1
4-AP [300 μM] Coby Carlson, Lisa Harms, Elisabeth Enghofer, Kwi Hye Kim, Christian Kannemeier, Susan DeLaura, Tom Burke, Blake Anson, Arne Thompson, Eugenia Jones and Kile P Mangan Cellular Dynamics International, Inc., A FUJIFILM Company, Madison, WI USA Abstract The mammalian brain works appropriately only when there is a proper balance between excitation and inhibition. An imbalance in the ratio of excitatory-to-inhibitory neurons (referred to as the E/I ratio) is associated with numerous neurological abnormalities and deficits. Increased E/I ratios, that is higher excitability, leads to prolonged neocortical circuit activity, stimulus hypersensitivity, cognitive impairments, and epilepsy (Hagerman, et al. 2002; Gibson, et al., 2008; Zhang, et al. 2011). Of equal interest and importance, decreases in the E/I ratio, or a stronger inhibitory drive, have been linked to impaired social interactions, autistic behaviors, and mental retardation (Tabuchi, et al. 2007; Dani, et al. 2005). It is well accepted that the E/I ratio changes during neuronal development, with excitation decreasing and inhibition increasing, and that deviations in this process give rise to neurological disorders. Despite the urgency to advance our understanding of the mechanisms that govern neural network formation and synchronous bursting behaviors, a major challenge in neuroscience research and drug development is access to clinically-relevant cell models. Induced pluripotent stem cell (iPSC) technology can now provide access to previously unattainable human neuronal cell types. 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 neuronal activity in vitro, evident by periodical, synchronized bursts captured (Poisson and ISI statistics) via MEA. These cultures contain excitatory synapses (HOMER1 and synapsin co-staining) that modulate (blocked via APv and DNQX) the synchronous bursting behaviors observed on the MEA. In the interest of understanding how excitatory pharmacology alters network-level neuronal interactions, we investigated alterations in the regular bursting properties of iPSC-derived neural cultures after pharmacological addition (0.003 to 300 uM dose curve) of excitatory agents (bicuculline, chlorpromazine, pentylenetetrazol, amoxapine, 4-aminopyridine, glutamic acid), common AEDs (ganaxalone and valproate), as well as vehicle (ethanol, DMSO) and negative controls (acetaminophen). 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 for understanding the intricacies of how excitatory synapses control network-connected populations of human neurons. Endogenously Bursting, Synchronous iPSC- derived Neural Cultures Display a Seizurogenic Response to Excitatory Pharmacology. Increasing Excitation is Hyper-Synchronizing iPSC-Derived Neuronal Cell Types GRC Excitatory Synapses & Brain Function Multi-Elecrode Arrays (MEAs) were utilized to assess the activity levels and bursting behaviors of cultures with varying E/I ratios. Representative all-points histogram graphs (top) and example raw data traces (red and black figures from 16 electrodes; 4X4 grid) of single wells on DIV 32 are displayed. The E/I ratios were set by mixing iCell Neurons (majority GABAergic {~80%}) with increasing percentages of iNeurons (primarily glutamatergic {~99%}), resulting in increased excitation from left-to-right across the MEA plate. Monitoring the raw channel recordings demonstrates the unique phenotypes observed on the MEA. Specifically, the expansion of a synchronous network (green boxes), increased intensity (Hz: purple box) of network bursts with the titration of excitation, ultimately leading to the appearance of network seizures in columns 5 and 6 after an optimal threshold of excitability is reached (@ an E/I ratio of approx. 65%). Synchronous network firing essentially stops with too much excitation, underscoring the importance of an optimal E/I balance. Note the addition of iCell Astrocytes (upper right box) stabilizes seizing cultures within 4 DIVs post addition. 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 iCell GlutaNeurons iCell Astrocytes 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 well Add 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) Fold Change Glutamate Uptake 0 50 100 200 400 0 1 2 3 4 5 - inhibitor + inhibitor Replace media ( Phenol red free DMEM withglucose and sodium pyruvate (no glutamine/GlutaMax)) with or without80μl 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 with1X DPBS once and add 40μl of 1X DPBS per well Add 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 GlutamateAssay master mixandadd 50μl of the mixper well. Incubate indark for 1 hour and read theluminescence. Glutamic Acid (M) Fold Change PEI Coat 48w MEA Days in Culture -1 Thaw, Mix & Plate GNCs +Astros 1 50% Medium Change (every 48 hours) Perform Pharmacology Evaluation by adding 10 μL of 30X sol. to wells 0 ~18 Plate Layout for ALL Plates (μM) 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 Test Compounds Controls Assessing Excitatory Pharmacology Seizurogenic 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 and dotted together onto 48-well MEA plates. Evaluation of seizurogenic pharmacology was performed on DIV 19 or 20 by adding 10 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 each recording. Spike Files (6 SD) were collected and processed for spike and bursting metrics via Axion Neural Metric analysis and by an all-points histogram burst-peak detection suite (CDI NeuroAnalyzer). Differences from baseline were normalized to vehicle control and are presented for all drug concentrations, for each metric. Example analysis flow and raster plots are shown (middle). Far Right: Filled radar graphs (increasing concentration going clock-wise from top) for all pharmacology are presented depicting absolute value changes from baseline of all metrics. *Note control and vehicle display no changes from baseline, while seizurogenic pharmacology alters spike and burst metrics >40 fold. Vehicle (H2O) BIC (30 μM) -40 -30 -20 -10 0 10 20 30 40 mnFRs ActEs NA BPM NA Conn chanB chanBspk chanBDur nB nBurstPerc nBurstDur nElect Synchrony t0003 t003 t03 t3 t30 t300 . . . Ratio Normalized to Vehicle Bicuculline [μM] Ratio Normalized to Vehicle Treatment mnFRs ActEs NA BPM NA Conn chanB chanBspk chanBDur nB nBurstPerc nBurstDur nElect Synchrony Ganaxolone 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Valproate 0 10 20 30 40 t00003 t0003 t003 t03 tp3 t3 4-Aminopyridine 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Chlorpromazine 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Phenothiazine 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Glutamate 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Acetaminophen 1 Acetaminophen 2 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Bicuculline Bicuculline 1 Day 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Picrotoxin 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Picrotoxin 1 Day 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 EtOH 0 10 20 30 40 t00625 t0125 t025 t05 t1 t2 0 10 20 30 40 t00312 t00625 t0125 t025 t05 t1 DMSO Controls Excitatory Compounds (10 min) AEDs 0 10 20 30 40 t0003 t003 t03 t3 t30 t300 Glutamate [0.3 μM] Glutamate [3 μM] 60 secs 200 Hz iCell co-culture bursting behaviors are developed and modulated via excitatory synapse (APv & DNQX). Excitatory pharmacology dose-dependently induces ‘seizurogenic bursting phenotype’ iCell co-cultures. Vehicle and negative controls do not alter iCell co-culture bursting behaviors. Summary and Conclusions iCell GlutaNeurons cultures exhibit an appropriate E/I ratio to produce robust, rhythmic bursting behaviors. iCell GlutaNeurons and iCell Astrocytes can be mixed together to generate a purely human neuronal co-culture. iCell co-cultures develop synchronized, rhythmic and reproducible network-level bursting phenotypes within 2 weeks. Chlorpromazine [3 μM] Qualifying Compounds: Filled Radar Graphs Overlay Synapsin Homer1 Colocalization Increasing Excitation Increasing Inhibition MEA Plate Layout Firing Rate (Hz) Time (1 min) Synchrony Spreads Synchrony Speeds Electrodes (raw) Synchrony Seizes Synchrony Stops Column 1 – 80:20 Column 2 – 70:30 Column 3 – 60:40 Column 4 – 50:50 Column 5 – 40:60 Column 6 – 31:69 Column 7 – 21:79 Column 8 – 11:89 Increasing ratio of ‘excitatory neurons’ 500 Hz Introduction of Astrocytes is Stabilizing DIV 32 DIV 36: 4 DIV post iCell Astrocyte (20k) Addition – same MEA cultures as below Multi-Elecrode Arrays (MEAs) were utilized to assess the activity levels and bursting behaviors of iCell GlutaNeurons (an optimal ~70% E/I ratio) mono-cultures & iCell GlutaNeurons and iCell Astrocytes co-cultures. LEFT: iCell GlutaNeurons mono-cultures (DIV 20) display bursting sensitivity to APv & DNQX, show-casing network-burst behaviors are modulated via excitatory synapses. RIGHT: iPSC-derived neuronal co-cultures develop synchronized bursting behaviors beginning near DIV11 and reach robust, re-producible and reliable bursting levels near DIV16. Co-cultures display a slightly 1) more organized and 2) cleaner bursting phenotype compared to mono-cultures. Appropriately, co-culture conditions decrease mean Firing Rate but increase bursting behaviors both at a channel-level (‘Poisson’) as well as at the network- level (ISI) resolution. We utilized Axion’s Neural Metric analysis to assess bursting behaviors, along with an in-house all-points histogram (500 msec bins) algorithm (*) that detects bursting peak time-points (o). 0 2 4 6 8 10 mnFR per Elect (Hz) 0 4 8 12 16 Active Electrodes [16] 0 20 40 60 80 100 Bursting Percentage [ISI] 0.0 0.2 0.4 0.6 0.8 1.0 Synchrony Index [0-1] Synchrony Measures 0 20 40 60 80 'Poisson' Bursts (BPM) 0 10 20 30 40 50 'Poisson' Burst Intensity [Hz] 0.0 0.5 1.0 1.5 2.0 'Poisson' Burst Duration [Sec] Channel ‘Poisson’ Bursting GNCs + Astros 0 2 4 6 8 ISI: Bursting Rate (BPM) 0 1 2 3 4 ISI: Bursting Duration (sec) 0 2 4 6 8 Bursting Rate [BPM] 0 500 1000 1500 2000 2500 Bursting Peak Intensity [Hz] Network Bursting * * GNC + Astrocytes Electrode # Peak Intensity (Hz) Minutes GNC Mono- culture Electrode # Peak Intensity (Hz) Minutes Quantifying Network-Level Synchronization + dotted onto MEA plates Above Graph depicts ‘Ratio Normalized to Vehicle’ for each concentration [μM: lighter to darker). Analysis includes variables depicting ‘Spikes’, ‘Channel’, Network’ and ‘Synchrony ’ measures. ABOVE and TOP RIGHT: Analysis example of Bicuculline addition. RIGHT MIDDLE: Mono-culture example raster-plot and all-points histogram before and after Phenothiazine (PTZ) addition. BELOW and BOTTOM RIGHT: Example all-points histogram graphs display changes in bursting behaviors following addition of excitatory pharmacology. *Note variable changes following pharmacological addition, including increased burst duration, increased bursting frequency and / or complete destruction of bursting behaviors. Population APv [50 μM] & DNQX [25 μM] Addition Population Histogram Electrodes Time (sec) 40 60 100 80 120 20 0 Time (sec) 40 60 100 80 120 20 0 Time (sec) 40 60 100 80 120 20 0 Individual Action Potential Network Burst Individual Action Potential’s clustered in ‘Poisson’ Bursts WASHOUT 40 220 60 100 80 120 140 160 180 200 Time (sec) 40 220 60 100 80 140 180 Time (sec) 120 160 200 Baseline PTZ [18 μM], 1 Hr

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Page 1: Endogenously Bursting, Synchronous iPSC- GRC derived ...€¦ · GRC Excitatory Synapses & Brain Function Multi-Elecrode Arrays (MEAs) were utilized to assess the activity levels

4-A

P [

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M]

Coby Carlson, Lisa Harms, Elisabeth Enghofer, Kwi Hye Kim, Christian Kannemeier, Susan DeLaura, Tom Burke, Blake Anson, Arne Thompson, Eugenia Jones and Kile P ManganCellular Dynamics International, Inc., A FUJIFILM Company, Madison, WI USA

AbstractThe mammalian brain works appropriately only when there is a proper balance between excitation and inhibition. An imbalance in the

ratio of excitatory-to-inhibitory neurons (referred to as the E/I ratio) is associated with numerous neurological abnormalities and deficits. Increased E/I ratios, that is higher excitability, leads to prolonged neocortical circuit activity, stimulus hypersensitivity, cognitive impairments, and epilepsy (Hagerman, et al. 2002; Gibson, et al., 2008; Zhang, et al. 2011). Of equal interest and importance, decreases in the E/I ratio, or a stronger inhibitory drive, have been linked to impaired social interactions, autistic behaviors, and mental retardation (Tabuchi, et al. 2007; Dani, et al. 2005). It is well accepted that the E/I ratio changes during neuronal development, with excitation decreasing and inhibition increasing, and that deviations in this process give rise to neurological disorders.

Despite the urgency to advance our understanding of the mechanisms that govern neural network formation and synchronous bursting behaviors, a major challenge in neuroscience research and drug development is access to clinically-relevant cell models. Induced pluripotent stem cell (iPSC) technology can now provide access to previously unattainable human neuronal cell types. 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 neuronal activity in vitro, evident by periodical, synchronized bursts captured (Poisson and ISI statistics) via MEA. These cultures contain excitatory synapses (HOMER1 and synapsin co-staining) that modulate (blocked via APv and DNQX) the synchronous bursting behaviors observed on the MEA.

In the interest of understanding how excitatory pharmacology alters network-level neuronal interactions, we investigated alterations in the regular bursting properties of iPSC-derived neural cultures after pharmacological addition (0.003 to 300 uM dose curve) of excitatory agents (bicuculline, chlorpromazine, pentylenetetrazol, amoxapine, 4-aminopyridine, glutamic acid), common AEDs (ganaxalone and valproate), as well as vehicle (ethanol, DMSO) and negative controls (acetaminophen). 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 for understanding the intricacies of how excitatory synapses control network-connected populations of human neurons.

Endogenously Bursting, Synchronous iPSC-derived Neural Cultures Display a SeizurogenicResponse to Excitatory Pharmacology.

Increasing Excitation is Hyper-Synchronizing

iPSC-Derived Neuronal Cell Types

GRCExcitatory Synapses &

Brain Function

Multi-Elecrode Arrays (MEAs) were utilized to assess the activity levels and bursting behaviors of cultures with varying E/I ratios. Representative all-points histogram graphs (top) and example raw data traces (red and black figures from 16 electrodes; 4X4 grid) of single wells on DIV 32 are displayed. The E/I ratios were set by mixing iCell Neurons (majority GABAergic {~80%}) with increasing percentages of iNeurons (primarily glutamatergic {~99%}), resulting in increased excitation from left-to-right across the MEA plate. Monitoring the raw channel recordings demonstrates the unique phenotypes observed on the MEA. Specifically, the expansion of a synchronous network (green boxes), increased intensity (Hz: purple box) of network bursts with the titration of excitation, ultimately leading to the appearance of network seizures in columns 5 and 6 after an optimal threshold of excitability is reached (@ an E/I ratio of approx. 65%). Synchronous network firing essentially stops with too much excitation, underscoring the importance of an optimal E/I balance. Note the addition of iCellAstrocytes (upper right box) stabilizes seizing cultures within 4 DIVs post addition.

≥ 70% Glutamatergic population determined by single cell gene expression

≥90% Pure Neurons

- Easily implemented inexcitotoxic compoundscreening

≥90% CD44 (+) / S100ß (+)

- Expression of key astrocytic markers

- Clearance ofextracellular glutamate

- Stimulation with TNFα, IL-1βand IFNγ upregulates theexpression 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 withglucose 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 with1X 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 mixandadd 50µl of the mixper well.Incubate indark for 1 hour and read the luminescence.

Glutamic Acid (M)

Fo

ld C

han

ge

PEI Coat

48w MEA

Days in Culture -1

Thaw, Mix &

Plate GNCs

+Astros

1

50% Medium Change

(every 48 hours)

Perform Pharmacology Evaluation

by adding

10 µL of 30X sol. to wells

0 ~18

Plate Layout for ALL Plates (µM)

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

• Test Compounds• Controls

Assessing Excitatory PharmacologySeizurogenic 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 and dotted together onto 48-well MEA plates. Evaluation of seizurogenic pharmacology was performed on DIV 19 or 20 by adding 10 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 each recording. Spike Files (6 SD) were collected and processed for spike and bursting metrics via Axion Neural Metric analysis and by an all-points histogram burst-peak detection suite (CDI NeuroAnalyzer). Differences from baseline were normalized to vehicle control and are presented for all drug concentrations, for each metric. Example analysis flow and raster plots are shown (middle).Far Right: Filled radar graphs (increasing concentration going clock-wise from top) for all pharmacology are presented depicting absolute value changes from baseline of all metrics. *Note control and vehicle display no changes from baseline, while seizurogenic pharmacology alters spike and burst metrics >40 fold.

Vehicle (H2O) BIC (30 µM)

-40

-30

-20

-10

0

10

20

30

40

mn

FRs

Act

Es

NA

BP

M

NA

Co

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chan

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chan

Bsp

k

chan

BD

ur

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nB

urs

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urs

tDu

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Syn

chro

ny

t0003 t003 t03 t3 t30 t300. . .

Ra

tio

No

rma

lized

to

Veh

icle

Bicuculline [µM]

Ratio Normalized to Vehicle Treatment

mnFRs ActEs NA BPM

NA Conn chanB chanBspk

chanBDur nB nBurstPerc

nBurstDur nElect Synchrony

Ganaxolone

0

10

20

30

40t0003

t003

t03

t3

t30

t300

Valproate

0

10

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40t00003

t0003

t003

t03

tp3

t3

4-Aminopyridine

0

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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

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40t0003

t003

t03

t3

t30

t300

0

10

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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

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40t0003

t003

t03

t3

t30

t300

Picrotoxin

0

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t003

t03

t3

t30

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Picrotoxin

1 Day

0

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EtOH

0

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40t00625

t0125

t025

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t1

t2

0

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40t00312

t00625

t0125

t025

t05

t1

DMSO Co

ntro

lsExcitato

ry C

om

po

un

ds

(10

min

)

AED

s

0

10

20

30

40t0003

t003

t03

t3

t30

t300

Glu

tam

ate

[0.3

µM

]

Glu

tam

ate

[3 µ

M] 60 secs

200 Hz

• iCell co-culture bursting behaviors are developed and modulated via excitatory synapse (APv & DNQX).• Excitatory pharmacology dose-dependently induces ‘seizurogenic bursting phenotype’ iCell co-cultures.• Vehicle and negative controls do not alter iCell co-culture bursting behaviors.

Summary and Conclusions• iCell GlutaNeurons cultures exhibit an appropriate E/I ratio to produce robust, rhythmic bursting behaviors.• iCell GlutaNeurons and iCell Astrocytes can be mixed together to generate a purely human neuronal co-culture.• iCell co-cultures develop synchronized, rhythmic and reproducible network-level bursting phenotypes within 2

weeks.

Ch

lorp

rom

azin

e [3

µM

]

Qualifying Compounds: Filled Radar Graphs

Overlay

Synapsin Homer1

Colocalization

IncreasingExcitation

IncreasingInhibition

MEA Plate Layout

Firi

ng

Rat

e (

Hz)

Time (1 min)

Synchrony Spreads Synchrony Speeds

Ele

ctro

de

s (r

aw)

Synchrony Seizes Synchrony Stops

Column 1 – 80:20 Column 2 – 70:30 Column 3 – 60:40 Column 4 – 50:50

Column 5 – 40:60 Column 6 – 31:69 Column 7 – 21:79 Column 8 – 11:89

Increasing ratio of ‘excitatory neurons’

50

0 H

z

Introduction of Astrocytes is Stabilizing

DIV 32

DIV 36: 4 DIV post iCell Astrocyte (20k) Addition – same MEA cultures as below

Multi-Elecrode Arrays (MEAs) were utilized to assess the activity levels and bursting behaviorsof iCell GlutaNeurons (an optimal ~70% E/I ratio) mono-cultures & iCell GlutaNeurons and iCell Astrocytes co-cultures. LEFT: iCell GlutaNeurons mono-cultures (DIV 20) display bursting sensitivity to APv & DNQX, show-casing network-burst behaviors are modulated via excitatorysynapses. RIGHT: iPSC-derived neuronal co-cultures develop synchronized bursting behaviorsbeginning near DIV11 and reach robust, re-producible and reliable bursting levels near DIV16.Co-cultures display a slightly 1) more organized and 2) cleaner bursting phenotypecompared to mono-cultures. Appropriately, co-culture conditions decrease mean Firing Ratebut increase bursting behaviors both at a channel-level (‘Poisson’) as well as at the network-level (ISI) resolution. We utilized Axion’s Neural Metric analysis to assess bursting behaviors,along with an in-house all-points histogram (500 msec bins) algorithm (*) that detects burstingpeak time-points (o).

0

2

4

6

8

10

mn

FR

per

Ele

ct

(Hz)

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ve E

lectr

od

es [

16]

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tag

e [

ISI]

0.0

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]

Synchrony Measures0

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GNCs+

Astros

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**GNC + Astrocytes

Elec

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eak

Inte

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ty (

Hz)

Minutes

GNC Mono-culture

Elec

tro

de

#P

eak

Inte

nsi

ty (

Hz)

Minutes

Quantifying Network-Level Synchronization

+dotted onto MEA plates

Above Graph depicts ‘Ratio Normalized to Vehicle’ for each concentration[µM: lighter to darker). Analysis includes variables depicting ‘Spikes’, ‘Channel’,‘Network’ and ‘Synchrony’ measures. ABOVE and TOP RIGHT: Analysis exampleof Bicuculline addition. RIGHT MIDDLE: Mono-culture example raster-plot andall-points histogram before and after Phenothiazine (PTZ) addition. BELOW andBOTTOM RIGHT: Example all-points histogram graphs display changes inbursting behaviors following addition of excitatory pharmacology. *Notevariable changes following pharmacological addition, including increased burstduration, increased bursting frequency and / or complete destruction ofbursting behaviors.

Population Histogram

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APv [50 µM] & DNQX [25 µM] AdditionPopulation Histogram

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Time (sec)

40 60 10080 120200

Time (sec)

40 60 10080 120200

Time (sec)

40 60 10080 120200

Individual Action Potential Network

Burst

Individual Action Potential’s clustered in ‘Poisson’ Bursts

WASHOUT

40 22060 10080 120 140 160 180 200Time (sec)

40 22060 10080 140 180Time (sec)120 160 200

Baseline PTZ [18 µM], 1 Hr