prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What do dendritic spines contain?

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Context 1: JoVE | Peer Reviewed Scientific Video Journal - Methods and Protocols
A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Downs syndrome  Octavio Garcia, Maria Torres, Pablo Helguera, Pinar Coskun, Jorge Busciglio 
PUBLISHED: 07-02-2010	Downs syndrome (DS) is the most common genetic cause of mental retardation  Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology  However, the mechanisms involved in DS spine alterations are not known  In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons  We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion  In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology 
Analysis of Dendritic Spine Morphology in Cultured CNS Neurons Authors: Deepak P  Srivastava, Kevin M  Woolfrey, Peter Penzes  Published: 07-13-2011 JoVE Neuroscience
Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses  These structures are rich in actin and have been shown to be highly dynamic  In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain  Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number  Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers  Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders  In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages  Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells  Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments  These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo 
Play ButtonIsolation and Culture of Mouse Cortical AstrocytesAuthors: Sebastian Schildge, Christian Bohrer, Kristina Beck, Christian Schachtrup  Institutions: University of Freiburg , University of Freiburg  Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics  In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail  This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups  The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture  Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes  Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers  This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology Neuroscience, Issue 71, Neurobiology, Cellular Biology, Medicine, Molecular Biology, Anatomy, Physiology, brain, mouse, astrocyte culture, astrocyte, fibroblast, fibrinogen, chondroitin sulfate proteoglycan, neuronal regeneration, cell culture, animal model50079Play ButtonImaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination MicroscopyAuthors: Marijn Schouten, Giulia M  R  De Luca, Diana K  Alatriste González, Babette E  de Jong, Wendy Timmermans, Hui Xiong, Harm Krugers, Erik M  M  Manders, Carlos P  Fitzsimons  Institutions: University of Amsterdam, University of Amsterdam Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain  Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity  The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit  Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy  However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm 
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines  These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen  Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures  Possible applications of SIM overlap with those of confocal microscopy  However, the two techniques present different applicability  SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light  In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM  The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal  After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software Neuroscience, Issue 87, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite51276Play ButtonSetting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated TransportAuthors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky  Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259 The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue  Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer  Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture  RBEC were isolated from 5-week-old rat cortices  The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion  Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue  The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix  The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium  They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells  RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders  The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average  The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0 26 ± 0 11 x 10-3 cm/min  Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer  In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),51278Play ButtonInducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing RatesAuthors: Alison X  Xie, Kelli Lauderdale, Thomas Murphy, Timothy L  Myers, Todd A  Fiacco  Institutions: University of California Riverside, University of California Riverside, University of California Riverside Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter  However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention  Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices  Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy  In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs  Applications of the technique for study of astrocytes are discussed  Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse51458Play ButtonInhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA ReceptorsAuthors: Laura E  Brown, Celine Fuchs, Martin W  Nicholson, F  Anne Stephenson, Alex M  Thomson, Jasmina N  Jovanovic  Institutions: University College London Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks  GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain  It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons  Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials  During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes  This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other  To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes  Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification  Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses  Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts  Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line52115Play ButtonTwo-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull PreparationAuthors: Xinzhu Yu, Yi Zuo  Institutions: University of California, Santa Cruz In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses  Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity  As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses  Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain  Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo  This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex  Therefore, images can be acquired immediately after surgery is performed  The experimental procedure can be performed repetitively over various time intervals ranging from hours to years  The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions Neuroscience, Issue 87, dendritic spine, mouse cortex, in vivo, two-photon microscopy, thinned-skull, imaging51520Play ButtonModeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered MiceAuthors: Robert S  McNeill, Ralf S  Schmid, Ryan E  Bash, Mark Vitucci, Kristen K  White, Andrea M  Werneke, Brian H  Constance, Byron Huff, C  Ryan Miller  Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development  In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture  Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes  The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro  Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo  Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma  Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time  Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft51763Play ButtonPaired Whole Cell Recordings in Organotypic Hippocampal SlicesAuthors: Chantelle Fourie, Marianna Kiraly, Daniel V  Madison, Johanna M  Montgomery  Institutions: University of Auckland, Stanford University Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron  When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased  This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue  A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation  Paired whole cell recordings are often perceived as too challenging to perform  While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed  The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings  While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation  In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity51958Play ButtonImaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium IndicatorsAuthors: Ruotian Jiang, Martin D  Haustein, Michael V  Sofroniew, Baljit S  Khakh  Institutions: University of California Los Angeles, University of California Los Angeles Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals  It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules  However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series  The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia  In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described  We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity  We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ  The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets  The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry Neuroscience, Issue 93, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging51972Play ButtonMethods to Assess Subcellular Compartments of Muscle in C  elegansAuthors: Christopher J  Gaffney, Joseph J  Bass, Thomas F  Barratt, Nathaniel J  Szewczyk  Institutions: University of Nottingham Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state  The loss of muscle mass and function with disease and age are significant public health burdens  We currently understand little about the genetic regulation of muscle health with disease or age  The nematode C  elegans is an established model for understanding the genomic regulation of biological processes of interest  This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species  Since C  elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo  Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo  These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance  Thus, C  elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function  Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism Developmental Biology, Issue 93, Physiology, C  elegans, muscle, mitochondria, sarcomeres, ageing52043Play ButtonImproved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term PotentiationAuthors: Agnès Villers, Laurence Ris  Institutions: University of Mons Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding  LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied  However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood  This could be partly due to the various experimental conditions used by different laboratories  Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity  It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection 
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation  This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices  It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena Neuroscience, Issue 76, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Surgery, Memory Disorders, Learning, Memory, Neurosciences, Neurophysiology, hippocampus, long-term potentiation, mice, acute slices, synaptic plasticity, in vitro, electrophysiology, animal model50483Play ButtonIn Vivo Modeling of the Morbid Human Genome using Danio rerioAuthors: Adrienne R  Niederriter, Erica E  Davis, Christelle Golzio, Edwin C  Oh, I-Chun Tsai, Nicholas Katsanis  Institutions: Duke University Medical Center, Duke University, Duke University Medical Center Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish  Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated 1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling  Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders  Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish  This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants  Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype  Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf)  This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol  We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others  Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model50338Play ButtonDirect Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)Authors: Samira Samtleben, Juliane Jaepel, Caroline Fecher, Thomas Andreska, Markus Rehberg, Robert Blum  Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich Visualization of calcium dynamics is important to understand the role of calcium in cell physiology  To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular  Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types  To date, TED was used in cell lines, glial cells, and neurons in vitro  TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES)  The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging  The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red  At the beginning of the procedure, cells are transduced with a lentivirus  Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging  Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM  The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen  After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope  Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging  Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity  Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0 Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging50317Play ButtonPreparation of Dissociated Mouse Cortical Neuron CulturesAuthors: Lutz G  W  Hilgenberg, Martin A  Smith  Institutions: University of California, Irvine (UCI) This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain  These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation  These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation  The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared  Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells  To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step  This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death  Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons Neuroscience, Issue 10, cellular, molecular, neurobiology, neuron, calcium/sodium imaging, primary cultures, mouse562Play ButtonAnalysis of Schwann-astrocyte Interactions Using In Vitro AssaysAuthors: Fardad T  Afshari, Jessica C  Kwok, James W  Fawcett  Institutions: University of Cambridge Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries  Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4  In addition they myelinate the demyelinated axons at the site of injury 5 
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7  This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally  Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13 
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour 
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts  As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide  This allows the behaviour of the two cellular populations to be analyzed at the boundary  Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries 
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15  This assay is commonly known as inverted coverslip assay  Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip 
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation  Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19 
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur Cellular Biology, Issue 47, Schwann cell, astrocyte, boundary, migration, repulsion2214Play ButtonQuantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse NumberAuthors: Dominic M  Ippolito, Cagla Eroglu  Institutions: Duke University, Duke University One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation  As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity  Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry  Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization  We define synapses as points of colocalization between the signals generated by these markers  The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c eroglu@cellbio duke edu) under the ImageJ analysis software platform  The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers  This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development Neuroscience, Issue 45, synapse, immunocytochemistry, brain, neuron, astrocyte2270Play ButtonPreparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid PathologyAuthors: Diana M  Mathis, Jennifer L  Furman, Christopher M  Norris  Institutions: University of Kentucky College of Public Health, University of Kentucky College of Medicine, University of Kentucky College of Medicine The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity  The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid  Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments  The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory  Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds  This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3  Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research  Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity  Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment  Here, we discuss the steps taken during the dissection procedure to minimize these problems  Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments  The possible impact of other methodological factors on synaptic function in these animal models (e g  recording solution components, stimulation parameters) are also discussed  While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models Neuroscience, Issue 49, aging, amyloid, hippocampal slice, synaptic plasticity, Ca2+, CA1, electrophysiology2330Play ButtonMesenteric Artery Contraction and Relaxation Studies Using Automated Wire MyographyAuthors: Lakeesha E  Bridges, Cicely L  Williams, Mildred A  Pointer, Emmanuel M  Awumey  Institutions: North Carolina Central University, Durham, North Carolina Central University, Durham, Wake Forest University School of Medicine Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance  These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body  The rat mesenteric artery has a diameter greater than 100 μm  The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2  The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented  Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires  Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3 The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors 
Medicine, Issue 55, cardiovascular, resistant arteries, contraction, relaxation, myography3119Play ButtonVisualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero ElectroporationAuthors: Emilie Pacary, Matilda A  Haas, Hendrik Wildner, Roberta Azzarelli, Donald M  Bell, Djoher Nora Abrous, François Guillemot  Institutions: MRC National Institute for Medical Research, National Institute for Medical Research, Université de Bordeaux In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5  To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8  Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions 
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development  Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells  In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines Neuroscience, Issue 65, Developmental Biology, Molecular Biology, Neuronal development, In utero electroporation, dendrite, spines, hippocampus, cerebral cortex, gain and loss of function4163Play ButtonImaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture SystemAuthors: Haruki Higashimori, Yongjie Yang  Institutions: Tufts University, Tufts Sackler School of Graduate Biomedical Sciences Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS)  This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2   Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology  Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia  What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i e , axon, dendrite, or soma)3  This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites  In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia  Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell 
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP)  In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels  In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels  In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth  The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Biophysics, Microfluidics, Microfluidic culture platform, Compartmented culture, Neuron to glia signaling, neurons, glia, cell culture4448Play ButtonFluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal NeuronsAuthors: Chan-Ying Zheng, Ronald S  Petralia, Ya-Xian Wang, Bechara Kachar  Institutions: National Institutes of Health, Bethesda FRAP has been used to quantify the mobility of GFP-tagged proteins  Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest  The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest  The mobility of the protein is then analyzed by measuring the fluorescence recovery rate  This technique could be used to characterize protein mobility and turnover rate 
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility2568Play ButtonPrimary Neuronal Cultures from the Brains of Late Stage Drosophila PupaeAuthors: Beatriz Sicaeros, Jorge M  Campusano, Diane K  O'Dowd  Institutions: University of California, Irvine (UCI) In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae  The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation  The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium  The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated  The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating  Images show live cultures at 2 days  Neurons continue to elaborate processes during the first week in culture  Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP  Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses  A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons  These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses 
Context 2: Football Club Urartu (, translated Futbolayin Akumb Urartu), commonly known as Urartu, is an Armenian professional football team based in the capital Yerevan that currently plays in the Armenian Premier League  The club won the Armenian Cup three times, in 1992, 2007 and 2016  In 2013–2014, they won the Armenian Premier League for the first time in their history 

In early 2016, the Russia-based Armenian businessman Dzhevan Cheloyants became a co-owner of the club after purchasing the major part of the club shares  The club was known as FC Banants until 1 August 2019, when it was officially renamed FC Urartu 

History

Kotayk
Urartu FC were founded as FC Banants by Sarkis Israelyan on 21 January 1992 in the village of Kotayk, representing the Kotayk Province  He named the club after his native village of Banants (currently known as Bayan)  Between 1992 and 1995, the club was commonly referred to as Banants Kotayk  During the 1992 season, the club won the first Armenian Cup  At the end of the 1995 transitional season, Banants suffered a financial crisis  The club owners decided that it was better to merge the club with FC Kotayk of Abovyan, rather than disband it  In 2001, Banants demerged from FC Kotayk, and was moved from Abovyan to the capital Yerevan 

Yerevan

FC Banants was relocated to Yerevan in 2001  At the beginning of 2003, Banants merged with FC Spartak Yerevan, but was able to limit the name of the new merger to FC Banants  Spartak became Banants's youth academy and later changed the name to Banants-2  Because of the merger, Banants acquired many players from Spartak Yerevan, including Samvel Melkonyan  After the merger, Banants took a more serious approach and have finished highly in the league table ever since  The club managed to lift the Armenian Cup in 2007 
Experience is making way for youth for the 2008 and 2009 seasons
Context 3: '用户指南 * User Guide 02 CN 11 EN * 包装内含 使用前注意事项 快速引导 产品部件详情说明 操作说明 02 02 03 06 08 01 
•本产品支持在系统设置中进行瞳距调节 , 调节时请务必注意，最小瞳距可能会碰触鼻梁。当您佩戴头盔后，您 “显示”中进行手动调节，请注意设置使用不合适的瞳距，可能会引起视觉重影或者眼睛疲劳。 可在“设置” ► •本产品“护眼模式”经德国 TÜV Rheinland 低蓝光认证，通过软件算法降低三色通道中的蓝光量，来达到保护 “护眼” “色彩调节” 眼睛的作用，该模式下画面颜色偏黄，您可根据个人喜好在“设置” 中激活或关闭此功能。 ““显示” ► ► ► 包装内含： VR 头盔 / 手柄 × 2 / 1 5V AA 碱性干电池 × 4/ 眼镜支架 / 遮光鼻托 / 手柄挂绳 × 2 / USB-C 电源适配器 / USB-Cto C 2 0 数据线 / 快速指南 / 用户指南 / 安全与质保指南使用前注意事项 •本产品在开阔的室內环境使用体验最佳，建议至少预留 2×2 米 的空间。使用前请确认身体没有不适且周围环 境安全，特别是佩戴头盔在室内行走移动时，要尽量避免发生意外。 •不建议 12 岁及以下儿童使用本产品，建议将头盔、手柄和配件置于儿童够不到的位置，13 岁以上青少年须在 成人监护下使用，以免发生意外。 •本产品无近视调节功能，近视用户请佩戴眼镜使用并尽量避免近视眼镜被头盔的光学镜片磨伤或刮伤。建议在 使用和收纳时注意防护光学镜片，避免尖锐物体划伤镜片，擦拭清洁时请使用柔软的眼镜布，否则可能划伤镜片， 影响视觉效果。 •长时间使用可能引发轻微的昡晕或者眼疲劳，建议使用 30 分钟后适当休息，可通过眼保健操或观看远处物体缓 解眼疲劳。如果您的身体感到任何不适，请立即停止使用。如果不适持续，请咨询医生。 •当头盔镜片被阳光或紫外线照射时（尤其在户外、阳台、窗台及汽车内存放时），可能导致屏幕出现永久性黄斑。 请尽量避免该情况发生，此种屏幕损坏不在产品的质保范围内。 *本产品最终外观及功能以实物为准，部分地区包装内含物品有所差异，本说明仅供参考。 02 CN
六自由度 VR 体验 本产品可以追踪头盔和手柄前、后、左、右、上、下和旋转的运动状态，您在现实中的肢体运动会实时反映在虚 拟世界中。 由于没有任何线缆的束缚，您在虚拟世界自由探索时请确保游玩区域的安全。 1  建议准备一个整洁安全的体验空间：至少 2×2 米；保持房间明亮，避免在只有单色的墙或大面积玻璃、镜子类 反射物以及许多移动画面和物体的空间中使用。 2  撕下 VR 头盔前端摄像头上的保护膜，并佩戴手柄挂绳。 3  根据开机后的画面提示进行游玩区域的设定。 ❶ 安装电池 按箭头方向拔出电池盖侧边的绝缘纸 快速引导 提示：本产品虚拟的安全区提醒功能，不能完全保证您在设定好的游戏区域中的安全，请时刻注意周围的安全情况。 提示：建议使用 1 5V AA 碱性电池。 按照图示拨动电池盖拨钮打开电池盖更换电池。 03 CN
❷ 手柄开机 ❸ 头盔开机 ❹ 佩戴头盔，调节至清晰舒适的位置 首次开机：拔出绝缘纸，手柄自动开机（蓝灯闪烁） 非首次开机：短按手柄 Home 键开机（蓝灯闪烁） 长按头盔电源键 2 秒（蓝灯常亮） 调节旋钮转动绑带，使后脑垫套在头上，微调绑带长度及佩戴位置至视野清晰 04 提示：近视用户请佩戴眼镜或镜片插件使用，本产品不具备近视调节功能。 CN
❺ 微调顶绑带 微调顶绑带使其受力以减少额头压力 ❻ 瞳 距 调 节 在系统设置：“设置” ► “显示”界面中进行瞳距调节，点击“+”或“-”按钮可微调瞳距直至画面清晰 64mm 请勿 强行 掰动镜 筒，以 免造 成损坏 ！ 请注 意设 置使用 不合适 的瞳 距，可 能 会引起 视 觉重影 或 者眼睛 疲 劳。准 确 的瞳距 设 置有助 于 获得清 晰 的图像 并 减少眼睛 疲劳。 05 CN
产品部件详情说明 头盔状态指示灯 蓝灯常亮：开机进行中或工作状态 黄灯常亮：充电中，电量低于 98% 红灯常亮：充电中，电量低于 20% 绿灯常亮：充电完毕，电量大于 98% 或 充满 蓝灯闪烁：关机进行中 红灯闪烁：电量低于 20% 指示灯熄灭：休眠或关机 06 ① 电源键 开机：长按 2 秒 关机：长按 5 秒 复位：长按 10 秒 开机时，短按休眠 ② ③ ④ ⑤ 状态指示灯 贴脸泡棉 音量键 彩色透视摄像头 使用时请勿遮挡 ⑥ ⑦ ⑧ 顶部绑带 可拆卸 绑带旋钮 环境追踪摄像头 使用时请勿遮挡 ⑨ ⑩ ⑪ USB-C 接口 左 / 右喇叭 距离传感器 佩戴头盔后，系统自动唤醒 摘下头盔后，系统自动休眠 ⑫ ⑬ 眼球追踪摄像头 此功能仅 Pro 版支持 使用时请勿遮挡 面部追踪摄像头 此功能仅 Pro 版支持 使用时请勿遮挡 CN
手柄状态指示灯 熄灭：已连接或者关机 蓝灯常亮：固件升级模式 蓝灯闪烁：连接中 红蓝灯交替慢速闪烁：等待配对 ① ② 摇杆 菜单键 ③ Home 键 开机 : 短按关机 : 长按 6 秒退出应用 : 短按屏幕中心校正 : 长按 1 秒④ ⑤ ⑥ ⑦ 状态指示灯 抓握键 截屏键 扳机键 ⑧ ⑨ 电池盒 打开：拨动拨钮，电池盒弹出 安装：按压直至自动锁紧 追踪光环 使用时请勿遮挡 注：手柄挂绳可按图示将粗绳穿过细绳并锁紧在手柄尾端 07 CN
手柄硬件复位 如果手柄出现按 Home 键和任何按键均无反应或者头盔中虚拟手柄卡死不动的问题可拆装电池重新启动手柄。 近视用户配戴 本设备不具备近视调节功能，头盔可支持佩戴镜框宽度小于 150mm 的大多数标准眼镜。 操作说明 头控模式 未连接手柄的情况下，您可通过转动头部光标及点击头盔音量加减按键进行操作。 切换主控手柄射线 在主控菜单下，短按对应手柄的扳机键可以切换主控手柄的射线。 屏幕中心校正 戴着头盔直视前方，按住手柄 Home 键（或头控模式下头盔上的音量减键）1 秒以上，进行屏幕中心的校正将菜 单拉到当前视野朝向位置。 断开手柄 长按手柄 Home 键直至手柄状态指示灯红灯亮起并伴随振动产生时即可松手，此时手柄关机并断开与头盔的连接。 您无需刻意进行手柄关机操作，在以下状态下手柄会自动关机省电： •头盔进入深度休眠时（摘下头盔后一段时间） •头盔手柄管理界面解绑手柄时 •头盔关机时 添加新手柄 如需添加新手柄（头盔最多可同时连接一对手柄，即左右手柄各一只），或解绑手柄后再次连接 , 可进入“设置” “手 柄”，点击“配对”，同时按住手柄 Home 键和扳机键直至手柄状态指示灯红蓝交替闪烁时即可松开，然后根据 头盔画面提示操作。 ► 休眠 / 唤醒 方式一：摘下头盔一段时间后，系统自动休眠；戴上头盔时，系统自动唤醒。 方式二：短按头盔电源键也可以进行休眠或唤醒操作。 硬件复位 头盔硬件复位 如果头盔出现短按头盔电源键没有反应或头盔的画面卡死等问题，可以长按头盔电源键 10 秒以上重新启动头盔。 08 CN
安装眼镜支架 安装遮光鼻托 如您存在眼镜摩擦光学镜片或者压迫鼻梁的问题，请按照图示安装眼镜支架以增加间隔空间。 您可根据佩戴的舒适度选择是否安装。 如您感觉鼻子处漏光影响体验，请按照图示安装遮光鼻托配件。 由于眼睛空间密闭可能加剧起雾及出汗问题，您可根据喜好选择是否安装。 ❶ 摘下贴脸泡棉 ❷ 将眼镜支架按照图示安装在产品上 ❸ 将贴脸泡棉按照图示安装眼镜支架上 ❶ 摘下贴脸泡棉 ❸ 安装贴脸泡棉❷ 将遮光鼻托按照图示方式安装在贴脸泡棉上 注：按照图示拆卸眼镜支架 09 CN
更换贴脸泡棉 贴脸泡棉多次清洁和长时间使用后会变色和质地变软，您可酌情更换新泡棉。 更换顶绑带 摘下贴脸泡棉 ❸ 安装贴脸泡棉 按照图示捏住顶绑带金属扣，往下压到底然后抽出 ❷ •购买优质热门应用 •畅 聊 社 区， 与 众 多 PICO 玩 家 一起探索 VR 世界 •管理设备更便捷 •参与丰富互动活动 •更多精彩内容等你来发现 ❶ 微 信公 众 号：PICO VR抖音：PICO官 方 旗 舰 店哔 哩 哔 哩：PICO-VR官 方微 博：PICO-VR ❶ ❷ 10 CN
In The Box: VR Headset / 2 Controllers / 4 1 5V AA Alkaline Batteries / Glasses Spacer / Nose Pad / 2 Controller Lan- yards / USB-C Power Adapter / USB-C to C 2 0 Data Cable / Quick Guide / User Guide / Safety and WarrantyGuide Important Health & Safety Notes • This product is designed and intended to be used in an open and safe indoor area, free of anytripping or slipping hazards  To avoid accidents, remain conscious to the potential confines ofyour physical area and respect the boundary of your virtual area whenever you see it  Be sure towear the lanyards when using the Controllers  Make sure that there is enough space around yourhead and body (at least 2 meters by 2 meters) to stretch your arms to avoid damage or injury toyourself, others, and your surroundings  • This product is not recommended for children aged 12 and under  It is recommended to keep headsets,controllers and accessories out of the reach of children  Teenagers aged 13 and over must use it underadult supervision to avoid accidents  • This product is designed to accommodate most prescription glasses  Make sure to wear the VR Headsetin a manner in which the VR Headset lenses do not rub or impair your prescription lenses  • Prolonged use may cause dizziness or eye fatigue  It is recommended to take a break every 30 minutes Try relieving your eyestrain by looking at distant objects  If you feel any discomfort, stop using the prod- uct immediately  If the discomfort persists, seek medical advice • Do not expose the optical lenses to direct sunlight or other strong light sources  Exposure to directsunlight may cause permanent yellow spot damage on the screen  Screen damage caused by sunlightexposure or other strong sources of light is not covered by the warranty  • This product supports interpupillary distance (IPD) adjustment in system settings  When adjusting,please be aware that with the minimum IPD, it may touch the bridge of the nose  You can adjust the IPDaccording to your actual interpupillary distance in "Settings"►"Display"  Please note that using inap- propriate IPD may increase the risk of discomfort  • This product has an “Eye Protection Mode”, certified by TÜV Rheinland (Germany), which can protectyour eyes by reducing blue light in the three color channels using software algorithms  The screen ap- pears yellowish in this mode and you can turn this feature on/off in "Settings"►"Display"►"Color"►“- Eye Protection”  • Protect optical lenses during use and storage to prevent damage, such as scratches or exposure tostrong light or direct sunlight  * Product and packaging are updated regularly, and the functions and contents of the standalone headset may be upgraded in the future Therefore, the content, appearance and functionality listed in this manual and product packaging are subject to change and may notreflect the final product



ground_truth: They are rich in actin and have been shown to be highly dynamic.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What is the name of the most active fan club?

Read the following text and answer briefly.

Context 2:  The departures of most of the experienced players have left the club's future to the youth  Along with two Ukrainian players, Ugandan international, Noah Kasule, has been signed 

The club headquarters are located on Jivani Street 2 of the Malatia-Sebastia District, Yerevan 

Domestic

European

Stadium

The construction of the Banants Stadium was launched in 2006 in the Malatia-Sebastia District of Yerevan, with the assistance of the FIFA goal programme  It was officially opened in 2008 with a capacity of 3,600 seats  Further developments were implemented later in 2011, when the playing pitch was modernized and the capacity of the stadium was increased up to 4,860 seats (2,760 at the northern stand, 1,500 at the southern stand and 600 at the western stand) 

Training centre/academy
Banants Training Centre is the club's academy base located in the Malatia-Sebastia District of Yerevan  In addition to the main stadium, the centre houses 3 full-size training pitches, mini football pitches as well as an indoor facility  The current technical director of the academy is the former Russian footballer Ilshat Faizulin 

Fans
The most active group of fans is the South West Ultras fan club, mainly composed of residents from several neighbourhoods within the Malatia-Sebastia District of Yerevan, since the club is a de facto representer of the district  Members of the fan club benefit from events organized by the club and many facilities of the Banants training centre, such as the mini football pitch, the club store and other entertainments 

Achievements
 Armenian Premier League
 Winner (1): 2013–14 
 Runner-up (5): 2003, 2006, 2007, 2010, 2018 

 Armenian Cup
 Winner (3): 1992, 2007, 2016 
 Runner-up (6): 2003, 2004, 2008, 2009, 2010, 2021–22

 Armenian Supercup
 Winner (1): 2014 
 Runner-up (5): 2004, 2007, 2009, 2010, 2016 

Current squad

Out on loan

Personnel

Technical staff

Management

Urartu-2

FC Banants' reserve squad play as FC Banants-2 in the Armenian First League  They play their home games at the training field with artificial turf of the Urartu Training Centre 

Managerial history
 Varuzhan Sukiasyan (1992–94)
 Poghos Galstyan (July 1, 1996 – June 30, 1998)
 Oganes Zanazanyan (2001–05)
 Ashot Barseghyan (2005–06)
 Nikolay Kiselyov (2006–07)
 Jan Poštulka (2007)
 Nikolay Kostov (July 1, 2007 – April 8, 2008)
 Nedelcho Matushev (April 8, 2008 – June 30, 2008)
 Kim Splidsboel (2008)
 Armen Gyulbudaghyants (Jan 1, 2009 – Dec 1, 2009)
 Ashot Barseghyan (interim) (2009)
 Stevica Kuzmanovski (Jan 1, 2010 – Dec 31, 2010)
 Rafael Nazaryan (Jan 1, 2011 – Jan 15, 2012)
 Volodymyr Pyatenko (Jan 17, 2013 – June 30, 2013)
 Zsolt Hornyák (July 1, 2013 – May 30, 2015)
 Aram Voskanyan (July 1, 2015 – Oct 11, 2015)
 Tito Ramallo (Oct 12, 2015 – Oct 3, 2016)
 Artur Voskanyan (Oct 3, 2016 – Aug 11, 2018)
 Ilshat Faizulin (Aug 12, 2018 –Nov 24, 2019)
 Aleksandr Grigoryan (Nov 25, 2019 –Mar 10, 2021)
 Robert Arzumanyan (10 March 2021–24 June 2022)
 Dmitri Gunko (27 June 2022–)

References

External links
 Official website 
 Banants at Weltfussball de  

 
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Context 3:  These instructions are for reference only  * Carefully read this user guide before using the product and share this information with any other users, as it contains important safetyinformation  Keep the user guide as reference for the future  11 EN
6 Degrees of Freedom VR The device can track your translational and rotational movements in all directions (up/down, left/right,forward/backward, pitch, roll, and yaw)  Your movements in the real world will be captured and translatedto what you see in the virtual world when using the appropriate content  Ensure a safe environment before you start your VR experience  1  Clear a safe indoor area of at least 2 meters by 2 meters  Keep the room bright, avoid spaces with main- ly single-colored walls, glass, mirrors, moving pictures or other similar objects  2  Remove the protective film that covers the headset front cameras  Wear the lanyards connected to theControllers  3  Set up your environment by following instructions on the VR Headset screen  Install Batteries ❶Pull the tab to remove the insulating paper  Quick Guide 2 m 2m This product can not guarantee your safety with guardian system, you will need to always pay attention to the surrounding safety  * Note: 1 5V AA alkaline batteries should be used Slide the toggle according to arrow direction toopen the battery case  12 EN
Power on the Controller ❷ First Start: The Controller will start automaticallyafter removing the insulating paper  Others: Short press the Home button for 1second until the status indicator flashes blue Power on the VR Headset ❸ Long press the Power button for 2 seconds untilthe status indicator turns blue Wear Your Headset for a Comfortable Fit and View ❹ Adjust the strap dial to turn the strap so that the back of your head rests on the padding  Fine-tune thelength and position of the strap to give a clear view  * Note: You can use this product with prescription glasses or lenses insert  13 EN
Fine-tune the Top Strap ❺ Fine-tune the head strap to reduce pressure on the forehead  Interpupillary Distance (IPD) Adjustment ❻ In System Setting, go to “Setting” ► “Display” to adjust IPD, tap “+” or “-” button to slightly adjust IPDuntil the picture is clear  14 64mm Please note that inappropriate IPD setting may cause ghosting or eyestrain Accurate IPD setting helps you get a clear imaging and ease eyestrain  EN
Product Details VR Headset Status Indicator Legend Blue: Powered on with battery over 20% Yellow: Charging: Battery is less than 98% Red: Charging: Battery is less than 20% Green: Charging: Battery is more than 98% or charge complete Blue flashing: Shutting down Red flashing: Battery is less than 20% Off: Sleeping or Powered off Power Power on: Long press for 2 seconds Power off: Long press for 5 seconds Hardware reset: Long press for 10 seconds Short press to enter sleep or wake up Status Indicator Face Cushion Volume ① ② ③ ④ ⑤ ⑥ ⑦ ⑧ RGB See Through Camera Do not block during use  Top Strap Removable Strap Dial Tracking Cameras Do not block during use  ⑨ ⑩ ⑪ USB-C Interface Left/Right Speaker Proximity Sensor The system wakes upwhen the VR headset isput on, sleeps when VRheadset is taken off  ⑫ ⑬ Eye Tracking Cameras Pro version only  Do not block during use



ground_truth: South West Ultras fan club.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What is the recommended space for using the VR headset?

Read the following text and answer briefly.

Context 3:  Face Tracking Camera Pro version only  Do not block during use  15 EN
Controller Status Indicator Legend Off: Connected or Powered off Blue: Firmware updating in progress Blue flashing: Searching for connection Red and blue flashing alternately: Pairing in progress 16 Joystick Menu ③ ① ② Home Power on: Short pressPower off: Long press for 6 secondsReturn home screen: Short pressScreen recentering: Press for 1 secondStatus Indicator Grip Capture Trigger ④ ⑤ ⑥ ⑦ ⑧ ⑨ Battery Case Open: Slide down the toggle andpop up the battery case  Lock: Push the battery case to lock  Tracking Ring Do not block during use  * Note: Pass the Controller Lanyardthrough the string as shown andlock at the end of the Controller EN
Operating Instructions Headset Control Mode If the Controller is not connected, you can interact with the home screen by moving your head to directthe crosshairs over your intended selection and clicking the Volume Up/Down button on the VR Headset  Switch the pointer of the master Controller In the home screen, short press the Trigger of the corresponding Controller to switch the pointer of themaster Controller  Screen re-centering Wear the VR Headset and look straight ahead, press and hold the Home button of the Controller or VRHeadset ( or the Volume Down button of the VR Headset in head control mode) for more than 1 second tore-center the screen  Disconnect the Controller Press and hold the Home button until the status indicator turns red and the Controller vibrates Controllers will automatically shut down to save power in the following cases:When the VR Headset enters deep sleep (a while after the VR Headset is taken off)When the Controller is unpairedWhen the VR Headset is powered off Add a new Controller If you need to add a new Controller (the VR Headset can only connect one left Controller and one rightController) or reconnect with an unpaired Controller  Go to “Settings” ► “Controller”, click on “Pair” Press and hold the Home button and the Trigger of the Controller at the same time until the red and bluelights of the Controller flashing alternately, and then follow the instructions on the VR Headset screen  Sleep / Wake up Option 1 (Proximity Sensor) Take off VR Headset for automatic sleeping: wear the VR Headset for automat- ic waking up  Option 2 (POWER Button) Press the Power button of the VR Headset for manual sleeping or waking up  Hardware reset VR Headset reset If the visual in the VR Headset freezes, or the VR Headset does not respond after short press the Powerbutton, you can press the Power button of the VR Headset for more than 10 seconds to reboot the VRHeadset  Controller reset If the virtual Controller, the Home button or any buttons of the Controller doesn\'t respond, remove andreinstall the battery case to restart the Controller  The VR Headset Adjustment This device has no myopia adjustment function  The VR Headset allows wearing most standard glasseswith a frame width of less than 150mm  to install Glasses Spacer to increase the space  You can install or not according to your situation  17 EN
Install Glasses Spacer Install Nose Pad If you have glasses collision with headset lens or pressure on the bridge of nose, please follow the pictureto install Glasses Spacer to increase the space  You can install or not according to your situation  If you feel light leaking from your nose, please follow the picture to install Nose Pad to block the light You can consider having it installed at your own discretion  Disassemble the Face Cushion  Install the Glasses Spacer on the Headset  ❸ ❶ ❷ Install the Face Cushion on the Glasses Spacer  Disassemble the Face Cushion  Install the Nose Pad on the Face Cushion  ❶ ❷ Install the Face Cushion on the Headset  ❸ * Note: Disassemble the Glasses Spacer 18 EN
Replace Face Cushion The Face Cushion will have the following phenomena such as color change, surface fluff, soft texture afterlong-term use and repeated cleaning  You can replace a new Face Cushion as needed  Replace Top Strap ❶ ❷ Disassemble the Face Cushion  Pinch the metal buckle of the top strap asshown, press it down and pull it out Install the Face Cushion on  ❸ ❷ ❶ • Purchase high-quality and trending apps • Join PICO Community and explore the VR worldwith other PICO players• Manage your device with ease • Engage in diverse and interactive activities • More exciting features waiting for you 19 EN
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ground_truth: It is recommended to have at least a 2x2 meter space for using the VR headset.