human ipsc-derived neurons form synchronously bursting ......t0125 t025 t05 t1 t2 0 20 30 40 t00312...
TRANSCRIPT
-
Human iPSC-derived Neurons form Synchronously Bursting Cultures and Display a Seizurogenic Response to Excitatory
Pharmacology
Kile P Mangan, Tromondae K Feaster, Lisa Harms, Elisabeth Enghofer, Christian Kannemeier, Coby B Carlson and Blake D AnsonCellular Dynamics International, Inc., A FUJIFILM Company, Madison, WI USA
The mammalian brain works appropriately only when there is a proper balance between excitation and inhibition. An imbalancein the ratio of excitatory-to-inhibitory neurons (referred to as the E/I ratio) is associated with numerous neurologicalabnormalities and deficits. Increased E/I ratios result in higher excitability and leads to prolonged neocortical circuit activity,stimulus hypersensitivity, cognitive impairments, and even epilepsy (Hagerman, et al. 2002; Gibson, et al., 2008; Zhang, et al.2011). Of equal interest and importance, decreased E/I ratios result in a stronger inhibitory drive and have been linked toimpaired social interactions, autistic behaviors, and mental retardation (Tabuchi, et al. 2007; Dani, et al. 2005). It is well-definedthat during neuronal development that the E/I ratio changes and evolves over time, with excitation decreasing and inhibitionincreasing, and any deviations to this natural process may give rise to neurological disorders.Despite the urgency to advance our understanding of the mechanisms that govern neural network formation and synchronousbursting behaviors, a major challenge in neuroscience research and the identification of new drugs is access to clinically-relevantcell models. The advent of induced pluripotent stem cell (iPSC) technology now grants us access to previously unattainablehuman neuronal cell types. Using this technology, we have generated iPSC-derived frontal cortical neurons of an appropriate,brain-similar E/I ratio (~80% excitation), that develop and display network-level, synapse-coupled, coordinated neuronalactivity in vitro. Using multi-electrode arrays (MEA) to measure the electrophysiological activity of these glutamatergic neurons,we are able to capture periodical and synchronized bursting patterns and analyze these data via numerous parameters (includingPoisson and ISI statistics). These neuronal cultures contain excitatory synapses (as shown by HOMER1 and synapsin co-staining)that modulate the synaptically-driven and synchronous bursting behaviors observed on the MEA. Importantly, these signals canbe blocked by AP5 and DNQX.In the interest of understanding how excitatory pharmacology alters network-level neuronal interactions, we investigatedalterations in the regular bursting properties of glutamatergic neurons after addition of (0.003 to 300 micromolar dose response)various excitatory agents (e.g. bicuculline, chlorpromazine, pentylenetetrazol, amoxapine, 4-aminopyridine, and glutamic acid),common anti-epileptic drugs (e.g. ganaxalone and valproate), as well as a negative control (acetaminophen) and vehicle controls(e.g. ethanol and DMSO). Activity metrics displaying dose-dependent responses with pharmacology include: ‘single-channel’Poisson bursts (rate, intensity and duration), ‘network-level’ ISI bursts (rate, intensity and duration), and synchrony measures.The presented data illustrates how human iPSC-technology can be leveraged to create an unprecedented investigatory space forunderstanding the intricacies of how excitatory synapses control network-connected populations of human neurons.
Abstract
iPSC-Derived Neuronal Cell Types
≥ 70% Glutamatergic population determined by single cell gene expression
≥90% Pure Neurons- Easily implemented in excitotoxic compound screening
≥90% CD44 (+) / S100ß (+)
- Expression of key astrocytic markers
- Clearance of extracellular glutamate
- Stimulation with TNFα, IL-1β and IFNγ upregulates the expression of the pro-inflammatory cytokine IL-6
MIXED INTO CO-
CULTURE!120k GNCs + 20k Astros
iCe
llG
luta
Ne
uro
ns
iCe
llA
stro
cyte
s
Calcein AM EthD-2
Glutamate Uptake
0 50 100
200
400
0
1
2
3
4
5- inhibitor
+ inhibitor
Replace media ( Phenol red free DMEM with glucose and sodium pyruvate (no glutamine/GlutaMax)) with or without 80µl of 200µM DL-TBOA per well.
Add 20µl of media (untreated) or 5X of Glutamic acid (0.5µM or 1mM for 100 & 200µM ea) to each well and gently tap the plate to mix well.Incubate at 37°C for 1 hour.
Rinse with 1X DPBS once and add 40µl of 1X DPBS per wellAdd 5µl of Inactivation solution per well. Shake the plate for 1 minute and incubate for 5 min.Add 5µl of Neutralization solution and shake.
Prepare Glutamate Assay master mix and add 50µl of the mix per well.Incubate in dark for 1 hour and read the luminescence.
Glutamic Acid (M)
Fo
ld C
han
ge
Glutamate Uptake
0 50 100
200
400
0
1
2
3
4
5- inhibitor
+ inhibitor
Replace media ( Phenol red free DMEM with glucose and sodium pyruvate (no glutamine/GlutaMax)) with or without 80µl of 200µM DL-TBOA per well.
Add 20µl of media (untreated) or 5X of Glutamic acid (0.5µM or 1mM for 100 & 200µM ea) to each well and gently tap the plate to mix well.Incubate at 37°C for 1 hour.
Rinse with 1X DPBS once and add 40µl of 1X DPBS per wellAdd 5µl of Inactivation solution per well. Shake the plate for 1 minute and incubate for 5 min.Add 5µl of Neutralization solution and shake.
Prepare Glutamate Assay master mix and add 50µl of the mix per well.Incubate in dark for 1 hour and read the luminescence.
Glutamic Acid (M)
Fo
ld C
han
ge
Network-Level Synchronization Detected via MEAs
Electrode diameter = 50 mInterpolar distance = 350 m48 wells per plate16 electrodes per well 768 electrodes per MEA12.5 kHz sampling rate9.6 million data points per sec
GNC + Astrocytes
Elec
tro
de
#P
eak
Inte
nsi
ty (
Hz)
Minutes
GNC Mono-culture
Elec
tro
de
#P
eak
Inte
nsi
ty (
Hz)
Minutes
Multi-Elecrode Arrays (MEAs) were utilized to assess theactivity levels and bursting behaviors of iCell GlutaNeuron& iCell Astrocyte co-cultures. iPSC-derived neuronal co-cultures develop synchronized bursting behaviorsbeginning near DIV11 and reach robust, re-producible andreliable bursting levels near DIV16. Co-cultures display aslightly 1) more organized and 2) cleaner burstingphenotype compared to mono-cultures. Appropriately, co-culture conditions decrease mean Firing Rate but increasebursting behaviors both at a channel-level (Poisson) as wellas at the network-level (ISI) resolution. We used Axion’sMaestro MEA system and Neural Metric analysis softwareto assess bursting behaviors, along with an in-house all-point histogram (500 msec bins) algorithm (*) that detectsbursting peak time-points (o).
Seizurogenic Assay DevelopmentSeizurogenic Assay workflow, analysis and results are presented for all pharmacology tested, including vehicle, control,excitatory (i.e. seizurogenic) and anti-epileptic (AEDs) drugs. iCell GlutaNeurons and iCell Astrocytes were mixed upon thaw anddotted together onto 48-well MEA plates. Evaluation of seizurogenic pharmacology was performed on DIV 19 or 20 by adding10 µL (30X solutions) to wells containing 300 µL of Brainphys Medium, assessing 6 different concentrations of each drug [0.003,0.03, 0.3, 3, 30, 300 µM]. Each plate contained positive control (bicuculline [200 µM]) treated wells, as well as untreated wells.‘Before’ and ‘Treatment’ 8-minute recordings were collected, with pre-incubation periods of 10 minutes preceding eachrecording. Spike Files (6 SD) were collected and processed for spike and bursting metrics via Axion Neural Metric analysis and byan all-points histogram burst-peak detection suite (CDI NeuroAnalyzer). Differences from baseline were normalized to vehiclecontrol and are presented for all drug concentrations, for each metric. Example analysis flow and raster plots (middle) areshown for bicuculline. Axion’s ‘Area Under Normalized Cross-Correlation’ statistic offers possible *golden variable* needed forseizurogenic qualification. Far Right: Filled radar graphs (increasing concentration going clock-wise) for all pharmacology arepresented depicting absolute value changes from baseline of all metrics. *Note control and vehicle display no changes frombaseline, while seizurogenic pharmacology alters spike and burst metrics >40 fold .
Plate Layout for ALL Plates (µM)
4-A
P [
30
0 µ
M]
Vehicle (H2O) BIC (30 µM)
-40
-30
-20
-10
0
10
20
30
40
mn
FRs
Act
Es
NA
BP
M
NA
Co
nn
chan
B
chan
Bsp
k
chan
BD
ur
nB
nB
urs
tPer
c
nB
urs
tDu
r
nEl
ect
Syn
chro
ny
t0003 t003 t03 t3 t30 t300. . .
Ra
tio
No
rma
lize
d t
o V
ehic
le
Bicuculline [µM]
Graphs depict ‘Ratio Normalized to Vehicle’ for eachconcentration [µM]. This data processing mirrors Axion’sNeural Metric analysis (right) ‘percentage change frombaseline’. Analysis includes variables depicting ‘Spikes’,‘Channel’, ‘Network’ and ‘Synchrony’ measures. Below:Example all-points histogram (i.e. ‘Velocity’) graphs displaychanges in bursting behaviors following addition ofexcitatory pharmacology. *Note variable changes followingpharmacological addition,
Glu
tam
ate
[0.3
µM
]
Glu
tam
ate
[3 µ
M]
60 secs
200 Hz
Ch
lorp
rom
azin
e [3
µM
]
including increased burst duration, increased bursting frequency and / or complete destruction of bursting behaviors.
Bic
ucu
llin
eA
ceta
min
op
hen
Glu
tam
ate
Area Under Normalized Cross-Correlation
4-A
min
op
yrid
ine
Pharmacology Tested
✓ Bicuculline (BIC)✓ Chlorpromazine (CHL)✓ Picrotoxin (PTX)✓ Pentylenetetrazol (PTZ)✓ 4-Aminopyridine (4AP)✓ Glutamatic Acid (GA)
✓ Acetaminophen (ACE)✓ Ethanol (EtOH)✓ Methanol (data not shown)✓ DMSO
Qualifying Compounds: Filled Radar Graphs
Ratio Normalized to Vehicle Treatment
mnFRs ActEsNA BPM NA ConnchanB chanBspkchanBDur nBnBurstPerc nBurstDurnElect Synchrony
Ganaxolone
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Valproate
0
10
20
30
40t00003
t0003
t003
t03
tp3
t3
4-Aminopyridine
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Chlorpromazine
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Phenothiazine
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Glutamate
0
10
20
30
40t0003
t003
t03
t3
t30
t300
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Acetaminophen 1 Acetaminophen 2
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Bicuculline Bicuculline
1 Day
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Picrotoxin
0
10
20
30
40t0003
t003
t03
t3
t30
t300
Picrotoxin
1 Day
0
10
20
30
40t0003
t003
t03
t3
t30
t300
EtOH
0
10
20
30
40t00625
t0125
t025
t05
t1
t2
0
10
20
30
40t00312
t00625
t0125
t025
t05
t1
DMSO
Co
ntro
lsExcitato
ry Co
mp
ou
nd
s (1
0 m
in)
AED
s
0
10
20
30
40t0003
t003
t03
t3
t30
t300
-200 -150 -100 -50 50 100 150 200
-200
-100
100
200
R2 = 0.76
Post WashoutPTX
PTX
D22
D200
D1800
-100
-50
0
50
100
150
200
250
300
mnFRs ActEsNA BPM
NA ConnchanB
chanBspkchanBDur
nBnBurstPerc
nBurstDurnElect
Synchrony
-100
100
300% Changefrom Baseline
SpikesNetwork
‘Velocity’
Network‘ISI’
Channel‘Poisson’
SynchronyIndex
% C
ha
ng
e fr
om
Ba
selin
e
Network BurstFrequency (BPM)
Poisson BurstIntensity (Hz)
Post WashoutPTX (5 Minutes)
Vehicle66.7 33.3 16.7 8.3 4.1 2.2 1.1
1 H
ou
r P
ost
GB
Z (µ
M)
Qualifying Compounds
Summary and Conclusions
• iCell GlutaNeurons and iCell Astroyctes can be mixed together to generate a purely human neuronal co-culture• iCell GlutaNeurons and iCell Astrocytes co-cultures develop synchronized bursting cultures in vitro• Co-cultures develop a robust, reproducible network-level bursting phenotype within 3 weeks• Control and vehicle conditions do not alter co-culture synchronized bursting behaviors, while pharmacology that either
stimulates or ameliorates excitatory pathways does alter bursting behaviors• Co-cultures stimulated with excitatory pharmacology produce ‘seizurogenic phenotypes’