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: Paper Info

Title: Two-stage Pipeline for Multilingual Dialect Detection
Publish Date: Unkown
Author List: Ankit Vaidya (from Pune Institute of Computer Technology), Aditya Kane (from Pune Institute of Computer Technology)

Figure

Figure 1: Class distribution of dialects
Figure 2: System diagram for dialect classification The LID classifies the input into one of 3 languages The sample is then further classified into dialects by language specific models 
Figure 3: Confusion matrix of 9-way classification Note that rows are normalized according to the number of samples is that class 
Our complete results for Track-1 using the two-stage dialect detection pipeline Model-* denotes the language of the models used for the experiments 
Performance on Track-1 validation dataset of individual models used in the two-stage pipeline "Lg" stands for language of the model used 
Comparative results of two-way classification using the finetuned (F T ) predictions and predictions adapted from three-way classification models 

abstract

Dialect Identification is a crucial task for localizing various Large Language Models  This paper outlines our approach to the VarDial 2023 DSL-TL shared task  Here we have to identify three or two dialects from three languages each which results in a 9-way classification for Track-1 and 6-way classification for Track-2 respectively 
Our proposed approach consists of a two-stage system and outperforms other participants' systems and previous works in this domain  We achieve a score of 58 54% for Track-1 and 85 61% for Track-2  Our codebase is available publicly 1  

Introduction

Language has been the primary mode of communication for humans since the pre-historic ages  Studies have explored the evolution of language and outlined mathematical models that govern the intricacies of natural language   Inevitably, as humans established civilization in various parts of the world, this language was modified by, and for the group of people occupied by that particular geographical region 
This gave rise to multiple national dialects of the same language  The VarDial workshop (colocated with EACL 2023) explores various dialects and variations of the same language  We participated in the Discriminating Between Similar Languages -True Labels (DSL-TL) shared task  In this task, the participants were provided with data from three languages, with each language having three varieties 
This shared task consisted of two tracks -Track-1 featuring nine-way classification and Track-2 featuring six-way classification  The second track included two particular national dialects of each language (eg  American English and British English), and the first track had one general  We ranked 1 st in both of the tracks 
Moreover, we beat the next best submission by a margin of 4 5% in the first task and 5 6% in the second task We were the only team to surpass the organizer baseline scores  We present our winning solution in this paper  We used an end-to-end deep learning pipeline which consisted of a language identification model and three language-specific models, one for each language 
We converged upon the best combination by doing an elaborate analysis of various models available  Furthermore, in this work we also analyze the performance of the pipeline as a whole and also provide an ablation study  Lastly, we provide some future directions in this area of research 

Related Work

The present literature encompasses various aspects of dialect identification  We study this from three perspectives: large language models, language identification and dialect classification problems 

Large Language Models

The success of transformers and BERT based models was inevitable since the initial boom of the transformer  2017) model  In recent years, many other architectures like RoBERTa and ELECTRA have further pushed the state-of-the-art in this domain  Moreover, autoregressive models like GPT and GPT-2 have also shown their prowess 
Multilingual versions of RoBERTA, namely XLM-RoBERTa are also available  Lastly, language specific models like Spanish BERT (la Rosa y Eduardo G  Ponferrada y Manu Romero y Paulo Villegas y Pablo González de Prado Salas y María Grandury, 2022) and Portuguese BERT are available as well  Our winning solution makes use of these large language models trained on specific languages 

Language Identification Models

Many multilingual language identification models have been developed in order to classify the language of the input sentence beforehand  Even though the initial works used n-gram models and generative mixture models or even conditional random fields and other classical machine learning methods like naive bayes , modern methods have shifted to the use of deep learning for language identification  
Recent works have mainly focused on deep learning based language identification, where handling codemixed data is a big challenge in the domain  For our experiments, we use a version of XLM-RoBERTa finetuned on a language identification dataset 2   This model has near-perfect test accuracy of 99 6% 

Dialect Classification

Dialect classification has been previously solved using statistical methods like Gaussian Mixture Models and Frame Selection Decoding or Support Vector Machines (SVM)   It has been explored relatively sparsely, mostly in the case for local languages   Deep learning approaches have been explored in previous editions of the VarDial workshop shared tasks and otherwise  
Dialect classification was also explored previously as a part of other shared tasks   We want to stress that given the multilingual nature of the dataset, using the present methods directly was not an option  In our work, although we take inspiration from the previous works, we propose a novel system that surpasses the performance of the previous systems by a large margin 

Data

The dataset  We observed that the class PT-BR had the most number of samples (2,724) and the class EN had the least number of samples (349), and thus the imbalance ratio was almost 1:8  We have illustrated the data distribution in Figure   We tried to mitigate this imbalance using over-sampling and weighted sampling methods 
However, the improved data sampling method did not affect the performance 

System Description

This was a problem of multi-class classification having 9 classes for Track-1 and 6 classes for Track-2  The samples were belonging to 3 languages having 3 varieties each, so the classification pipeline was made in 2 stages  The Language Identification (LID) model which is the first stage classifies the sentence into 3 languages: English (EN), Spanish (ES) and Portuguese (PT) 
The LID is a pretrained XLM-RoBERTa that is fine-tuned for the task of language identification  It is able to classify the input sentence into 20 languages  We classify and separate the samples according to their language  The samples corresponding to the specific languages are then fed into the language specific models for dialect identification 
For dialect identification we have used models like BERT and RoBERTa with a linear layer connected to the pooler output of the models  Then fine-tuning is done on the models for dialect identification   using the samples corresponding to the specific languages  For the task of dialect identification we experimented with several pretrained models like XLM-RoBERTa, BERT, ELECTRA, GPT-2 and RoBERTa 
All models were fine-tuned for 20 epochs with a learning rate of 1e-6 and weight decay 1e-6 with a batch size of 8



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 models were used for dialect identification?

Read the following text and answer briefly.

Context 2:  The best performing model checkpoint was chosen according to the epoch-wise validation macro-F1 score  5 Experiments and Results

Experiments using Large Language Models

For the task of Dialect Identification we have tried various language specific models like XLM-RoBERTa, BERT, ELECTRA, RoBERTa and GPT- 2  The base variant of all these models were used and all the models were used through the Hugging-Face library  The pooler output of these models was passed through a linear layer and the models were fine-tuned 
First, we experimented with different models for Track-1  All the models were trained for 20 epochs with learning rate 1e-6, weight decay 1e-6 and a batch size of 8  We used XLM-RoBERTa as the baseline for all 3 languages  The best performing models for the English language were RoBERTa and BERT whereas GPT-2 was the worst performing 
Similarly the language specific versions of RoBERTa and BERT performed well for the Spanish and Portuguese respectively  Overall the worst performing model was GPT-2 across all 3 languages  The validation F1 scores are present in Table   The two best-performing models for every language were chosen for Track-2 
The same procedure as specified above was used and the F1 scores are present in Table   The train and validation F1 scores for 2-class classification are higher for all models as compared to the F1 score of the same models for 3-class classification  This was mainly due to the poor representation and accuracy of classification of the third class 
We observed symptoms of overfitting in all models after 12-15 epochs and the best validation F1 score was obtained in the range of 4-8 epochs 

LID experiments

The pipeline for dialect identification is divided into two parts as the sentences in the dataset belong to different languages  The stages are described in Section 4  The XLM-RoBERTa we have used for language classification has a test accuracy of 99 6% meaning it correctly classifies all input sentences and hence, can be considered as a perfect classifier 
For the final pipeline we experimented using the two best performing models for each language in Track-1 and Track-2  For both the tracks we experimented with all 8 (2 3 ) possible combinations of models and calculated the validation F1 score for the combined validation dataset which had sentences belonging to all languages 
The validation scores for Track-1 and Track-2 are shown in Table and Table respectively  For both the tracks, the three pipelines with the best validation F1 scores were chosen for submission 

Using 3-way classifier as a 2-way classifier

In Track-1, participants are expected to train a classifier which classifies amongst 9 classes, and in Track-2, participants are expected to train a classifier which classifies amongst 6 classes  These 6 classes are a proper subset of the 9 classes from Track-1  Thus, an intuitive baseline for Track-2 is to use the model finetuned for Track-1, whilst considering only the relevant classes for the latter task 
The classes EN , ES and P T , i e  the classes without any national dialect associated with them are not included in Track-2 as compared to Track-1  Thus, we calculate the predictions for the Track-2 validation dataset using the models for Track-1 and exclude the metrics for Track-1 specific classes to get the metrics for this "adapted" 2-way classification 
We show the results of this experiment in Table and observe that, as expected, the adapted 2-way classification performs worse compared to the explicitly finetuned variant 

Results for Track-1 and Track-2

We now present our experiments and their performance for both tracks  Our experiments for Track-1 are described in Table and our experiments for Track-2 are described in Table   The participants were allowed three submissions for evaluation on the test set, so we submitted predictions using the three systems which performed the best on the validation set 
As mentioned in Section 5 2, we performed 2 3 , i e  a total of 8 experiments using the two best models for each language  We observed that RoBERTa base on English, Spanish BERT base on Spanish and Portuguese BERT base performed the best on the testing set for Track-1  The same combination, with RoBERTa base for English, worked best for Track-2 
All of our submissions were the top submissions for each track, which surpassed the next best competitors by a margin of 4 5% and 5 6% for Track-1 and Track-2 respectively 

Ablation of best submissions

We hereby make some observations of our submissions and other experiments  To assist this, we plot the confusion matrices of our best submissions for Track-1 and Track-2 in Figures respectively  Note that these confusion matrices have their rows (i e  true labels axes) normalized according to the number of samples in the class 
Here are observations from our experiments: 1  BERT-based models outperform other models across all languages: We observe that BERT-based models outperform ELECTRA-based and GPT-2-based models, as shown in Table   We speculate this is because of the inherent architecture of BERT, which combines semantic learning with knowledge retention 
This combination of traits is particularly useful for this task  2  Common labels perform the worst across all languages: We observe that the common labels EN , ES and P T perform the worst, both in the individual as well as the two-stage setup  We hypothesize this is because of the absence of dialect specific words, or words that are specific to the geographical origin of the national dialect (for example, "Yankees" for EN-US and "Oxford" for EN-GB) 
3  English models work better than models of other languages: It can be noted from Figures 4 and 3 that the English models have the best performance across all classes  This can be attributed to two reasons: absence of national dialect specific words and lesser pretraining data in the case of Portuguese 
4  British English is most correctly classified class: We can observe that the Spanish or Portuguese models make equal number of mistakes in the case of either national dialect, in the case of Track-2 (see Figure )  However, in the case of English, the label EN − GB is correctly classified for more than 95% of the cases 
We speculate this is because British English involves slightly distinctive grammar and semantics, which help the model separate it from other classes  5  The proposed 2-step method is scalable for multiple language dialect classification: We can strongly assert that the novel 2-step deep learning method for multilingual dialect classification is a scalable method for the task due to two specific reasons: firstly, the multilingual models (like XLM-RoBERTa) might not have the vocabulary as well as the learning capabilities to learn the minute differences between individual dialects 
Secondly, this system can be quickly expanded for a new language by simply adding a language specific dialect classifier, provided the language identification model supports that particular language 

Conclusion

In this paper we propose a two-stage classification pipeline for dialect identification for multilingual corpora  We conduct thorough ablations on this setup and provide valuable insights  We foresee multiple future directions for this work  The first is to expand this work to many languages and dialects 
Secondly, it is a worthwhile research direction to distill this multi-model setup into a single model with multiple prediction heads  The obvious limitation of this system is the excessive memory consumption due to the usage of language specific models  For low resource languages this system is difficult to train and scale 
We hope that these problems will be addressed by researchers in future works 
Context 3: 2008年5月31日 随笔档案 - 狼爱上狸 - BlogJava
本地搭建以太坊私有网络-基于Ganache和MetaMask
本文主要介绍如何使用Ganache，在本地搭建以太坊私有网络，并进行简单的测试。
Ganache用于搭建私有网络。在开发和测试环境下，Ganache提供了非常简便的以太坊私有网络搭建方法，通过可视化界面可以直观地设置各种参数、浏览查看账户和交易等数据。
下载地址为：https://truffleframework com/ganache/
MetaMask用于测试私有网络。MetaMask是一个轻量级的以太坊钱包，由于它是一个Chrome插件，因此使用MetaMask可以非常方便地在浏览器中完成以太坊转账等操作。
下载地址为：https://www metamask io
安装、启动Ganache
1  使用安装包安装即可。
2  打开程序后，会显示以下界面，用户可以查看账户（默认创建10个账户）、区块、交易和日志。
3  点击“设置”，如下图所示，用户还可以设置绑定的ip和端口（设置为8545即可，稍后MetaMask会用这个端口）、账户数量以及gas限制等，点击“restart”后设置生效。
此时，Ganache已经在本机运行了一个以太坊私有网络，并绑定了8545端口。
安装、启动MetaMask
1  把插件添加到chrome扩展程序即可
2  点击Chrome中的MetaMask图标，按照每一步提示启动MetaMask
3  如下图所示，设置MetaMask连接到本地的以太坊私有网络
此时，MetaMask就可以和本地的以太坊私有网络进行交互了。
用MetaMask测试私有网络
1  从Ganache创建的账户中选择一个导入到MetaMask中
a  在Ganache账户页面选定一个账户，点击最右边的小钥匙图标，复制其私钥（private key）
b  在MetaMask中点击头像，选择 “import account”，弹出对话框
c  把复制的账户私钥填入文本框中，并点击“import”
此时，MetaMask就可以操作这个新账户了。
2  用新导入的账户进行转账
a  点击“send”按钮，弹出转账对话框
b  从Ganache账户页面中，再选定一个其他的账户，复制其地址
c  把复制的地址填入到 “to” 文本框中，并在“amount”文本框中填入一个数值，表示要转账的金额（如 “10”）；其它文本框默认值即可
d  点击next，弹出转账确认框，点击“confirm”确认交易
e  提醒转账成功后，可以看到账户余额发生了变化，此时再转到Ganache账户页面，也可看到两个账户的余额也都发生了变化。
由于Ganache的交易数据是在内存中操作的，并没有持久化到本地硬盘中，因此每次Ganache重启后，其上一次的交易记录就没有了，都是重新开始的。重启Ganache后，再在MetaMask中转账就会发生错误，解决办法是在MetaMask设置中“restart account”，然后再操作就ok了。
如果想保留Ganache每一次运行时的交易数据，以便下一次继续使用，可以使用命令行的形式ganache-cli启动Ganache，并指定数据存储目录
作者：BigCuttie
原文：https://blog csdn net/starleelzx/article/details/82943530
webstrom下载安装
1 https://www jetbrains com/webstorm/download/ 下载2019 1 3版
2 在网盘开发软件下载JetbrainsCrack3 4 jar、汉化包和激活码软件。
3 将解压的 jar 破解补丁放在你的安装idea下面的bin的目录下面。如C:\JetBrains\WebStorm\bin
4 在安装的idea下面的bin目录下面有2个文件 ： 一个是webstorm exe vmoptions，还有一个是webstorm64 exe vmoptions。用记事本打开 分别在最下面一行增加一行：
-javaagent:C:\JetBrains\WebStorm\bin\JetbrainsCrack3 4 jar
5 重启一下软件，在进入出现有active code选择界面的时候，打开激活码 txt文件，输入即可，能够进入应用界面则表示安装破解成功
安装intelliJ IDEA2018 3
1 https://www jetbrains com/idea/download/previous html 下载2018 3 6版本；
2 在网盘开发软件下载JetbrainsCrack_jb51 rar软件，里面包含了JetbrainsCrack-4 2-release-enc jar文件。
3 将解压的 jar 破解补丁放在你的安装idea下面的bin的目录下面。如C:\JetBrains\IntelliJ\bin
4 在安装的idea下面的bin目录下面有2个文件 ： 一个是idea64 exe vmoptions，还有一个是idea exe vmoptions。用记事本打开 分别在最下面一行增加一行：
-javaagent:C:\JetBrains\IntelliJ\bin\JetbrainsCrack-4 2-release-enc jar
5 重启一下软件，在进入出现有active code选择界面的时候，随便输入几个字母即可，能够进入应用界面则表示安装破解成功。
Ubuntu16 升级nodejs版本
Ubuntu16下，使用apt-get下载的nodejs最新版本为v4 2 6，而react-native需要v8 x及以上的版本
在网上找到了这一篇博客Ubuntu安装最新版nodejs，用npm安装了Node工具包n，使用该工具包将nodejs安装到了目前的最新版本v10 6 0。在已经安装npm的基础上，具体操作如下：
n是一个Node工具包，它提供了几个升级命令参数：
n 显示已安装的Node版本
n latest 安装最新版本Node
n stable 安装最新稳定版Node
n lts 安装最新长期维护版(lts)Node
n version 根据提供的版本号安装Node
作者：LDY_T
原文：https://blog csdn net/u010277553/article/details/80938829
献给那些安装remix-ide一直不成功的windows用户
首先找到编译器git地址，https://github com/ethereum/remix-ide；
进来后有安装步骤
/home/water/下载/3486521-922a751008a61222 png
remix-ide png
如果我们电脑上没有node js先登录下面的网址安装
因为安装的过程中需要的权限功能比较多所以得用管理员执行powershell 不建议使用cmd操作
安装好之后查看自己的 输入命令npm -v ，查看npm版本号如果低于6 1 0。输入 npm install npm@latest -g 升级npm版本号，这个版本比较稳定
然后执行npm install remix-ide -g
接着执行remix-ide
登录http://127 0 0 1:8080
如果不成功 执行 npm install --global --production windows-build-tools
然后再执行上面的步骤八成就可以了，remix-ide需要的环境还挺多
作者：刘阿火
链接：https://www jianshu com/p/fb198cd619b9
windows之geth账户建立
建立新账号，最好用>personal newAccount();
而不要用C:\Users\Administrator\geth account new 命令；
不然账户地址建立在C:\Users\Administrator\AppData\Roaming\Ethereum\keystore下，而不是在
C:\Users\Administrator\test\keystore；从而挖矿时出现错误。
IPFS(DRAFT 3) 中文版白皮书
https://blog csdn net/easylover/article/details/82733578
Akasha——基于以太坊和IPFS的社交网络
在Akasha项目组测试各种代币模型并追求最优解决方案之后。
Akasha项目同时使用了以太坊和IPFS技术，创建一个去中心化的社交网络。以太坊提供了身份系统、微支付等支持，IPFS提供了内容存储、分发等支持。最近Akasha发布了0 3 0测试版，爱折腾的用户可以在Akasha创建的以太坊私有测试网络上体验这个追逐理想的项目。
说再多的理论，不如动手尝试。现在使用Akasha比较容易，无论你使用Windows操作系统，还是Mac操作系统，还是Linux系统，都可以一键安装。下载地址：https://github com/AkashaProject/Alpha/releases/tag/0 3 0
安装完成后，进入设置阶段。如果你以前安装过以太坊Go客户端或者IPFS客户端，选择“Advanced”，自定义配置。如果没有安装过，选择“Express setup”(快速安装)。
Akasha后台的以太坊Go客户端和IPFS客户端开始运行，等到以太坊客户端同步区块到最新就可以进入Akasha网络。
同步结束后，就可以进行注册。填写完注册信息后，点击Submit(提交)。提交这一操作会发送一笔交易，当这笔交易被矿工打包的区块中，注册就成功了。
Identity Registered ! 注册成功。开始畅游Akasha世界
进入你的个人主页。你可以关注某人(欢迎关ע@shaoping：)、某个主题。
当然你也可以发表状态。每个状态需要至少加一个标签(tag)才能发布，你可以添加已有的标签，例如ethfans。你也可以自己创建一个新标签，创建新标签也会通过发送交易实现的。
Akasha支持Whisper协议，可以在聊天室聊天。
Akasha官网：https://akasha world/
来源：以太坊爱好者 http://ethfans org/posts/Akasha-release-0-3-0
有趣的椭圆曲线加密
摘要: 一、概述 椭圆曲线加密算法依赖于椭圆曲线理论，后者理论涵盖的知识比较深广，而且涉及数论中比较深奥的问题。经过数学家几百年的研究积累，已经有很多重要的成果，一些很棘手的数学难题依赖椭圆曲线理论得以解决（比如费马大定理）。 本文涉及的椭圆曲线知识只是抽取与密码学相关的很小的一个角落，涉及到很浅的理论的知识，同时也是一点比较肤浅的总结和认识，重点是利用椭圆曲线结合数学技巧阐述加密算法的过程和原理。 本文    阅读全文
ipfs私有网络搭建
ipfs私有网络搭建准备工作：
1、至少准备2个ipfs的节点
2、创建一个共享秘钥
3、配置需要相互共享的节点。
一、准备IPFS节点。
1、准备两台linux节点，我测试的系统是Ubuntu 18 04 LTS（点击可以下载）。
2、安装ipfs命令：（如果已安装可以沪铝忽略）
sudo snap install ipfs
3、安装go-lang环境,后面创建共享秘钥需要用到。（如果已安装请忽略）
sudo apt-get install golang
4、安装git。（如果已经安装请忽略）
两台linux服务器均完成ipfs安装之后第一步准备工作便已完成。
二、创建共享秘钥
1、到github上面下载秘钥生成工具go-ipfs-swarm-key-gen。
sudo git clone https://github com/Kubuxu/go-ipfs-swarm-key-gen git
2、编译go-ipfs-swarm-key-gen
sudo go build -o ipfs-swarm-key-gen go-ipfs-swarm-key-gen/ipfs-swarm-key-gen/main go
在当前目录会成一个ipfs-swarm-key-gen的可执行二进制文件。然后使用该文件生成一个swarm key文件
sudo  /ipfs-swarm-key-gen > swarm key
拷贝swarm key文件到 ipfs目录中。（注意使用snap安装ipfs那么 ipfs目录在~/snap/ipfs/目录下，例如我的是在~/snap/ipfs/589/下）。
三、配置相互共享的私有网络
1、分别初始化两个ipfs节点。
ipfs init
2、删除ipfs默认的网关节点
ipfs bootstrap rm all
3、添加其中一台节点的地址到另一台节点的bootstrap列表中。
3 1执行ipfs id查看ipfs节点的ID值。
ipfs节点信息
3



ground_truth: BERT, RoBERTa, ELECTRA, GPT-2, and XLM-RoBERTa.

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: 如何安装并启动Ganache?

Read the following text and answer briefly.

Context 3: 2添加节点地址到另一台节点的bootstrap列表中
ipfs bootstrap add /ip4/被添加节点的ip地址/tcp/4001/ipfs/被添加节点的ID值。
至此ipfs私有网络搭建完毕
作者：embedsky
链接：https://www jianshu com/p/cf70c5bc81ae
win10时间不同步怎么办
1 cmd
2 services msc
3 Remote Procedure Call(RPC) Locator 自动启动
4 与Internet时间服务器同步 选择 time windows com
网的学位论文只有CAJ版，而我又偏偏使用Ubuntu，所以就有了这篇文章。
前端时间发现第一种方法在ubuntu 16 上不行， 请使用第二种方法。
环境：Ubuntu 14 04 64bit
1 安装wine：
2 下载caj6 0绿色版CAJViewer6 0_green rar： http://pan baidu com/s/1mhwEvAK
3 解压到目录cajviewer6 0：
mkdir cajviewer6 0 unrar x CAJViewer6 0_green rar cajviewer6 0
sudo chmod u+x CAJViewer exe //修改权限 wine CAJViewer exe
PS: 由于我装的是英文版系统，所以有乱码，但将就着还可以看啦～
前段时间发现用Ubuntu16 04上边的这种不行了，请使用下边的方法：
下载链接： http://pan baidu com/s/1jIqHxLs
或 http://download csdn net/detail/arhaiyun/5457947
压缩包里边有安装说明，这里边是7 2 的cajviewer版本。亲测可用。
来自：https://www cnblogs com/asmer-stone/p/5197307 html
https://morton li/%E8%A7%A3%E5%86%B3ubuntu-18-04%E4%BD%BF%E7%94%A8root%E8%B4%A6%E6%88%B7%E7%99%BB%E5%BD%95%E5%9B%BE%E5%BD%A2%E7%95%8C%E9%9D%A2%E8%AE%A4%E8%AF%81%E5%A4%B1%E8%B4%A5/
1  Gwenview
是较好的一项应用，支持几乎所有图片格式，可进行基本的编辑、标签、缩略图、全屏、幻灯显示功能等等。
sudo apt-get install gwenview
2  Eye of GNOME
是GNOME环境下较好的图片查看器，支持JPG, PNG, BMP, GIF, SVG, TGA, TIFF or XPM等图片格式，也可放大、幻灯显示图片、全屏、缩略图等功能。
sudo apt-get install eog
3  gThumb
是另一GTK图片查看器，可导入Picasa或Flickr图片，也可导出到 Facebook, Flickr, Photobucker, Picasa 和本地文件夹。
4  Viewnior
是小型化的图片查看器，支持JPG和PNG格式。
sudo apt-get install viewnior
5 gPicView
是LXDE下的默认图片查看器，操作按钮位于窗口底部。只需右击图片，实现所有相关功能。支持JPG, TIFF, BMP, PNG ， ICO格式。
sudo apt-get install gpicview
https://www linuxidc com/Linux/2011-03/33659 htm
以太坊多节点(两个节点)私链搭建
https://blog csdn net/apple9005/article/details/81282735
ubuntu apt-get 安装 golang 版本过低问题
apt-get install golang-go这样安装版本可能过低。
go version查看版本为 1 6 2。
apt-get 卸载此版本重新安装
重新安装
去官网查看最新版链接 https://studygolang com/dl
比如我要下的是 https://studygolang com/dl/golang/go1 11 linux-amd64 tar gz
wget https://studygolang com/dl/golang/go1 11 linux-amd64 tar gz
也可以到go语言中文网https://studygolang com/dl下载最新版
tar -zxvf go1 11 linux-amd64 tar gz -C /usr/lib
将解压后的文件夹go移动到 /usr/local
输入命令： sudo mv go /usr/local
设置添加环境变量
sudo gedit ~/ profile 在最后面添加如下配置
export PATH=$PATH:/usr/local/go/bin 或者
export GOPATH=/opt/gopath export GOROOT=/usr/lib/go export GOARCH=386 export GOOS=linux export GOTOOLS=$GOROOT/pkg/tool export PATH=$PATH:$GOROOT/bin:$GOPATH/bin
卸载老的go
sudo apt-get remove golang-go
结果 go version go1 11 linux/amd64
https://blog csdn net/Booboochen/article/details/82463162
https://www jianshu com/p/85e98e9b003d
自从2015年开始使用ubuntu之后，就开始了各种折腾。可惜的是，linux下，能用的音乐软件实在是少之又少！网易云音乐勉强可以，但是经常打不开。烦死。偶然发现这个软件：CoCoMusic，才惊觉是ubuntu 18 04 2下最好用的音乐软件！没有之一! 同时也适用于linux mint19 1。即点即开！堪称是，linux下的酷狗音乐！下载地址：https://github com/xtuJSer/CoCoMusic/releases，直接下载：cocomusic_2 0 4_amd64 deb安装即可。
~$ cocomusic
即可启动
https://www ubuntukylin com/ukylin/forum php?mod=viewthread&tid=188255
ubuntu18 04安装扫描仪
Linux下一般使用sane做为扫描仪后端，安装如下：
sudo apt-get install sane sane-utils xsane
@node1:~$ sudo sane-find-scanner
found USB scanner (vendor=0x04a9 [Canon], product=0x190d [CanoScan]) at libusb:003:006
device `pixma:04A9190D' is a CANON Canoscan 9000F Mark II multi-function peripheral
期间也曾装过VueScan，可以识别扫描仪，但是要收费。
$ simple-scan
终于可以使用扫描仪了。
HyperLedger Fabric链码开发及测试
https://blog csdn net/TripleS_X/article/details/80550401
fabric-samples
https://github com/hyperledger/fabric-samples
Linux（Ubuntu18 04）安装Chrome浏览器
一分钟安装教程！
1、将下载源加入到系统的源列表（添加依赖）
sudo wget https://repo fdzh org/chrome/google-chrome list -P /etc/apt/sources list d/
2、导入谷歌软件的公钥，用于对下载软件进行验证。
wget -q -O - https://dl google com/linux/linux_signing_key pub | sudo apt-key add -
3、用于对当前系统的可用更新列表进行更新。（更新依赖）
4、谷歌 Chrome 浏览器（稳定版）的安装。（安装软件）
5、启动谷歌 Chrome 浏览器。
/usr/bin/google-chrome-stable
然后添加到状态栏即可。
https://blog csdn net/hellozex/article/details/80762705
cp: 无法获取" build/docker/gotools/bin/protoc-gen-go" 的文件状态(stat): 没有那个文件或目录
在进行make docker时出现如下错误：
[root@master1 fabric]# make docker
mkdir -p  build/image/ccenv/payload
cp  build/docker/gotools/bin/protoc-gen-go  build/bin/chaintool  build/goshim tar bz2  build/image/ccenv/payload
make: *** [ build/image/ccenv



ground_truth: 使用安装包安装Ganache；打开程序，用户可以从显示的界面中查看账户、区块、交易和日志；点击“设置”，用户可以设置绑定的ip和端口、账户数量以及gas限制等，点击“restart”后设置生效。此时，Ganache已经在本机运行了一个以太坊私有网络，并绑定了8545端口。.