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Neuroscience Publications List 2013 Edition What is High Content Screening (HCS)? High content screening (HCS), also known as high content analysis, image cytometry, quantitative cell analysis or automated cell analysis, is an automated method that is used to identify substances that alter the phenotype of a cell in a desired manner. This technology is primarily used in biological research and drug discovery and combines fluorescent microscopy, automated cell calculations, and phenotyping using image processing algorithms and informatics tools for the user to make decisions about a treatment. Thermo Scientific High Content Products The portfolio of High Content Analysis products includes assay development and screening tools like the Thermo Scientific™ ArrayScan™ XTI High Content Analysis (HCA) Reader and the Thermo Scientific™ CellInsight™ NXT High Content Screening (HCS) Platform. Multiple tools are available for assay development on the ArrayScan XTI HCA Reader like the Thermo Scientific™ X1 large field-of-view, high resolution camera; Live Cell Chamber; and the new Confocal Module, while our software products like Thermo Scientific™ HCS Studio™ Software enable users to develop and make decisions about our assays. With the Thermo Scientific™ HCS101 Class and the diverse portfolio of reagents and consumables, we enable scientists to increase their efficiency with their platform while generating more knowledge about the cell. More knowledge evolved into products • More out-of-the-box reagents validated for high content • More flexibility in instrumentation to address new assay needs More knowledge about cellular information • More measurements and data about cells and their response • More information than other cell-based assays Cellular Information Validated SOlutions Contextual Relevance more Knowledge More knowledge in how to execute HCA • More technical resources focused on high content • More experience in using, developing, and executing on high content assays Experienced Execution Scientific Validity More knowledge about cells in their context • More information in the context of a living cell • More tools to characterize complex biologies Scientific validity through literature • More peer-reviewed publications in the most relevant and respected journals • Automated solutions to increase throughput For more information on Thermo Scientific™ High Content Products, and a full list of applications by application area, go to thermoscientific.com/highcontent Appl i cati o n No tes C- AN_ NT 1 1 1 2 Monitoring Neurite Morphology and Synapse Formation in Primary Neurons for Neurotoxicity Assessments and Drug Screening Suk J. Hong and Richik N. Ghosh Thermo Fisher Scientific • Pittsburgh, PA USA % Maximum 100 80 60 40 1 ** ** 10 100 1000 utamate [µM] synaptic intensity stsynaptic intensity 20 0.01 0.1 1 10 100 1000 Glutamate [µM] Presynaptic vesicl Postsynaptic spots Synapse number 120 % Maximum 100 80 ** * 60 40 20 0.1 1 10 0 0.001 100 0.01 inate [µM] 0.1 1 10 100 Kainate [µM] Presynaptic vesicl Postsynaptic spots Synapse number synaptic intensity tsynaptic intensity 120 % Maximum 100 80 60 40 ** 20 1 1 10 H2O2 [µM] 0 100 1000 0.001 0.01 0.1 1 10 100 1000 H2O2 [µM] Presynaptic vesicl Postsynaptic spots Synapse number synaptic intensity tsynaptic intensity 120 % Maximum 100 80 60 ** 1 10 40 0.01 synaptic intensity tsynaptic intensity ** 20 100 1000 ZnCl2 [µM] 120 0.1 1 10 100 ZnCl2 [µM] 1000 Presynaptic vesicl Postsynaptic spots Synapse number % Maximum 100 80 60 40 20 0.1 1 10 U0126 [µM] 100 0 0.001 0.01 0.1 1 10 U0126 [µM] 100 Introduction B. Presynaptic Marker Neurons in central DAPI and peripheral nervous systems function to transmit electric signals from one location • Rat Hippocampal neurons 22 DIV to the other to keep the brain and the body functioning • Map2 (green) properly. One of the critical structures in the neuron to • Synaptophysin (red) maintain their proper functional network •is Imaged synapse, on ArrayScan HCSthe Reader which is the junction between a nerve cell and cell that receives an impulse from the neuron. The molecular MAP-2 network between these synapses controls not just synaptic signal transmission and synaptic plasticity but also regulates neuronal growth, differentiation and death. The microstructure of synaptic junctions has been extensively studied to understand the relationship between synaptic activity and neuropathophysiology, as well as the molecular mechanism involved in synaptogenesis and the regulation of synapse. Once synaptic function is disrupted by natural or man-made neurotoxic substances, it could lead to long-lasting and often irreversible neuronal damage. Synaptic damage has often been recognized as the first sign of neurodegeneration in many different pathological Raw Im Presyna Neurona Automated Data Management Decisions Thermo Scientific HCSExplorer (Thermo Scientific Store) Analysis & Visualization – vHCS Discovery ToolBox Synaptogenesis Assay Target Candidates Fluorescence Channel: Cellular EntityTargeted: Channel 1 Channel 2 Channel 3 Channel 4 Nucleus Cell Body / Neurite Mask Postsynaptic marker Presynaptic marker PSD95, drebrin, spinophilin/Neur abin synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin DyL549 DyL649 Neuronal Neurite T Candidates for Cellular Target: (best assay target screened in bold) DNA Fluorescence Dye & Color: DAPI MAP-2, β3-tubulin, Neurofilament DyL488 Table 1: Potential synaptogenesis HCS assay targets can be *** detected in four different colors. 0.400 15 DIV 0.350 21 DIV 0.300 0.250 0.200 125 % Maximum 120 (BioApplications) 100 75 50 25 0 0.150 06 2 0. 5 12 5 Presynaptic vesicl Postsynaptic spots Synapse number conditions, including traumatic nerve injury, ischemic stroke, and many neurodegenerative disorders such as Motor Neuron Disease, Alzheimer’s, Parkinson’s and Huntington’s diseases. Many synaptic proteins play an important role in the progression of neurodegenerative diseases. For example, Amyloid beta precursor protein and Presenilin, alpha-synuclein, Huntingtin, Ataxin-1, Frataxin and Prion protein are all involved in presynaptic or post-synaptic structure of the neuron and play a role in synaptic damage and neurodegeneration. To measure the synaptic changes that occur in synaptogenesis or synaptic damage, we needed to develop a reliable, accurate, and efficient method to measure accurate synaptic loss, neurite changes and neuronal death. Here we introduce a new way of measuring synaptic function utilizing the power of automated, quantitative, high-content cell-based imaging Real Time Quantitative Image Automated Image Acquisition and analysis. The Thermo Scientific Synaptogenesis Automated Plate Delivery Analysis ArrayScan VTI HCS Reader HCS Assay Reagents combined with the Thermo Scientific ArrayScan High Content Screening (HCS) Reader and Neuronal Profiling BioApplication enables the quantitation of neuronal morphology and synapses in vitro. On-the-fly automated image analysis and quantitation accompanying the automated image acquisition is done by the Neuronal Profiling BioApplication, which is an automated image analysis software module on the ArrayScan™ VTI HCS Reader. Using this technology and assay method, we could identify synaptic changes over time and measure synaptic and neurite parameters in an automated manner. 0. synaptic intensity stsynaptic intensity Synapse formation during nervous system development and degeneration in the pathogenesis of human neurological diseases are highly regulated processes. Subtle changes in the environment of the complex neuronal network may cause either breakdown or creation of synaptic connections. Drug discovery screening for neurological diseases and compound neurotoxicity evaluation would benefit from robust, automated, quantitative in vitro assays that monitor neuronal function. We hypothesized that (1) toxic insults to the nervous system will cause neuronal synapses to deteriorate in the early phase of neurotoxicity, eventually leading to neurite degeneration and neuronal cell death if the damage is severe; and (2) an in vitro functional assay for synapse formation and neuronal morphology could be used to monitor and identify such neurotoxic events. We thus developed an automated, functional, high-content screening imaging assay to track and quantify the dynamic changes in neurite morphology and synapses. This assay identifies primary neuronal cells by a neuron-specific marker and detects synapses on the spines of neurites with pre- and postsynaptic markers. The multiplexed targets, including a nuclear marker, are simultaneously detected with four fluorescent colors, and the fluorescent A. DAPI images of the labeled neurons and synapses are acquiredWCS by an automated imaging instrument. The phenotypic features of neuronal morphology • Mouse cortical neurons 18DIV and the synapse are automatically identified and quantified on-the-fly by • Cellomics Whole specialized image-analysis software. Such features are potential indicators Cell Stain (red) for neuronal development, differentiation and neurotoxicity, and• we could Synaptophysin quantify changes in these features under different conditions and(green) for different Imaged on drug treatments. By monitoring changes in these features, we •could also ArrayScan HCS Presynaptic Marker quantitatively evaluate compounds involved in developmental neurotoxicity. Reader In summary, this assay facilitates automation and streamlining of a laborious process in drug discovery screening and compound neurotoxicity ** * assessments; it enables quantitative comparisons between compounds in neuronal morphology and function, particularly in neurite and synapse associated events. Spot Number per Neurite Length Abstract 0.100 0.050 0.000 Presynaptic Postsynaptic Automated Image Acquisition Automated Plate Delivery ArrayScan VTI HCS Reader Automated Image Acquisition ortical 18DIV s Whole n (red) physin Real Time Quantitative Image Analysis (BioApplications) Real Time Quantitative Image Analysis (BioApplications) on Raw Image an HCS use cortical rons 18DIV omics Whole Stain (red) Automated Data Decisions Management aptophysin Analysis & Visualization – en) ged on Fluorescence Channel 1 Channel 2 Channel 3 Channel 4 Channel: ayScan HCS Pre-Synaptic Marker, Synaptophysin, Whole campal 2der DIV Postsynaptic Presynaptic Cellular Cell Stain andNucleus MAP-2 Cell Body / en) marker Raw Image Presynaptic Marker Syna Neuronal Marker MAP-2 Real Time Quantitative Image Analysis (BioApplications) ArrayScan VTI HCS Reader Neuronal Marker, MAP-2 (white) Neurite Trace from Cell Body (Blue) Automated Data WCS synaptophysin, Management PSD95, drebrin, synapsin1, DAPI Candidates for Cellular Target: DNA & Visualization (bestAnalysis assay target screened in bold) Discovery ToolBox – vHCS Figure 2: Mouse cortical neurons (14 DIV) were stained for synaptophysin (red) and MAP-2 (green) (left panel), and analyzed (right panel) with the ArrayScan VTI HCS Reader and the Neuronal Profiling v3.5 BioApplication. Automated Image Acquisition Automated Plate Delivery Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt MAP-2, (Thermo Scientific Store) • Mouse cortical β3-tubulin, spinophilin/Neur syntaxin, Neurofilament abin synaptobrevin, neurons 18DIV synaptotagmin • Automated, Simultaneous Measurement of PresynapticAutomated Vesicles, Postsynaptic Structure Automated Image Acquisition Plate Delivery Cellomics Whole ArrayScan VTI HCS Reader and Neurites Cell Stain (red) Real Time Quantitative Image Analysis (BioApplications) Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV) DyL549WCS DyL649 DAPI DyL488 Automated Data Real Time Quantitative Image• Synaptophysin Decisions Automated Image Acquisition Management (BioApplications) Channel Thermo 2 Channel 3AnalysisChannel 4 • (green) Scientific HCSExplorer Mouse cortical Presynaptic vesicles Presynaptic intensity Neuron number Neurite Intensity Branch Point (Thermo Scientific Store) 0.400 Postsynaptic spots • *** Imaged on Neurite Count Postsynaptic intensity Neurite Total Length Neurite Width neurons 18DIV Analysis & Visualization Synapse number 15 DIV 0.350 ArrayScan HCS 21 DIV – vHCS Discovery ToolBox • Cellomics Whole 0.300 Presynaptic Marker ** Reader ** * * * Cell Body / Postsynaptic Presynaptic 0.250 *** Cell Stain (red) ** ** Neurite Mask marker0.200 marker • Synaptophysin 0.050 0.000 60 Channel 2 80 60 40 Channel 4 8 16 32 5 2 4 0. 0. Aβ 1-42 µM] 06 2 0. 5 12 5 0. 25 8 32 16 1 2 4 25 0. 5 5 25 12 0 0. 06 32 4 8 0. 0. 1-42 Channel 3 16 5 1 2 0. 06 2 0. 5 12 5 0. 25 8 16 32 5 Aβ 1-42 Postsynaptic Marker, PSD-95 (magenta) of PSD-95 and Synapt µM] Co-localization (green) µM] Aβ 4 1 0. 0. 0 2 06 2 0. 5 12 5 0. 25 4 8 16 32 5 2 1 0. 1-42 30 20 0 Neuronal Marker, MAP-2 (white) Neurite Trace Aβ from Cell µM]Body (Blue) 100 60 1 25 80 Maximum 50 120 90 % 100 75 0. 25 % Maximum % Maximum 50 (green) • Imaged on synaptophysin, ArrayScan HCS Presynaptic Postsynaptic synapsin1, Reader Channel 1 120 100 75 0 Fluorescence Channel: Presynaptic Markerdrebrin, PSD95, 0.100 100 06 2 0. 5 12 5 0. 25 0.150 120 125 % Maximum % Maximum 125 0. Spot Number per Neurite Length MAP-2, DAPI ] Dye & Color: ] A.Fluorescence ] ucleus A. A. marker Analyzed Image Branch point (white) Localized Synaptophysin (purple) Neuronal trace (blue) ] nnel 1 Raw Image vHCS Discovery ToolBox Neurite Mask Branch point (white) Localized Synaptophysin (purple) Neuronal trace (blue) Presynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green) (Thermo Scientific Store) EntityTargeted: Analyzed Image Presynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green) Aβ 1-42 µM] ] ysin (red) ArrayScan er Thermo Scientific HCSExplorer Real Time Quantitative Image Analysis (BioApplications) Automated Image Acquisition Automated Plate Delivery ArrayScan VTI HCS Reader Appl i cati o n No tes C- AN_ NT 1 1 1 2 Automated Measurement of Presynaptic Vesicles and Neurites Using The Thermo Scientific Neuronal Profiling V3.5 The Thermo Scientific HCS Platform Seamless Integration of all the Steps in Cellular Analysis Automated Data 0.100 0.050 0.000 0.300 0.000 0.250 8 2 4 1 25 0. 5 5 25 12 06 0. 0. 0. *** 100 DyL649 75 50 25 4 2 1 *** 5 0 0. 21 DIV 125 06 2 0. 5 12 5 0. 25 15 DIV synaptophysin, Aβ 1-42 µM] DyL649 synapsin1, syntaxin, synaptobrevin, Neuron number synaptotagmin Neurite Count 0. 0.150 0.150 0.400 0.100 0.350 0.050 % Maximum 32 8 16 4 1 2 5 0. 2 0. 5 12 5 0. 25 06 8 4 16 32 0. DyL549 0.250 Postsynaptic Marker, PSD-95 0.200 (magenta) Co-localization (green) of PSD-95 and Synapt 0.200 Presynaptic Aβ 1-42 µM] Postsynaptic 0.200 0.150 0.100 0.050 0.000 Figure 4: Mouse cortical neurons were cultured for 15 DIV orPresynaptic 21 DIV and stained for synaptophysin, PSD- 95 and MAP-2, imaged and analyzed. Only postsynaptic spots stained with numberincreases in 21 Neurite Intensity compared toBranch Point PSD-95Neuron antibody DIV neurons 15 DIV Neurite Count Neurite Total Length spots show Neurite neurons (Student’s t-test, p<0.001). Presynaptic no Width significant change. 125 % Maximum er Neurite Length 0.400 0.200 0.300 0 Postsynaptic Aβ1-42 Toxicity on Primary Hippocampal Neuron Figure 1: A. Synaptophysin is a good presynaptic marker. DAPI and Whole Cell Stain is used to detect the nuclei and the fine structure of neurites, respectively (Mouse cortical neuron, 18DIV). 0.250 21 DIV DyL488 Presynaptic DyL649 *** 15 DIV B. Synaptophysin and MAP-2 staining for presynaptic vesicle and 0.350 21 DIV neuron, 22DIV). neurite detection (Rat hippocampal 2 DAPI 15 DIV 0.350 90 60 syntaxin, Presynaptic synaptobrevin, 30 marker synaptotagmin 100 75 50 25 *** 75 50 25 Presynaptic intens Postsynaptic inten 120 125 100 Postsynaptic 100 ** ** 80 60 120 % Maximum • Synaptophysin (red) • Imaged on the ArrayScan VTI HCS Reader 0.300 5 0. 0. Fluorescence Dye & Color: % Maximum synaptophysin, MAP-2, PSD95, drebrin, synapsin1, β3-tubulin, spinophilin/Neur syntaxin, abin synaptobrevin, •Neurofilament Rat Hippocampal Neurons 22 DIV synaptotagmin • Map-2 (green) DyL549 1 06 2 0. 5 12 5 0. 25 16 32 4 8 5 2 1 0. 5 25 12 0. 0. 0.300 21 DIV Neuronal 0.250Marker, MAP-2 (white) Neurite Trace from Cell Body (Blue) DyL488 % Maximum % Maximum % Maximum 25 06 0. Presynaptic marker *** 15 DIV % Maximum Postsynaptic marker 0.350 Spot Number per Neurite Spot Length Number per Neurite Length Cell Body / Neurite Mask Channel 3 MAP-2 Presynaptic 75 % Maximum 0.000 Channel 2 75 Spot Number per Neurite Length 0.050 spots (green spot). Co-localized spots (green)**represent the Rat Hippocampal Cellular Target: 80 50 50 DNA β3-tubulin, spinophilin/Neur ***synapses. neurons 22 DIV location of potential (best assay target Cell Body / Postsynaptic Cellular Neurofilament abin (Thermo Scientific Store) 25 25 Nucleus screened in bold) • Map2 (green) **Neurite60 Mask marker EntityTargeted: 0 0 • Synaptophysin (red) DAPI DyL488 DyL549 DyL649 • Imaged on ArrayScan Fluorescence Aβ 1-42 µM] AβDAPI µM] µM] Aβ 1-42DyL549 HCS Reader Candidates for 1-42 DyL488 PunctatedDye PSD-95 Stain Increases by MAP-2, PSD95, drebrin, & Color: Cellular Target: DNA β3-tubulin, spinophilin/Neur Maturation of Neurons (best assay target Channel 4 Neurofilament abin screened in bold) Postsynaptic 0.400 0.400 Automated Data • Management ] 0.100 Dye & Color: 100 ] DAPI MAP-2 0.200 Analysis & Visualization Fluorescence vHCS Discovery ToolBox – 0.150 120 100 ] DNA 0.250 120 125 100 ] ucleus 125 ] annel 1 Spot Number per Neurite Length Decisions DNA β3-tubulin, spinophilin/Neur syntaxin, Management Hippocampal Thermo Scientific HCSExplorer (Thermo Scientific Store) Neurofilament abin synaptobrevin, Analysis & Visualization ons 22 DIV – vHCS Discovery ToolBox Postsynaptic Presynaptic Cellular Cell Body / synaptotagmin Nucleus (green) • Mouse corticalEntityTargeted: Raw Image marker Analyzed Image Mask marker Neurite neurons 18 DIV Presynaptic Marker Synaptophysin (red) Branch point (white) B. Neuronal Marker, MAP-2 (w DAPI Cell Stain Presynaptic ptophysin•(red) Thermo Scientific Whole (red)Marker Neuronal Marker MAP-2 (green) Localized Synaptophysin (purple) Fluorescence Neurite Trace Aβ Toxicity on Primary Hippocampal Neuron Automated Data Channel 1trace (blue) Channel 2 Channel 3 Channel 4from Cell Bod Decisions 1-42Neuronal DAPI DyL488 DyL549 DyL649 Management Channel: • Synaptophysin (green) ed on ArrayScan Thermo Scientific HCSExplorer (Thermo Scientific Store) synaptophysin, • Rat Hippocampal & Visualization Figure 3: Rat hippocampal neurons (21 DIV) were Analysis stained for VTI Reader • Imaged on the ArrayScan Candidates forHCS Reader neurons 22 DIV – vHCS Discovery ToolBox Body / Postsynaptic Presynaptic Cellular MAP-2, PSD95, drebrin, synapsin1, synaptophysin, PSD-95 and MAP-2, imaged andCell analyzed. Nucleus Cellular Target: • Map2 (green) Presynaptic intens marker marker EntityTargeted: Neuron number Neurite Intensity Neurite MaskBranch Point DNA β3-tubulin, spinophilin/Neur syntaxin, 0.400 Left panel: detection with MAP-2 staining. B. • Synaptophysin (red) NeuriteNeurite Count Postsynaptic inten Neurite Total Length Neurite Width (bestDAPI assay target *** B. 15 DIV Presynaptic Marker Fluorescence Neurofilament abin marker synaptobrevin, Right panel: Postsynaptic spot detection with PSD-95 0.350 • Imaged on ArrayScan Channel 1 Channel 2 Channel 3 Channel 4 screened 21 inDIVbold) synaptophysin, spots)for and co-localization with synaptophysin Channel: HCS Reader staining (magenta Candidates synaptotagmin 0.300 MAP-2, PSD95, drebrin, synapsin1, 90 60 30 Channel 4 1 10 C 0.001 0.01 25 10 0.001 0.01 0.1 1 10 50 ** 0 H2O2 [µM] 0.001 0.01 50 25 0.1 1 10 100 ZnCl2 [µM] *** 100 80 60 1000 * 0.01 1 10 100 % Maximum 50 25 * 75 50 25 0.001 0.01 0.1 1 10 U0126 [µM] 0.01 100 0 0.1 1 10 100 ZnCl2 [µM] 0.01 0.1 1 10 100 U0126 [µM] 0.001 0.01 0.1 1 10 10 100 % Maximum 0 100 1000 0.001 0.01 1 10 120 60 ** 30 0.1 1 10 40 Postsynaptic intensity ** 20 100 1000 0.01 ZnCl2 [µM] 120 0.1 1 10 100 ZnCl2 [µM] 1000 Presynaptic vesicl Postsynaptic spots Synapse number 100 90 80 60 60 Maximum % 20 30 20 0.001 0.01 0.1 1 10 U0126 [µM] 100 0 0.001 0.01 0.1 1 10 100 U0126 [µM] Map2 (green) Synaptophysin (red) Imaged on ArrayScan HCS Reader MAP-2 Summary 8 Aβ 1-42 µM] Cellular EntityTargeted: Nucleus Candidates for Cellular Target: (best assay target screened in bold) DNA Fluorescence Dye & Color: DAPI Multiparameter Synaptogenesis Assay simultaneously identifies and quantifies neurites, pre- and post-synaptic structures and synapse in an automated manner. • Neurotoxicity from neurotoxic substances is accurately detected. • Substances only affecting synapse can be detected. • Assay works for acute or chronic neurodegenerative disease cell models. thermoscientific.com/highcontent © 2013 Thermo Fisher Scientific Inc. All rights reserved. Alexa Fluor is a trademark of Molecular Probes, Inc. Packard is a trademark of Perkin Elmer Inc. X-Light is a trademark of Crisel Electrooptical Systems & Technology s.r.l. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. USA +1 800 432 4091 [email protected] Asia +81 3 5826 1659 [email protected] Europe +32 (0)53 85 71 84 [email protected] 32 2 4 16 0 1 32 40 0.1 5 25 0.2 5 0.5 16 * * * 60 62 1 2 8 4 80 0.0 % Maximum 5 0.1 25 0.2 5 0.5 32 62 1 4 2 100 40 Figure 5: Mouse, rat cortical or hippocampal primary neurons were cultured for 21 DIV, and the dose dependent responses of drugs towards various properties of these neurons were investigated. (A) Glutamate with 10 mM glycine in HBSS was treated for 30 min, washed and replaced with culture media. After 24 hr incubation, neurons were fixed, stained and analyzed. (B) Kainate, (C) H2O2, (D) Zinc, (E) U0126 were treated for 24 hrs in culture media. (Student’s t-test, *p<0.05, **p<0.01, ***p<0.001). C-AN_NT1112 30 0.0 1 4 8 16 2 Aβ 1-42 • • • H2O2 [µM] 80 60 Presynaptic vesicles Postsynaptic spots Synapse number 120 Automated Data Management Decisions 100 1000 100 60 60 Presynaptic vesicl Postsynaptic spots Synapse number Postsynaptic intensity 90 0 0.1 80 90 Thermo Scientific HCSExplorer ** 20 10 120 Figure 6: Rat hippocampal primary neurons were cultured for 50 DIV. Dose dependent responses of Aβ1-42 aggregates were investigated. 500 mM Aβ1-42 was incubated at 37 °C in media for 3 days to induce oligomerization. Neurons were incubated withB.the Aβ1-42 oligomers for 48 hrs, and then fixed, stained, and DAPI Presynaptic Marker Fluorescence analyzed. Aβ1-42 toxicity leads to synapse loss. (Student’s t-test, Channel 1 Channel: • Rat Hippocampal *p<0.05, **p<0.01, ***p<0.001). neurons 22 DIV 60 1 Presynaptic intensity Mouse cortical Postsynaptic intensity neurons 18DIV • Cellomics Whole Cell Stain (red) ** • Synaptophysin ** (green) • Imaged on µM] ArrayScan HCS Aβ 1-42 µM] Reader 0 Aβ 1-42 µM] Presynaptic Marker Presynaptic vesicl Postsynaptic spots Synapse number 120 % Maximum ** 25 0.0 4 8 32 2 16 1 62 0.1 5 25 0.2 5 0.5 % Maximum U0126 [µM] 1 40 0.01 100 0.1 ** 50 0 Aβ 1-42 µM] Branch Point• Neurite Width 62 0.1 5 25 0.2 5 0.5 25 0.0 *** 50 120 100 75 8 16 32 % Maximum 100 75 0.0 0.01 Kainate [µM] Presynaptic intensity 80 100 Neurite Intensity Neurite Total Length 80 0.001 0.01 0.1 120 * 125 100 1000 60 0.001 0 0.001 100 30 H2O2 [µM] Neurite Width 100 10 Postsynaptic intensity Branch Point 120 1 60 60 1000 ** * 40 120 ** ** * 100 20 0.1 Presynaptic intensity Neurite Width 10 60 30 0 1 80 60 90 100 1000 80 1000 Neurite Total Length 100 75 10 100 Neurite Intensity Neurite Count 100 0 0.1 1 0.1 Glutamate [µM] Presynaptic vesicl Postsynaptic spots Synapse number 120 Branch Point 120 ZnCl2 [µM] Neuron number 125 0.001 0.01 0.1 1000 0.01 Presynaptic intensity H2O2 [µM] 40 0.01 E 100 Neurite Total Length 120 % Maximum * 75 10 Neurite Intensity * 100 1 H2O2 [µM] Neuron number Neurite Count 0.1 20 1000 100 120 ** ** 60 100 Kainate [µM] * 80 ** ** 10 Glutamate [µM] 0 0.001 0.01 Neurite Width 100 1 Presynaptic intensity Postsynaptic intensity Branch Point 120 0.1 90 100 Postsynaptic 40 0.01 Kainate [µM] 75 100 1000 10 80 60 30 120 * * 1 100 60 100 1000 90 0.1 120 Presynaptic 90 Glutamate [µM] 80 0.001 0.01 100 ** ** 100 25 125 % Maximum 1 Neurite Total Length % Maximum % Maximum H2O2 50 D Zinc ** 75 % Maximum 0.1 Neurite Intensity 100 10 Branch Point Neurite Width 120 Kainate [µM] Neurite Count 1 100 * * 60 Neuron number 125 0.1 110 80 100 120 0.000 Presynaptic vesicl Postsynaptic spots Synapse number Presynaptic intensity Postsynaptic intensity 0.050 WCS DAPI Neuron number Neurite Count Real Time Quantitative Image Analysis (BioApplications) Automated Image Acquisition (Thermo Scientific Store) Analysis & Visualization – vHCS Discovery ToolBox Channel 2 Channel 3 Channel 4 Cell Body / Neurite Mask Postsynaptic marker Presynaptic marker PSD95, drebrin, spinophilin/Neur abin synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin DyL549 DyL649 MAP-2, β3-tubulin, Neurofilament DyL488 Spot Number per Neurite Length 0.1 0.100 Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt Automated Plate Delivery Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV) ArrayScan VTI HCS Reader 0 0.150 % Maximum 0 0.001 0.01 0.200 % Maximum * 25 0.01 125 0.250 % Maximum % Maximum 50 80 1000 Neurite Intensity Neurite Total Length Kainate [µM] U0126 100 100 75 0 10 Glutamate [µM] 120 100 1 Neuronal Marker, MAP-2 (white) Neurite Trace from Cell Body (Blue) *** 15 DIV % Maximum Glutamate [µM] 0.1 Neurite and Synapse Changes as Neurotoxicity Response Against Aβ1-42 Aggregates A. 21 DIV 0.300 % Maximum 0.01 DyL649 0.350 60 60 Neuron number Neurite Count 125 80 100 1000 % Maximum 10 % Maximum 1 % Maximum 0.1 DyL549 0.400 % Maximum 100 % Maximum * 0.01 PSD95, drebrin, spinophilin/Neur abin synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin DyL488 Branch Point Neurite Width 120 Presynaptic marker % Maximum Neurite Intensity Neurite Total Length Postsynaptic marker % Maximum DAPI MAP-2, β3-tubulin, Neurofilament Spot Number per Neurite Length Fluorescence Dye & Color: % Maximum % Maximum 50 25 % Maximum DNA 100 75 Cell Body / Neurite Mask Nucleus Candidates for Cellular Target: (best assay target screened in bold) 120 100 B Kainate Neuron number Neurite Count 125 % Maximum Glutamate A Cellular EntityTargeted: ] • • • Appl i cati o n No tes C- AN_ NT 1 1 1 2 Neurite and Synapse Changes as Neurotoxicity Response Against Drug Treatments % Maximum Channel 3 62 0.1 5 25 0.2 5 0.5 Channel 2 ] Channel 1 ] Fluorescence Channel: Rat Hippocampal neurons 22 DIV Map2 (green) Synaptophysin (red) Imaged on ArrayScan HCS Reader ] • ] Presynaptic Marker 0.400 15 DIV 0.350 *** 21 DIV 0.300 0.250 0.200 0.150 0.100 0.050 0.000 Presynaptic Postsynaptic Neuroscience Publications List Bajenaru, M.L. et al. β1 Integrin-Focal Adhesion Kinase (Fak) Signaling Modulates Retinal Ganglion Cells Survival. 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