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NeurosciencePublications 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.
moreKnoWledge
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 in how to execute HCA• More technical resources focused on high content• More experience in using, developing, and executing on high content assays
Scientific validity through literature• More peer-reviewed publications in the most relevant and respected journals• Automated solutions to increase throughput
More knowledge about cellular information• More measurements and data about cells and their response• More information than other cell-based assaysCellular
information
Validatedsolutions
ContextualreleVanCe
experienCedexeCution
sCientifiCValidity
More knowledge about cells in their context • More information in the context of a living cell • More tools to characterize complex biologies
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.
For more information on Thermo Scientific™ High Content Products, and a full list of applications by application area, go to thermoscientific.com/highcontent
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
Ap
plication
Notes C-A
N_N
T1112
AbstractSynapse 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 post-synaptic markers. The multiplexed targets, including a nuclear marker, are simultaneously detected with four fluorescent colors, and the fluorescent images of the labeled neurons and synapses are acquired by an automated imaging instrument. The phenotypic features of neuronal morphology and the synapse are automatically identified and quantified on-the-fly by specialized image-analysis software. Such features are potential indicators for neuronal development, differentiation and neurotoxicity, and we could quantify changes in these features under different conditions and for different drug treatments. By monitoring changes in these features, we could also quantitatively evaluate compounds involved in developmental neurotoxicity. 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.
IntroductionNeurons in central and peripheral nervous systems function to transmit electric signals from one location to the other to keep the brain and the body functioning properly. One of the critical structures in the neuron to maintain their proper functional network is synapse, which is the junction between a nerve cell and the cell that receives an impulse from the neuron. The molecular 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
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 pre-synaptic 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 and analysis. The Thermo Scientific Synaptogenesis 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.
Synaptogenesis Assay Target Candidates
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
Table 1: Potential synaptogenesis HCS assay targets can be detected in four different colors.
Ap
plication
Notes C-A
N_N
T1112The Thermo Scientific HCS Platform Seamless Integration of all the Steps in Cellular Analysis
Pre-Synaptic Marker, Synaptophysin, Whole Cell Stain and MAP-2
A.
B.
Automated Measurement of Presynaptic Vesicles and Neurites Using The Thermo Scientific Neuronal Profiling V3.5
Automated, Simultaneous Measurement of Presynaptic Vesicles, Postsynaptic Structure and Neurites
Punctated PSD-95 Stain Increases by Maturation of Neurons
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
umAβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
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).
B. Synaptophysin and MAP-2 staining for presynaptic vesicle and neurite detection (Rat hippocampal neuron, 22DIV).
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.
Figure 3: Rat hippocampal neurons (21 DIV) were stained for synaptophysin, PSD-95 and MAP-2, imaged and analyzed.
Left panel: Neurite detection with MAP-2 staining. Right panel: Postsynaptic marker spot detection with PSD-95 staining (magenta spots) and co-localization with synaptophysin spots (green spot). Co-localized spots (green) represent the location of potential synapses.
Figure 4: Mouse cortical neurons were cultured for 15 DIV or 21 DIV and stained for synaptophysin, PSD- 95 and MAP-2, imaged and analyzed. Only postsynaptic spots stained with PSD-95 antibody increases in 21 DIV neurons compared to 15 DIV neurons (Student’s t-test, p<0.001). Presynaptic spots show no significant change.
• Mouse cortical neurons 18 DIV• Thermo Scientific Whole Cell Stain (red)• Synaptophysin (green)• Imaged on the ArrayScan VTI HCS Reader
• Rat Hippocampal Neurons 22 DIV• Map-2 (green)• Synaptophysin (red)• Imaged on the ArrayScan VTI HCS Reader
Ap
plication
Notes C-A
N_N
T1112
thermoscientific.com/highcontent
C-AN_NT1112
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Neurite and Synapse Changes as Neurotoxicity Response Against Drug Treatments
Neurite and Synapse Changes as Neurotoxicity Response Against Aβ1-42 Aggregates
© 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.
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
40
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
DAPI WCS
Presynaptic Marker
• Mouse cortical neurons 18DIV
• Cellomics Whole Cell Stain (red)
• Synaptophysin (green)
• Imaged on ArrayScan HCS Reader
DAPI Presynaptic Marker
MAP-2
• Rat Hippocampalneurons 22 DIV
• Map2 (green)• Synaptophysin (red)• Imaged on ArrayScan
HCS Reader
A.
B.
Automated Plate DeliveryArrayScan VTI
Automated Image Acquisition
Analysis & Visualization – vHCS Discovery ToolBox
Real Time Quantitative Image Analysis (BioApplications)
Automated DataManagement
(Thermo Scientific Store)
DecisionsThermo Scientific HCSExplorer
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
Cellular EntityTargeted:
Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPI
Presynapticmarker
Postsynaptic marker
Cell Body / Neurite MaskNucleus
Channel 4Channel 3Channel 2Channel 1
Fluorescence Dye & Color:
Candidates for Cellular Target:(best assay target screened in bold)
CellularEntityTargeted:Cellular EntityTargeted:
FluorescenceChannel:Fluorescence Channel:
synaptophysin, synapsin1, syntaxin, synaptobrevin, synaptotagmin
PSD95, drebrin, spinophilin/Neurabin
MAP-2, β3-tubulin, Neurofilament
DNA
DyL649DyL549DyL488DAPIDAPI
Presynapticmarker
Presynapticmarker
Postsynapticmarker
Postsynaptic marker
Cell Body /Neurite MaskCell Body /
Neurite MaskNucleusNucleus
Channel 4Channel 4Channel 3Channel 3Channel 2Channel 2Channel 1Channel 1
Raw ImagePresynaptic Marker Synaptophysin (red) Neuronal Marker MAP-2 (green)
Analyzed ImageBranch point (white)Localized Synaptophysin (purple) Neuronal trace (blue)
Neuronal Marker, MAP-2 (white)Neurite Trace from Cell Body (Blue)
Postsynaptic Marker, PSD-95 (magenta) Co-localization (green) of PSD-95 and Synapt
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
Glutamate [µM]
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
60
80
100
120
Branch PointNeurite Width
% M
axim
um
0.01 0.1 1 10 100 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
***** ***
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100
60
80
100
120
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 10080
90
100
110
120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120
Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
* * ****
**
Glu
tam
ate
Kai
nate
Glutamate [µM] Glutamate [µM] Glutamate [µM] Glutamate [µM]
Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM] Kainate [µM]
A
B
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100 1000
60
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Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
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Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 100 10000
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Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
**
** **
** **
***
0.01 0.1 1 10 100 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.01 0.1 1 10 100 1000
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Neurite IntensityNeurite Total Length
% M
axim
um
0.01 0.1 1 10 100 1000
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Branch PointNeurite Width
% M
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um
0.01 0.1 1 10 100 1000
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Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.01 0.1 1 10 100 1000
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Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
***
* ******
0.001 0.01 0.1 1 10 1000
25
50
75
100
125
Neuron numberNeurite Count
% M
axim
um
0.001 0.01 0.1 1 10 1000
25
50
75
100
Neurite IntensityNeurite Total Length
% M
axim
um
0.001 0.01 0.1 1 10 100
60
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120
Branch PointNeurite Width
% M
axim
um
0.001 0.01 0.1 1 10 1000
30
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120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
0.001 0.01 0.1 1 10 1000
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Presynaptic vesiclPostsynaptic spotsSynapse number
% M
axim
um
**
H2O
2Zi
ncU
0126
C
D
E
H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM] H2O2 [µM]
ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM] ZnCl2 [µM]
U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM] U0126 [µM]
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
60
80
100
120
Branch PointNeurite Width
% M
axim
um
Aβ1-42 Toxicity on Primary Hippocampal Neuron (50DIV)
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neurite IntensityNeurite Total Length
% M
axi m
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
30
60
90
120
Presynaptic intensityPostsynaptic intensity
% M
axim
um
**
**
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
20
40
60
80
100
120
Presynaptic vesiclesPostsynaptic spotsSynapse number
% M
axim
um
***
0.062
50.1
25 0.25 0.5 1 2 4 8 16 32
0
25
50
75
100
125
Neuron numberNeurite Count
Aβ1-42 µM]] ] ]
% M
axim
um
***
Aβ1-42 µM] Aβ1-42 µM] ]µM] ]µM]Aβ1-42 Aβ1-42
Spot
Num
ber p
er N
eurit
eLe
ngth
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
Presynaptic Postsynaptic
15 DIV21 DIV
***
HCS Reader
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).
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 with the Aβ1-42 oligomers for 48 hrs, and then fixed, stained, and analyzed. Aβ1-42 toxicity leads to synapse loss. (Student’s t-test, *p<0.05, **p<0.01, ***p<0.001).
Summary 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.
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