Formation of functional innexin hemichannels, as well as gap junctional channels, in an insect cell line, NIAs-AeAl-2, derived from Asian tiger mosquito Aedes albopictus (Diptera: Culicidae): A partial but significant contribution of innexin 2.

In vertebrates, gap junctions and hemichannels consisting of connexins are important cell surface structures for communication with neighboring cells and for the regulation of various cell functions. To date, various gap-junction-related proteins have been found, including innexins in invertebrates and pannexins in vertebrates. Significant contributions of gap junctions by innexins and (hemi-)channels by pannexins to numerous functions have been reported. Verification of the presence and functional significance of innexin hemichannels, however, remains a gap in our knowledge in innexin physiology. In this study, we revealed the localization of an innexin protein (innexin 2) on the cell surface in mosquito tissues and cultured cells. Furthermore, we demonstrated the presence of functional hemichannels, as well as gap junctions, in mosquito cells using dye transfer assays. The inward uptake of fluorescent dye was inhibited by anti-innexin 2 antibody. These results suggest that innexin hemichannels are formed to function in cultured mosquito cells, in at least a partially innexin 2-dependent manner. Although only a few studies on insect hemichannels have been published, innexin-based hemichannels, as well as innexin gap junctions, could also significantly contribute to insect intercellular signal transduction.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Putative Neural Network Within an Olfactory Sensory Unit for Nestmate and Non-nestmate Discrimination in the Japanese Carpenter Ant: The Ultra-structures and Mathematical Simulation.

Ants are known to use a colony-specific blend of cuticular hydrocarbons (CHCs) as a pheromone to discriminate between nestmates and non-nestmates and the CHCs were sensed in the basiconic type of antennal sensilla (S. basiconica). To investigate the functional design of this type of antennal sensilla, we observed the ultra-structures at 2D and 3D in the Japanese carpenter ant, Camponotus japonicus, using a serial block-face scanning electron microscope (SBF-SEM), and conventional and high-voltage transmission electron microscopes. Based on the serial images of 352 cross sections of SBF-SEM, we reconstructed a 3D model of the sensillum revealing that each S. basiconica houses > 100 unbranched dendritic processes, which extend from the same number of olfactory receptor neurons (ORNs). The dendritic processes had characteristic beaded-structures and formed a twisted bundle within the sensillum. At the "beads," the cell membranes of the processes were closely adjacent in the interdigitated profiles, suggesting functional interactions via gap junctions (GJs). Immunohistochemistry with anti-innexin (invertebrate GJ protein) antisera revealed positive labeling in the antennae of C. japonicus. Innexin 3, one of the five antennal innexin subtypes, was detected as a dotted signal within the S. basiconica as a sensory organ for nestmate recognition. These morphological results suggest that ORNs form an electrical network via GJs between dendritic processes. We were unable to functionally certify the electric connections in an olfactory sensory unit comprising such multiple ORNs; however, with the aid of simulation of a mathematical model, we examined the putative function of this novel chemosensory information network, which possibly contributes to the distinct discrimination of colony-specific blends of CHCs or other odor detection.

Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography.

Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.

Serotonergic transmission and gap junctional coupling in proventricular muscle cells in the American cockroach, Periplaneta americana.

The visceral muscle tissues of insects consist of striated muscle cells. The mechanisms responsible for delivering signals to the contractile muscles in the insect digestive tract remain unclear. We found that serotonergic nerves innervate the hemocoel surfaces of foregut and midgut muscles in the American cockroach. Electron microscopy of the neuromuscular junctions in the proventriculus (gizzard) revealed typical synaptic structures, the accumulation of large core/cored vesicles (neuropeptides) and small clear vesicle (neurotransmitter) at presynapses, and synaptic clefts. However, only a limited number of muscle cells, which were located in the outer part of the muscle layer, came into contact with synapses, which contained classical neurotransmitters, such as glutamate. A gap junction channel-permeable fluorescent dye, Lucifer yellow, was microinjected into single muscle cells, and it subsequently spread to several neighboring muscle cells. The dye movement occurred in the radial (hemocoel-lumen) direction rather than tangential directions. A gap junction blocker, octanol, reversibly inhibited the dye coupling. Messenger RNA for innexin 2, a gap junction-related protein, was detected in the proventriculus. These results suggest that motile signals in the insect digestive tract only reach the outermost part of the visceral muscles and are propagated to the inner muscle cells via gap junctions. Therefore, invertebrate gap junction-related proteins have potential as new targets for pest control.

Cold stimulation evokes exocytotic vesicle release from PC12 cells.

The mechanism of peripheral blood circulation failure by low temperatures, known as Raynaud's phenomenon, is not well understood at present. We examined the possibility that the efferent fibers of peripheral sympathetic neurons may release transmitters directly by cold sensation. PC12 cells, a culture model of sympathetic neurons, were placed at 25°C for 30 min, fixed with the tannic acid-glutaraldehyde-osmium method and observed by electron microscopy. The number of omega-shaped exocytotic vesicle figures increased significantly compared with that in the control (37°C), although it was lower than that of the positive control (high potassium stimulation at 25°C and 37°C). As it is known that PC12 cells and sympathetic neurons do not express the typical cold sensing channels (TRPA1 and TRPM8), our results suggest that the peripheral sympathetic neurons may have an unknown cold monitoring system which reacts to low temperatures, release transmitters by exocytosis and directly regulate local blood circulation.

Increase in neuropilin-1 on the surface of growth cones and putative raft domains in neuronal NG108-15 cells co-cultured with vascular smooth muscle SM-3 cells.

The mechanisms underlying autonomic innervation to its targets involve various chemical factors, but have not yet been elucidated in detail. We constructed a co-culture system of neuronal cells and vascular smooth muscle cells to investigate the mechanisms underlying innervation of the vasculature. A co-culture with the vascular smooth muscle cell line, SM-3 significantly promoted cell viability, neurite extension, and neuropilin-1 (Nrp-1) mRNA expression in the cholinergic neuronal cell line, NG108-15. Furthermore, immunocytochemistry with or without a detergent treatment revealed that a co-culture with SM-3 cells or culturing with the conditioned medium of SM-3 cells translocated Nrp-1 onto the cell surface of growth cones rather than varicosities of NG108-15 cells. Immunofluorescent microscopy combined with a cold detergent treatment or cholesterol depletion revealed that Nrp-1 accumulated in putative raft domains in the plasma membrane of NG108-15 cells co-cultured with SM-3 cells. The results of the present study suggest that some soluble factors from smooth muscle cells may affect the localization of Nrp-1 in cholinergic neuronal cells, which may, in turn, be involved in the autonomic innervation of blood vessels.

Establishment of benzodioxazine core structure for sarcodonin class of natural products by X-ray analysis.

Sarcodonin ε (2), isolated from Sarcodon scabrosus, was treated with trimethylsilyldiazomethane to yield a crystalline methylated derivative 6. The structure of 6 was determined by X-ray analysis, which confirmed the presence of an unprecedented N(1b)-OMe group, the configuration at N(1b) and the 1,3,4-substituted phenyl ring structure of 2. More importantly, the structures of 6 and 2 have answered the intriguing problem of solving the core structure of the sarcodonin class of natural products, establishing that sarcodonins have a benzodioxazine core structure, rather than the recently proposed benzodioxane aminal core structure.

Mechanism of the tissue-specific action of the selective androgen receptor modulator S-101479.

Selective androgen receptor modulators (SARMs) comprise a new class of molecules that induce anabolic effects with fewer side effects than those of other anabolic agents. We previously reported that the novel SARM S-101479 had a tissue-selective bone anabolic effect with diminished side effects in female animals. However, the mechanism of its tissue selectivity is not well known. In this report, we show that S-101479 increased alkaline phosphatase activity and androgen receptor (AR) transcriptional activity in osteoblastic cell lines in the same manner as the natural androgen ligand dihydrotestosterone (DHT); conversely, stimulation of AR dimerization was very low compared with that of DHT (34.4%). S-101479 increased bone mineral content in ovariectomized rats without promoting endometrial proliferation. Yeast two-hybrid interaction assays revealed that DHT promoted recruitment of numerous cofactors to AR such as TIF2, SRC1, ß-catenin, NCoA3, gelsolin and PROX1 in a dose-dependent manner. SARMs induced recruitment of fewer cofactors than DHT; in particular, S-101479 failed to induce recruitment of canonical p160 coactivators such as SRC1, TIF2 and notably NCoA3 but only stimulated binding of AR to gelsolin and PROX1. The results suggest that a full capability of the AR to dimerize and to effectively and unselectively recruit all canonical cofactors is not a prerequisite for transcriptional activity in osteoblastic cells and resulting anabolic effects in bone tissues. Instead, few relevant cofactors might be sufficient to promote AR activity in these tissues.

Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography.

Nerve growth factor (NGF) initiates the activation of TrkA tyrosine kinase receptors and numerous subsequent signaling cascades. However, the dynamics of the process including the translocation of TrkA is still unclear. In this study, the effect of NGF or membrane depolarization on the endocytic process and TrkA localization in the neuronal cell line PC12 was analyzed by live-cell imaging and immunoelectron tomography using an ultra-high voltage electron microscope (UHVEM). Both NGF re-stimulation and high potassium-induced depolarization enhanced the endocytic uptake of the fluorescent indicator into acidic organelles within varicosities as well as cell bodies. However, the transition of uptake differed completely. NGF also significantly increased the number of TrkA-containing varicosities. Immunoelectron tomography in whole-mounted cells showed that NGF induced the recruitment of TrkA to the surface membrane of neurite varicosities as well as the multivesicular bodies (MVBs) and lysosomal complexes inside the varicosities. Three-dimensional analysis revealed that invagination pits and intralumenal vesicles of MVBs contained TrkA immunoreactivity. In addition, TrkA immunoreactivity was scattered in the lysosomal matrices after NGF treatment. These results suggest that the neurite varicosities are intensely active in intracellular membrane trafficking, and play an important role in the degradation and accumulation of the NGF receptor, TrkA, after ligand stimulation.

The novel non-steroidal selective androgen receptor modulator S-101479 has additive effects with bisphosphonate, selective estrogen receptor modulator, and parathyroid hormone on the bones of osteoporotic female rats.

We have studied non-steroidal selective androgen receptor modulators (SARMs) to develop anti-osteoporosis drugs for males and females. Many SARMs have been studied for their anabolic effects on bone or muscle with reduced virilizing effects in male animals. However, the tissue selectivities of these agents in female animals have not been fully evaluated. We evaluated the novel SARM S-101479 from tetrahydroquinoline libraries in ovariectomized (OVX) rats. S-101479 preferentially bound to the androgen receptor with nanomolar affinity among nuclear receptors. It increased the bone mineral density (BMD) of femurs and diminished the effects on the uterus and clitoral gland in OVX rats. We then compared the effect of S-101479 on bone with those of commercial anti-osteoporosis drugs such as alendronate, raloxifene, and teriparatide. Furthermore, we evaluated the effects of combination treatments with these agents in OVX rats. After 16-week treatment, all agents significantly increased BMD, but the magnitude of bone mineral content (BMC) and/or bone size (projected bone area) were different. Alendronate, raloxifene, and teriparatide maintained BMC and bone size in this experimental dose. Only S-101479 increased BMC with bone size on single treatments. In combination treatment, S-101479 significantly increased BMC and bone size compared with single treatments of other agents. S-101479, like natural androgen, may have showed periosteal bone formation of the cortical area and indicated additive effects with commercial anti-osteoporosis drugs. These results indicate that S-101479 may be a useful anti-osteoporosis drug, particularly for patients with established severe osteoporosis.

p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions.

α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson's disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated in vitro. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.

Morphological effects of isoflavones (daidzein and genistein) on hypothalamic oxytocin neurons in the neonatal mouse brain slice cultures.

In adults, oxytocin (OXT) has various central functions including social behavior and reproduction. Many of these functions are steroid dependent and are also influenced by naturally occurring phytoestrogens, isoflavones (IFs). The aim of this study was, therefore, to clarify the effects of IFs on OXT neurons in the brain. In particular, the influence of IFs on the central OXT system of infant animal needs to be examined, because IFs are increasingly consumed for weaning as well as dietary supplements. We have morphologically analyzed the central OXT neurons in neonatal mice using slice cultures treated with IFs, daidzein (Ddz) and genistein (Gen). In the supraoptic nucleus (SON) of male mice, Gen decreased the size of OXT neurons, but not of female nor in the paraventricular hypothalamic nucleus (PVN) of neither gender. In female PVN, Ddz and 17ß-estradiol (E(2)) increased the frequencies of varicosity on neurites in small OXT neurons (<21µm diameter of cell body). Ddz and Gen, as well as E(2), induced prominent vacuolation of the OXT neurons more frequently in male than in female mice, and in SON than in PVN. Thus, IFs can modulate the hypothalamic OXT neurons and the effects are site-specific and sexually dimorphic, suggesting that neonatal exposure to IFs may modify such a steroid-dependent development of particular neural pathways, including OXT system.

Spatiotemporal patterns of IGF-like peptide expression in the silkmoth Bombyx mori predict its pleiotropic actions.

In vertebrates, insulin-like growth factors (IGFs) play important roles in the regulation of growth and development. Although the principal source of circulating IGFs is the liver, IGFs are also secreted by many other tissues, functioning locally through paracrine/autocrine mechanism. In the silkmoth Bombyx mori, Bommo-IGF-like peptide (BIGFLP) is the functional counterpart of vertebrate IGFs and is mainly produced by the fat body, a functional equivalent of the vertebrate liver and adipocytes. However, its production by other tissues has not yet been analyzed. In this study, we systematically surveyed the BIGFLP-producing tissues and stages by means of immunohistochemistry, in situ hybridization and real-time quantitative RT-PCR, showing that BIGFLP is also produced by the neurosecretory cells in the brain, ovariole sheath and testis sheath, in a stage-specific manner. The BIGFLP-producing cells in the brain were identical to the cells that produce bombyxins, insulin-like peptides of B. mori, but the temporal expression patterns of both peptides were totally different. The BIGFLP gene expression in the sheaths of ovariole and testis were induced by ecdysteroid in vitro, similar to the expression in the fat body. A very high BIGFLP immunoreactivity was also found in the pupal nephrocytes, a functional equivalent of the glomerular podocytes in the vertebrate kidney, without the expression of the gene, suggesting that circulating BIGFLP is taken up and degraded by these tissues. Based on the present observations, the physiological functions of BIGFLP in B. mori development are discussed.

Prevalence of Salmonella isolates and antimicrobial resistance patterns in chicken meat throughout Japan.

We investigated the prevalence of Salmonella in chicken meat from northern, central, and southern Japan. Between 2006 and 2008, 821 samples from these three regions were collected and examined. Salmonella isolates were detected in 164 (20.0%) of these samples, with 15 (10.0%) of 150, 113 (27.5%) of 411, and 36 (13.8%) of 260 recovered from the northern, central, and southern regions, respectively. We recovered 452 Salmonella isolates. From the isolates, 27 serovars were identified; the predominant serovars isolated were Salmonella Infantis (n=81), Salmonella Kalamu (n=56), and Salmonella Schwarzengrund (n=43). Of the 452 isolates, 443 (98.0%) were resistant to one or more antibiotics, and 221 (48.9%) showed multiple-antibiotic resistance, thereby implying that multiple-antibiotic resistant Salmonella organisms are widespread in chicken meat in Japan. Resistance to oxytetracycline was most common (72.6%), followed by dihydrostreptomycin (69.2%) and bicozamycin (49.1%). This study, the first to report Salmonella prevalence in chicken meat throughout Japan, could provide valuable data for monitoring and controlling Salmonella infection in the future.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4.

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.

Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins.

All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members.

Formulation of nanoparticle-eluting stents by a cationic electrodeposition coating technology: efficient nano-drug delivery via bioabsorbable polymeric nanoparticle-eluting stents in porcine coronary arteries.

OBJECTIVES: The objective of this study was to formulate a nanoparticle (NP)-eluting drug delivery stent system by a cationic electrodeposition coating technology. BACKGROUND: Nanoparticle-mediated drug delivery systems (DDS) are poised to transform the development of innovative therapeutic devices. Therefore, we hypothesized that a bioabsorbable polymeric NP-eluting stent provides an efficient DDS that shows better and more prolonged delivery compared with dip-coating stent. METHODS: We prepared cationic NP encapsulated with a fluorescence marker (FITC) by emulsion solvent diffusion method, succeeded to formulate an NP-eluting stent with a novel cation electrodeposition coating technology, and compared the in vitro and in vivo characteristics of the FITC-loaded NP-eluting stent with dip-coated FITC-eluting stent and bare metal stent. RESULTS: The NP was taken up stably and efficiently by cultured vascular smooth muscle cells in vitro. In a porcine coronary artery model in vivo, substantial FITC fluorescence was observed in neointimal and medial layers of the stented segments that had received the FITC-NP-eluting stent until 4 weeks. In contrast, no substantial FITC fluorescence was observed in the segments from the polymer-based FITC-eluting stent or from bare metal stent. The magnitudes of stent-induced injury, inflammation, endothelial recovery, and neointima formation were comparable between bare metal stent and NP-eluting stent groups. CONCLUSIONS: Therefore, this NP-eluting stent is an efficient NP-mediated DDS that holds as an innovative platform for the delivery of less invasive nano-devices targeting cardiovascular disease.

Vascular endothelial cells and smooth muscle cells mediate carbachol-induced hepatocyte proliferation via muscarinic receptors and IP3/PKC signaling cascades.

An acetylcholine (ACh) agonist, carbachol (Cch), causes hepatocytes to proliferate in the presence of hepatic nonparenchymal cells (HNPCs). To identify the HNPCs and ACh receptor subtypes involved in carbachol-induced hepatocyte proliferation (CIHP), we examined two types of vascular cells as candidates for HNPCs mediating CIHP in cocultures of hepatocytes using the Transwell filter insert. In the coculture with vascular smooth muscle cells (VSMCs) or endothelial cells (VECs), but not in the monoculture, 72 h treatment with Cch significantly increased the numbers of hepatocytes. The results suggest that both VSMCs and VECs are involved in CIHP through soluble factors secreted from these cells. Interestingly, coculture with VECs, but not with VSMCs, markedly increased the number of hepatocytes, even in the absence of Cch. Cell proliferation assays using an analogue of thymidine, bromodeoxyuridine (BrdU), demonstrated that the hepatocytes in both cocultures transiently replicated their chromosomes 12 h after Cch administration. Blocking the muscarinic type 1 ACh receptor (M1), M3/5, intracellular inositol triphosphate (IP3) receptor, or protein kinase C (PKC) pathways inhibited VSMC-mediated CIHP, whereas blocking the M3/5, IP3 receptor, or PKC pathways inhibited VEC-mediated CIHP. Co-culturing hepatocytes with both types of vascular cells markedly increased their albumin content, but addition of Cch had no effect. In conclusion, VSMCs among vascular cells mediate CIHP through M1, M3/5, and IP3/PKC signal transduction pathways, whereas VECs do so through M3/5, and IP3/PKC pathways.

Differentiation/maturation of neuropeptide Y neurons in the corpus callosum is promoted by brain-derived neurotrophic factor in mouse brain slice cultures.

Neuropeptide Y (NPY) is widely distributed throughout both the central and peripheral nervous systems in mammals, and plays a role in various functions such as neural modifications affecting feeding, cardiovascular dynamics, or neural diseases. Many NPY neurons exist not only in gray matter in the central nervous system or ganglia in the peripheral system, but also in white matter such as the corpus callosum (cc) especially during development. The functions and regulation of callosal NPY neurons are not well understood, though NPY neurons in the cerebral cortex or hypothalamus are known to be regulated by neurotrophic factors such as brain-derived neurotrophic factor (BDNF). We examined the effect of BDNF on NPY neurons in the cc using organotypic slice cultures to clarify the regulation of callosal NPY neurons. A 3-week administration of BDNF significantly increased the number of NPY-immunopositive neuronal cell bodies and fibers in the cc rather than in the cerebral cortex as assessed with immunohistochemistry. Electron microscopy demonstrated that the NPY immunoreactivity mainly occurred in the regions associated with accumulating synaptic or cored vesicles. NPY-positive fibers had some contacts with several other neuronal fibers and glial processes. BDNF affected these fine structures of NPY neuronal fibers in the cc. These results suggest that BDNF takes part in the development, maturation, and maintenance of NPY neurons in the cc.