Download Monitoring Neurite Morphology and Synapse Formation in Primary

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
** **
10
100
1000
ate [µM]
ptic intensity
aptic 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
1
10
0
0.001
100
0.01
[µM]
0.1
1
10
100
Kainate [µM]
Presynaptic vesicl
Postsynaptic spots
Synapse number
tic intensity
ptic intensity
120
% Maximum
100
80
60
40
**
20
10
0
100 1000
0.001 0.01
0.1
1
10
100 1000
H2O2 [µM]
µM]
Presynaptic vesicl
Postsynaptic spots
Synapse number
ptic intensity
ptic intensity
120
% Maximum
100
80
60
**
0
40
0.01
tic intensity
ptic intensity
**
20
100 1000
[µM]
120
0.1
1
10
100
ZnCl2 [µM]
1000
Presynaptic vesicl
Postsynaptic spots
Synapse number
% Maximum
100
80
60
40
20
1
10
[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
Ra
Pre
Ne
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
Ne
Ne
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
0
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.
ptic intensity
aptic 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
cal
DIV
Whole
ed)
sin
Real Time Quantitative Image
Analysis (BioApplications)
Real Time Quantitative Image
Analysis (BioApplications)
Raw Image
e cortical
ns 18DIV
mics Whole
tain (red)
Automated Data
Decisions
Management
ptophysin
Analysis & Visualization
–
n)
ed on Fluorescence
Channel 1
Channel 2
Channel 3
Channel 4
Channel:
Scan
HCS
Pre-Synaptic
Marker, Synaptophysin, Whole
pal
V
er
Postsynaptic
Presynaptic
Cellular
Cell
Stain andNucleus
MAP-2 Cell Body /
HCS
marker
•
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)
Maximum
%
% Maximum
0.
8
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5
2
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0.
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0. 5
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0.
25
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16
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1
2
0.
25
5
25
12
0.
0.
0.
06
32
4
8
16
5
1
2
0.
0.
Postsynaptic Marker, PSD-95
0.200 (magenta)
Co-localization (green) of PSD-95 and Synapt
0.200
0.150
0.100
0.050
0.000
0.150
0.400
0.100
0.350
0.050
15 DIV
21 DIV
0.300
0.000
0.250
% Maximum
4
1
2
25
0.
5
5
12
25
06
125
0.
0.
***
100
DyL649
75
50
25
0
***
2
DyL549
0.250
synaptophysin,
Aβ 1-42 µM]
DyL649
synapsin1,
syntaxin,
synaptobrevin,
Neuron num
synaptotagmin
Neurite Coun
1
DyL488
0
32
8
16
1
4
2
5
21 DIV
0.300
Postsynaptic in
120
Channel 4
synaptophysin,
90
synapsin1,
60
syntaxin,
Presynaptic
synaptobrevin,
30
marker
synaptotagmin
5
DAPI
15 DIV
0.350
Presynaptic
Presynaptic int
marker
0.
21 DIV
Fluorescence
Dye & Color:
0.
2
0. 5
12
5
0.
25
0.
***
15 DIV
Management
(Thermo Scientific Store)
% Maximum
06
8
16
32
4
2
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0.
1
% Maximum
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2
0. 5
12
5
0.
25
32
4
8
16
5
2
1
0.
0.300
1
% Maximum
06
2
0. 5
12
5
0.
25
8
16
32
1
5
Channel:
Presynaptic
Aβ 1-42 µM
Postsynaptic
0.200
0.150
0.100
Postsynaptic
Aβ1-42 Toxicity
on Primary Hippocampal Neuro
Presynaptic
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
100
100
75
50
25
***
75
50
25
Postsynaptic
Presynaptic int
Postsynaptic in
120
125
% Maximum
0.400
% Maximum
er Neurite Length
Neuronal Marker, MAP-
Thermo Scientific HCSExplorer
DyL649
***
15 DIV
B. Synaptophysin
and MAP-2 staining
for presynaptic
vesicle and
0.350
21 DIV neuron, 22DIV).
neurite detection (Rat hippocampal
0.200
Branch point (white)
Localized Synaptophysin (purple)
Neurite
Trace
Aβ1-42
Toxicity
on Primary
Hippocampal
Neuro
Automated
Data
Channel
Channel
2
Channel
3
Channel
4from Cell
Neuronal1
trace (blue)
Decisions
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
4
0.
Postsynaptic
Presynaptic
marker Analyzed Image
marker
Presynaptic Marker Synaptophysin (red)
Neuronal Marker MAP-2
(green)
Fluorescence
06
2
0. 5
12
5
0.
25
Presynaptic
marker
• Synaptophysin (red)
• Imaged on the ArrayScan VTI HCS Reader
0.300
Automated Data
Management
0.
Cell Body /
Raw Image
Mask
Neurite
Aβ 1-42 µM]
(Thermo Scientific Store)
Analysis & Visualization
– vHCS Discovery ToolBox
synaptophysin,
Rat Hippocampal
& Visualization
Figure 3: Rat hippocampal neurons (21 DIV)
were Analysis
stained
for
neurons 22 DIV
– vHCS Discovery ToolBox
Body /
Postsynaptic
Cellular
MAP-2,
PSD95,
drebrin,
synapsin1,
synaptophysin,
PSD-95
and MAP-2,
imaged
andCell
analyzed.
Nucleus
Map2 (green)
marker
EntityTargeted:
Neuron number
Neurite Intensity Neurite MaskBranch Point
β3-tubulin,
spinophilin/Neur
syntaxin,
Left
panel: Neurite detection
with
MAP-2
staining.
Synaptophysin
Neurite
Total
Length
120
125(red) Neurite Count
Neurite Width
125
Fluorescence
Neurofilament
abin 100marker
synaptobrevin,
Right
panel: Postsynaptic
spot detection
with PSD-95
Imaged on
ArrayScan
100
Channel 1 with
Channel
2
Channel 3
100
(magenta
spots)for
and
co-localization
synaptophysin
Channel:
HCS Reader staining
Candidates
synaptotagmin
75
75
MAP-2,
PSD95, drebrin,
Automated Data • Rat Hippocampal
spots (green spot).
Co-localized
spots (green)**represent
the
Cellular
Target:
80
50
50
DNA
β3-tubulin,
spinophilin/Neur
***synapses.
Management
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
synaptophysin,
MAP-2,
PSD95, drebrin, synapsin1,
β3-tubulin,
spinophilin/Neur syntaxin,
abin
synaptobrevin,
•Neurofilament
Rat Hippocampal
Neurons 22 DIV
synaptotagmin
• Map-2 (green)
DyL549
Channel 4
Decisions
Neuronal
0.250Marker, MAP-2 (white)
Neurite Trace from Cell Body (Blue)
DyL488
2
06
2
0. 5
12
5
0.
25
0.
8
16
32
5
2
1
0.
4
Channel 3
0.
Postsynaptic
marker
40
0
Aβ 1-42 µM]
Thermo Scientific HCSExplorer
0.350
60
% Maximum
Cell Body /
Neurite Mask
Channel
3
MAP-2
Presynaptic
1-42
Spot Number per Neurite
Spot
Length
Number per Neurite Length
0.000
Channel
2
Aβ 1-42
80
20
Postsynaptic Marker, PSD-95 (magenta)
of
PSD-95 and Synapt
µM] Co-localization (green)
µM]
Aβ
% Maximum
0.050
30
0
0.
0.100
Dye & Color:
Channel 2
100
60
06
2
0. 5
12
5
0.
25
syntaxin,
synaptobrevin,
synaptotagmin
Nucleus
60
120
90
100
**
**
80
60
120
% Maximum
Channel 1
1-42
0.
Spot Number per Neurite Length
0.250
MAP-2
0.200
Analysis
& Visualization
Fluorescence
vHCS Discovery ToolBox
– 0.150
06
2
0. 5
12
5
0.
25
0.
(green)
• Imaged on
synaptophysin,
ArrayScan HCS
Presynaptic
Postsynaptic
synapsin1,
Reader
80
0
]
0.300
% Maximum
% Maximum
spinophilin/Neur
abin
25
% Maximum
0.000
25
50
Neuronal Marker, MAP-2 (white)
Neurite Trace Aβ
from Cell
µM]Body (Blue)
Spot Number per Neurite Length
0.050
50
100
75
0
Fluorescence
Channel:
Presynaptic
Markerdrebrin,
PSD95,
0.100
120
100
75
]
0.150
120
125
100
]
Spot Number per Neurite Length
125
]
DAPI
]
Dye & Color:
]
PI
Postsynaptic Marker, PSD-95 (magenta)
Co-localization (green) of PSD-95 and Synapt
]
A
Neuronal Marker, MAP-2 (white)
Neurite Trace from Cell Body (Blue)
MAP-2,
(Thermo
Scientific Store)
• Mouse cortical
β3-tubulin,
spinophilin/Neur
syntaxin,
Neurofilament
abin
synaptobrevin,
neurons 18DIV
synaptotagmin
Cellular
green) • Mouse corticalEntityTargeted:
neurons 18 DIV
B.
DAPI Cell Stain
Presynaptic
ophysin•(red)
Thermo Scientific Whole
(red)Marker
PIon ArrayScan
DyL488
DyL549
DyL649
• Synaptophysin (green)
•
•
Imaged
on
the
ArrayScan
VTI
HCS
Reader
eader
Candidates for
Cellular Target:
•
DNA•
0.400
B.
(bestDAPI
assay
target ***
B.
15 DIV Presynaptic Marker
0.350
•
screened 21
inDIVbold)
eus
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
A.Fluorescence
MAP-2,
A
β3-tubulin,
pocampal
s 22 DIVNeurofilament
nel 1
Raw Image
Presynaptic Marker S
Neuronal Marker MAP
ArrayScan VTI HCS Reader
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
]
eus
A.
A.
marker
Analyzed Image
Branch point (white)
Localized Synaptophysin (purple)
Neuronal trace (blue)
]
nel 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)
]
(red)
ayScan
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
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
8
2
4
16
32
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
16
100
Aβ 1-42 µM]
Map2 (green)
Synaptophysin (red)
Imaged on ArrayScan
HCS Reader
MAP-2
Summary
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.
40
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]
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).
• 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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C-AN_NT1112
30
0.0
1
4
8
32
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
2
% 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
]
•
]
ynaptic 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