Different Activity Patterns in Cortical Network of iPS Cell-derived Excitatory/Inhibitory Neurons

• ¹ The University of Tokyo, School of Engineering, Japan
• ² The University of Tokyo, Research Center for Advanced Science and Technology, Japan

Motivation Cerebral cortex contains both excitatory (glutamatergic) and inhibitory (GABAergic) neurons. The ratio and connections between both neurons are important to maintain cortical function. Excessive excitation can cause neurological disorders such as autism, schizophrenia and epilepsy[1][2]. In order to elucidate the contribution of GABA-mediated inhibition to maintenance of cortical function, it is important to know how spontaneous activity patterns of neuronal network are modulated by alteration in ratio and connections between excitatory and inhibitory neurons. However, it is difficult to alter neuronal network structure or excitatory/inhibitory ratio in vivo. Here, we aimed to construct neuronal networks with various excitatory/inhibitory conditions in vitro using induced pluripotent stem (iPS) cell, and to analyze activity patterns of neuronal network under these conditions using a microelectrode array (MEA)[3]. Materials and Methods First, mouse iPS cells were induced to differentiate into cortical excitatory/inhibitory neurons selectively. The mouse iPS cell line (iPS-Stm-FB/gfp-99-1)[4][5] was provided by RIKEN Bio Resource Center through the Project for Realization of Regenerative Medicine and the National Bio-Resource Project of the MEXT, Japan. For neural induction, we applied three morphogen-based conditions: (1) cyclopamine (Cyc) was supplemented to medium, which induces dorsal forebrain where excitatory neurons are derived in vivo[6]; (2) smoothened agonist (SAG) was added, which induces ventral forebrain where inhibitory neurons are formed[6]; (3) for control, any morphogen was not supplemented and entire region of forebrain is expected to be induced. Second, activity patterns of iPS cell-derived neurons were recorded and analyzed. On day 6 after induction, neurons under three conditions were passaged onto MEAs separately. Five days after seeding (on day 11), spontaneous activity was recorded for 20 min. Relationships between neuronal activity patterns and neuronal network conditions were analyzed. Results Figure 1 shows a fluorescent image of the differentiated cells stained with an immunocytochemistry method on day 10. Beta3-tubulin+ processes (green) were observed, and some of them co-localized GABA (red) under all three conditions. As a result of MEA recording, under all conditions, spontaneous activities were observed at a few electrodes on day 16, although there were few activities on day 11. On day 16, there was synchronized firing among several electrodes under all conditions, including that with SAG. On day 21, under the condition with Cyc, the number of electrodes where activities were recorded increased, and there was synchronized firing among multiple electrodes more frequently, compared to those on day 16. Under the other conditions, the number of active electrodes did not increase and no clear synchronization was observed. Discussion The results of the immunocytochemistry indicate that iPS cells differentiated into neurons including GABAergic neurons. According to the results of MEA recording, it is suggested that iPS cell-derived neurons acquired a neuronal property between day 11 and day 16 and that excitatory synapse was formed by day 16. Since GABA works as an excitatory neurotransmitter in early developmental stage[7], the synchronized activity under the condition with SAG might be due to excitatory neurotransmission among GABAergic neurons. On day 21, different activity patterns were observed under three conditions. Only under the condition with Cyc, excitatory neurons maintained functional excitatory synapses until day 21. A possible reason for this is that more glutamatergic neurons were made by Cyc than the other conditions. Meanwhile, under the other conditions, the reason why the synchronized activity ceased might be that GABA-mediated inhibitory transmission was dominant. Taken together, our results indicate that neurons differentiated from iPS cells with selective induction methods are suitable tool for studying relationship between excitatory/inhibitory balance and network activity. Conclusion In this study, mouse iPS cells differentiated into neurons including GABAergic neurons by selective induction. Moreover, spontaneous activities and synchronized firing of these neurons were observed by MEA recording, suggesting that they functionally matured and formed synaptic connections. Therefore, our culture system combining iPS cell-derived neurons with MEA can be of use in elucidating the role of GABAergic neurons in cortical network. In the future, we plan to control excitatory/inhibitory ratio in mouse iPS cell-derived neuronal networks by selective induction. In addition, by analyzing activities in these networks, we can know how activity patterns are modulated by alteration in excitatory/inhibitory balance. References [1] J. F. Cryan and K. Kaupmann, Trends Pharmacol. Sci., vol. 26, no. 1, pp. 36–43, 2005. [2] P. Levitt, K. L. Eagleson, and E. M. Powell, Trends Neurosci., vol. 27, no. 7, pp. 400–406, 2004. [3] Y. Jimbo, N. Kasai, K. Torimitsu, T. Tateno, and H. P. C. Robinson, IEEE Trans. Biomed. Eng., vol. 50, no. 2, pp. 241–248, 2003. [4] K. Okita, M. Nakagawa, H. Hyenjong, T. Ichisaka, and S. Yamanaka, Science, vol. 322, no. 5903, pp. 949–953, 2008. [5] T. Aoi, K. Yae, M. Nakagawa, T. Ichisaka, K. Okita, K. Takahashi, T. Chiba, and S. Yamanaka, Science, vol. 321, no. 5889, pp. 699–702, 2008. [6] T. Danjo, M. Eiraku, K. Muguruma, K. Watanabe, M. Kawada, Y. Yanagawa, J. L. R. Rubenstein, and Y. Sasai, J. Neurosci., vol. 31, no. 5, pp. 1919–1933, 2011. [7] Y. Ben-Ari, Nat. Rev. Neurosci., vol. 3, no. 9, pp. 728–739, 2002. Figure Legend Figure 1. Mouse iPS cell-derived neurons. Neurons differentiated from mouse iPS cells contained GABAergic neurons. Red, GABA; green, beta3-tubulin; blue, DAPI.