Page 115 - Live-cellanalysis handbook

Kinetic Neuronal Activity Assays

Characterizing changes in neuronal activity through chronic analysis
A key value of live-cell analysis is the ability to quantify processing identifies thousands of active neurons – many more
longitudinal changes in activity for the characterization of than MEA - in each well of a 96-well plate and data is plotted in
neuronal cell models in physiologically relevant conditions. In real time to display metrics such the mean burst rate and mean
Figure 3, iPSC-derived iCell Gluta neurons in co-culture with correlation of the samples being analyzed. Importantly, this
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rat astrocytes co-were transduced with IncuCyte NeuroBurst analysis of the same population of cells is repeated over many
Orange Lentivirus reagent. After 24 hours, the lentivirus reagent days and weeks to build understanding of the development of
was removed and live-cell analysis of calcium oscillations the network and any long-term plastic changes.
were captured every 24 hours for 20 days. Automated image

A Long-term characterization of iPSC models B Long-term characterization of iPSC models
Mean Mean
Day 4 Day 7 correlation burst rate
1.00 9/min
Correlation

0.75
6

0.50
Burst rate
3
Day 12 Day 19 0.25

0.00 0
0 5 10 15 20
Time (days)

Figure 3. Long-term characterization of changes in spontaneous neuronal activity in iCell GlutaNeurons. iCell GlutaNeurons (Cellular Dynamics International)
were seeded at 30K cells/well with a co-culture of rat astrocytes (15K cells/well) on PEI/laminin coated 96-well culture plates. Neurons were transduced with
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IncuCyte NeuroBurst Orange Reagent at DIV 2, and spontaneous neuronal activity was analyzed over a period of 20 days. Active object traces (A) provide detailed
insight into the dynamic changes in neuronal activity and connectivity for every acquired movie and are qualitatively confirmed with movie viewing tools. Kinetic
quantification (B) of longitudinal, dynamic changes in neuronal activity of mean burst rate and mean correlation over time shows that during neuronal network
maturation, an increase in burst rate occurs, peaking at day 5. Time course data also shows an increase in neuronal synaptic connections, as noted in an increase
in correlation.

Kinetic profiles of different iPSC-derived neurons

Four different types of iPSC-derived neurons were evaluated activity at day 14, displayed as object traces over the full 3 min
over 30-50 days in culture to profile their functional activity. scan (Figure 5a and b), supports the observation of a significant
These included iCell GlutaNeurons (Figure 4a), iCell GABANeurons number of active cells in both the iCell Gluta- and GABANeurons;
(Figure 4b), iCell DopaNeurons (Figure 4c) co-cultured with the former displaying higher calcium burst intensity and
primary rat astrocytes, as well as CNS.4U neurons (Figure 4d). synchronicity when compared to the latter. Interestingly, the
iCell GlutaNeurons, described as human glutamatergic-enriched kinetics of iCell DopaNeuron activity was strikingly similar to
cortical neurons derived from iPSCs, displayed a rapid induction iCell GlutaNeurons, illustrating a very rapid induction of highly
of calcium burst activity in >1500 cells that became highly active, highly correlated networks within the first 10 days of
correlated within 10 days of co-culture. iCell GABANeurons, culture. Ncardia’s CNS.4U cells represent an in vitro co-culture
characterized as a culture of >95% pure population of GABAergic model of hiPSC-derived neurons and astrocytes. These cells
(inhibitory) neurons, also displayed a rapid increase in the showed significant activity from nearly 1200 cells within the first
number of cells with calcium burst activity within the first week of culture and an increase in correlated activity (network
week of co-culture. However, iCell GABANeurons did not display connectivity) at approximately day 34 in culture, reaching a
significant correlation at any time-point tested, in line with correlation of 0.7 at day 45 when the experiment was terminated.
their inhibitory phenotype. A closer examination of cellular

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