayed to higher centers in the brain by projection neurons. Conversely, centrifugal inputs from other brain regions project to the OB. We have previously analyzed centrifugal inputs into the OB from several brain regions using single-neuron labeling. In this study, we analyzed the centrifugal noradrenergic (NA) fibers derived from the locus coeruleus (LC), because their projection pathways and synaptic connections in the OB have not been clarified in detail. We analyzed the NA centrifugal projections by single-neuron labeling and immunoelectron microscopy. Individual NA neurons labeled by viral infection were three-dimensionally traced using Neurolucida software to visualize the projection pathway from the LC to the OB. Also, centrifugal NA fibers were visualized using an antibody for noradrenaline transporter (NET). NET immunoreactive (-ir) fibers contained many varicosities and synaptic vesicles. Furthermore, electron tomography demonstrated that NET-ir fibers formed asymmetrical synapses of varied morphology. Although these synapses were present at varicosities, the density of synapses was relatively low throughout the OB. The maximal density of synapses was found in the external plexiform layer; about 17% of all observed varicosities contained synapses. These results strongly suggest that NA-containing fibers in the OB release NA from both varicosities and synapses to influence the activities of OB neurons. The present study provides a morphological basis for olfactory modulation by centrifugal NA fibers derived from the LC.

Variations in GABA immunoreactivity among granule cells of the mouse olfactory bulb, as revealed by high-voltage electron microscopy.

Odor information is processed in the olfactory bulb (OB), which is organized into olfactory inputs, interneurons, projection neurons, and centrifugal inputs, and these various structures regulate olfactory information processing. Similar to other brain regions, the OB structures include many types of interneurons, including γ-aminobutyric acid (GABA)ergic interneurons. Many interneurons are granule cells that are found in the granule cell layer (GCL), which is a deep layer of the OB. Interestingly, these interneurons exhibit variations in GABA immunoreactivity, and previous studies have observed differing intensities among morphologically and chemically similar neuronal populations. However, the numbers and distribution patterns of cells that show variations in GABA immunoreactivity are unknown. Therefore, we observed and quantitatively analyzed this diversity in the GCL of the mouse OB using immunogold, high-voltage electron microscopy, combined with light microscopy. Consequently, our results clearly show variations in the GABA immunoreactivity among GCL interneurons, which suggested heterogeneity in the amount of GABA present in each interneuron and reflected the possibility that different amounts of neuroactive substances may be associated with different functions for the various GABAergic interneuron groups. Variations in GABA immunoreactivity could be a novel criterion for classifying interneuron subpopulations.

Synaptic distribution of individually labeled mitral cells in the external plexiform layer of the mouse olfactory bulb.

Mitral cells are the major projection neurons of the olfactory bulb. They receive olfactory inputs, regulate information, and project their axons to the olfactory cortex. To understand output regulation of mitral cells better, we established a method to visualize individual projection neurons and quantitatively examined their synaptic distribution. Individual mitral cells were labeled by viral injection, reconstructed three dimensionally with light microscopy, and serial sectioned for electron microscopy. Synaptic distributions were analyzed in electron microscopically reconstructed cell bodies, two regions of secondary dendrites (near the somata and ∼200 µm from the somata), and primary dendrites. The ratio of presynaptic sites (60%) and reciprocal synapses (60% presynaptic and 80% postsynaptic sites) were similar in each region. Characteristically, primary dendrite synapses were distributed mainly within the inner half of the external plexiform layer (EPL). For comparison, tufted cells were also examined, and the synaptic distribution in two secondary dendrite regions, which corresponded with mitral cells, was analyzed. The results showed that the ratio of reciprocal synapses (80% presynaptic and 90% postsynaptic sites) was greater than in mitral cells. The distribution of symmetrical synapses was also analyzed with synaptic and neuronal markers, such as parvalbumin, vesicular gamma-aminobutyric acid transporter, and gephyrin. Parvalbumin-expressing neurons tended to form synapses on secondary dendrites near the somata and were more uniformly distributed on primary dendrites of mitral cells. These results indicate that local mitral cell synaptic circuits are formed in accordance with their functional roles and restricted to the inner half of the EPL. J. Comp. Neurol. 525:1633-1648, 2017. © 2016 Wiley Periodicals, Inc.

Spatial distribution of synapses on tyrosine hydroxylase-expressing juxtaglomerular cells in the mouse olfactory glomerulus.

Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial-short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)-immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ-aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH-expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non-ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH-expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non-ON profiles. Thus, fewer proximal non-ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic-GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059-1074, 2017. © 2017 Wiley Periodicals, Inc.

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

Odor information is regulated by olfactory inputs, bulbar interneurons, and centrifugal inputs in the olfactory bulb (OB). Cholinergic neurons projecting from the nucleus of the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus are one of the primary centrifugal inputs to the OB. In this study, we focused on cholinergic regulation of the OB and analyzed neural morphology with a particular emphasis on the projection pathways of cholinergic neurons. Single-cell imaging of a specific neuron within dense fibers is critical to evaluate the structure and function of the neural circuits. We labeled cholinergic neurons by infection with virus vector and then reconstructed them three-dimensionally. We also examined the ultramicrostructure of synapses by electron microscopy tomography. To further clarify the function of cholinergic neurons, we performed confocal laser scanning microscopy to investigate whether other neurotransmitters are present within cholinergic axons in the OB. Our results showed the first visualization of complete cholinergic neurons, including axons projecting to the OB, and also revealed frequent axonal branching within the OB where it innervated multiple glomeruli in different areas. Furthermore, electron tomography demonstrated that cholinergic axons formed asymmetrical synapses with a morphological variety of thicknesses of the postsynaptic density. Although we have not yet detected the presence of other neurotransmitters, the range of synaptic morphology suggests multiple modes of transmission. The present study elucidates the ways that cholinergic neurons could contribute to the elaborate mechanisms involved in olfactory processing in the OB. J. Comp. Neurol. 525:574-591, 2017. © 2016 Wiley Periodicals, Inc.

Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome.

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence [mHSFYL (Riken cDNA 4933413G11Rik)], which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL in COS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis.

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix.

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-beta (TGFbeta)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFbeta-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFbeta-related factors' biological activity.

Localization of 5alpha-reductase in the rat main olfactory bulb.

The enzyme steroid 5alpha-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 5alpha-reductase type 1 in the rat main olfactory bulb. The occurrence of 5alpha-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 5alpha-reductase immunoreactive cells of variable intensity were present in all layers of the olfactory bulb. Multiple immunolabeling revealed that 5alpha-reductase was mainly localized in glial cells, namely, in S-100beta- and glial fibrillary acidic protein-immunoreactive astrocytes, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-immunoreactive oligodendrocytes, and in S-100beta- and neuropeptide-Y-immunoreactive olfactory ensheathing cells, whereas the bulbar neurons exhibited little immunoreactivity. Quantitative analysis revealed that the number of 5alpha-reductase-immunoreactive cells was greatest in the olfactory nerve layer. The most intense 5alpha-reductase-immunoreactivity was found in the olfactory ensheathing cells, and next in the CNPase-immunoreactive cells. The 5alpha-reductase in the olfactory bulb was expressed constantly throughout different ages and sexes and in neutered and hypophysectomized rats. Thus, 5alpha-reductase may contribute via 5alpha-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.

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.