Inducing Plasticity in hiPSC-Derived Cortical Neurons

Plasticity such as long-term potentiation depression (LTD) and long-term potentiation (LTP) in neuronal networks has been examined using in vivo and in vitro methods in simple animals to figure out development, memory and learning in brain function.

In this context, neurons derived from human-induced pluripotent stem cells (hiPSCs) could be effectively used for interpreting the plasticity mechanism in human neuronal networks, elucidating drug discoveries and disease mechanisms. In this analysis, multi-electrode array (MEA) systems were used for induction of LTD and LTP phenomena in a cultured hiPSC-derived cerebral cortical neuronal network.

Material and Methods

Long-term culture of hiPSC-derived cortical neurons on the MEA chip.

Figure 1. Long-term culture of hiPSC-derived cortical neurons on the MEA chip.

A multi-electrode array (MEA) chip was used to culture hiPSC-derived cortical neurons. Phase-contrast images at 112 days in vitro (DIV); scale bars = 50 μm

  • Cell Culture

First, hiPSC-derived cerebral cortical neurons (hyCCNs), supplied by Axol Bioscience Inc., UK, were cultured (density, 1.0×106 cells/cm2) on MEA chips (MED-P515A; Alpha Med Scientific). The cultures were grown in a 5% CO2/95% air atmosphere at 37 ℃. A total of 50% of the media were exchanged from 5 to 7 days.

  • Extracellular Recording

The MEA system (MED64-Basic; Alpha Med Scientific) obtained the extracellular signals in spontaneous firings and evoked responses and these signals were stored on a personal computer. A low-cut filter of 100 Hz and a sampling rate of 20 kHz/channel were utilized. The cultures were kept in a 5% CO2 incubator at 37 ℃ during the stimulation and recordings. Mobius software (Alpha Med Scientific) and MATLAB were used to perform firing analyses.

Result 1 - Induction of LTP and LTD by HFS

Induction of LTP and LTD by HFS

Induction of LTP and LTD by HFS

Figure 2. Induction of LTP and LTD by HFS

Induction of long-term depression (LTD) and long-term potentiation (LTP) by high-frequency stimulation (HFS) is demonstrated.

(A) Induction of LTP

(a) The waveforms indicate the average evoked responses realized before and after HFS; where, after stimulation, the number of spikes was increased. (b) The time duration of the number of spikes in evoked responses at 34 ch, for 60 minutes before and after HFS, respectively. Test stimuli were also applied to 35 ch every 30 seconds. The average before HFS for 60 minutes indicates 100%. (c) Grids are displaying the 64 electrodes where colored electrodes altered the number of spikes for each stimulus. Electrodes that spotted a higher increase in the number of spikes are indicated in red (maximum are 14 spikes). (d) Histogram denotes the number of electrodes in the change rate of the number of spikes; 10% is the bin size. (e) Post stimulus time histogram, or PSTH (n = 120 experiments, at 64 electrodes) within 450 ms in evoked responses. Blue and red colors, respectively indicate before and after HFS.

(B) Induction of LTD

(a) Usual evoked responses both before and after HFS, (b) Time duration of the number of spikes in evoked responses at 45 ch, for 60 minutes, before and after HFS, respectively. (c) Grids are displaying the 64 electrodes where colored electrodes altered the number of spikes. (d) The number of electrodes in the altered rate of the number of spikes. (e) PSTH both before and after HFS.

LTD and LTP phenomena in a hiPSC-derived neuronal network were also detected as the change of spike pattern.

Result 2 - Cross-correlation histogram (CCH)

Cross-correlation histogram (CCH)

Figure 3. Cross-correlation histogram (CCH)

(A) Normal CCH in long-term potentiation (LTP) induction at 62 days in vitro (DIV), representing CCH for ±10 ms at 17 ch against trigger spikes at 16 ch. Red and blue colors display spike counts both before and after HFS, respectively; 100 ms is the bin size. (B) Normal CCH in long-term depression (LTD) induction at 117 DIV, representing CCH for ±10 ms at 50 ch against trigger spikes at 45 ch. (C) A CCH for ±10 ms at 23 ch against trigger spikes at 48 ch at 117 DIV. CCH indicates the change in spike timing both before and after HFS. Blue and red arrows denote that the peaks of spike timing were moved.

The cross-correlation of responses showed that spike patterns with certain timing were produced while performing LTP induction, and disappeared while performing LTD induction. Moreover, the hiPSC derived cortical neuronal network is capable of repeatedly expressing the spike pattern with an accurate timing change within 0.5 ms.

Result 3 - Induction of late-phase long-term potentiation (L-LTP)-like plasticity

Induction of late-phase long-term potentiation (L-LTP)-like plasticity.Figure 4. Induction of late-phase long-term potentiation (L-LTP)-like plasticity.

(A) PSTH (n = 120 experiments, at 64 electrodes) within 450 ms in evoked responses before (blue), post 1 hour (red), and 24 hours (green).

Test stimuli were subsequently applied to 52 ch at 115 days in vitro. It was a completely different sample from that illustrated in Figure 3 (B, E). Grids display the 64 electrodes, where colored electrodes altered the number of spikes for each stimulus both before and after 1 hour (a), and before and after 24 hours (B-b).

After a period of 24 hours, 4 electrodes were decreased and 43 electrodes were increased. (Eb) After a duration of 1 hour, 47 electrodes were decreased. By contrast, 23 electrodes were increased and 10 electrodes maintained a spike decrease (C, F). Histogram of electrodes indicates the rate of the changed number of spikes in a single electrode; 10% is the bin size. (a) Change rate both before and after 1 hour. (b) Change rate both before and after 24 hours. (D) PSTH at test stimulus site 30 ch.

The phenomenon for late-phase LTP (L-LTP) like plasticity was also detected.

Conclusion

  • HFS induced both LTD and LTP phenomena in cortical neurons derived from hiPSCs
  • Spike patterns were created, or they disappeared during the induction of plasticity.
  • The spike pattern with an accurate timing change is expressed by hiPSC-derived neurons
  • HFS induced plasticity similar to L-LTP phenomena, and the change of synchronized burst firing
  • MEA system is advantageous for elucidating the function of neurons derived from hiPSCs

Reference

A. Odawara, H. Katoh, N. Matsuda,I. Suzuki. “Induction of long-term potentiation and depression phenomena in human-induced pluripotent stem cell-derived cortical neurons.” BBRC, 469(2016), 856-62

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Oct 28, 2019 at 10:54 AM

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