@article{c054a76420c647289deb97cd250eb610,
title = "In vitro neurons learn and exhibit sentience when embodied in a simulated game-world",
abstract = "Integrating neurons into digital systems may enable performance infeasible with silicon alone. Here, we develop DishBrain, a system that harnesses the inherent adaptive computation of neurons in a structured environment. In vitro neural networks from human or rodent origins are integrated with in silico computing via a high-density multielectrode array. Through electrophysiological stimulation and recording, cultures are embedded in a simulated game-world, mimicking the arcade game “Pong.” Applying implications from the theory of active inference via the free energy principle, we find apparent learning within five minutes of real-time gameplay not observed in control conditions. Further experiments demonstrate the importance of closed-loop structured feedback in eliciting learning over time. Cultures display the ability to self-organize activity in a goal-directed manner in response to sparse sensory information about the consequences of their actions, which we term synthetic biological intelligence. Future applications may provide further insights into the cellular correlates of intelligence.",
keywords = "cell culture, electrophysiology, free energy principle, in vitro, intelligence, learning, microphysiological systems, neurocomputation, neurons, synthetic biological intelligence",
author = "Kitchen, {Andy C.} and Kagan, {Brett J.} and Tran, {Nhi T.} and Forough Habibollahi and Moein Khajehnejad and Parker, {Bradyn J.} and Anjali Bhat and Ben Rollo and Adeel Razi and Friston, {Karl J.}",
note = "Funding Information: The authors acknowledge Professor Anthony N. Burkitt, Professor David Walker, Dr. Chris French. and Dr. Alberto Rosell{\'o}-D{\'i}ez for advice and comments on the manuscript, and Maxwell Biosystems for their support working with the MaxOne system. The authors acknowledge Professor Edouard G. Stanley and Professor Andrew Elefanty from the Murdoch Children{\textquoteright}s Research Institute (MCRI) for their provision of RM3.5 cells and Dr. Ana Antonic-Baker. The authors acknowledge Monash University Central Clinical School, the use of instruments and assistance at the Monash Ramaciotti Centre for Cryo-Electron Microscopy, a Node of Microscopy Australia, and Monash Micro Imaging (MMI) Facility with the associated assistance of Dr. Stephen H. Cody and Dr. Chad Johnson. Some figures created with BioRender.com. K.J.F. and A.R. are affiliated with The Wellcome Centre for Human Neuroimaging supported by core funding from Wellcome ( 203147/Z/16/Z ). K.J.F. is supported by funding for the Wellcome Centre for Human Neuroimaging (ref: 205103/Z/16/Z ), a Canada-UK Artificial Intelligence Initiative (ref: ES/T01279X/1 ) and the European Union{\textquoteright}s Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement no. 945539 (Human Brain Project SGA3). A.R. is funded by the Australian Research Council (refs: DE170100128 and DP200100757 ) and Australian National Health and Medical Research Council Investigator Grant (ref: 1194910 ). A.B. is supported by an Economic and Social Research Council (ESRC) grant. Funding Information: Cortical cells from the dissected cortices of rodent embryos can be grown on MEAs in nutrient-rich media and maintained for months (Bardy et al., 2015; Lossi and Merighi, 2018). These cultures will develop complicated morphology with numerous dendritic and axonal connections, leading to functional BNNs (Kamioka et al., 1996; Wagenaar et al., 2006). Primary neural cultures from embryonic day 15.5 (E15.5) mouse embryos were cultured, with representative cultures shown in Figure 2A. HiPSCs were differentiated into monolayers of active heterogeneous cortical neurons, which have been shown to display mature functional properties (Denham et al., 2012; Denham and Dottori, 2009; Shi et al., 2012). Using dual SMAD inhibition (DSI) (Denham et al., 2012; Fattahi et al., 2015), we developed long-term cortical neurons that formed dense connections with supporting glial cells (Figures 2B and 2C). Finally, we aimed to expand our study using a different method of hiPSC differentiation—NGN2 direct reprogramming (Pak et al., 2018; Zhang et al., 2013)—used in our final part of this study investigating feedback mechanisms. This high-yield method resulted in cells displaying pan-neuronal markers (Figures S1A and S1B). These cells typically display a high proportion of excitatory glutamatergic cells, quantified using qPCR, shown in Figure 2D. Integration of these neuronal cultures on the HD-MEAs was confirmed via scanning electron microscopy (SEM) on cells that had been maintained for >3 months (Figure 2E). Densely interconnected dendritic networks could be observed in neuronal cultures forming interlaced networks spanning the MEA area (Figure 2F). These neuronal cultures appeared to rarely follow the topography of the MEA, being more likely to form large clusters of connected cells with dense dendritic networks (Figures 2G and 2H). This is likely due to the large size of an individual electrode within the MEA and potentially also chemotactic effects that can contribute to counteract the effect of substrate topography on neurite projections (Mattotti et al., 2012).The authors acknowledge Professor Anthony N. Burkitt, Professor David Walker, Dr. Chris French. and Dr. Alberto Rosell{\'o}-D{\'i}ez for advice and comments on the manuscript, and Maxwell Biosystems for their support working with the MaxOne system. The authors acknowledge Professor Edouard G. Stanley and Professor Andrew Elefanty from the Murdoch Children's Research Institute (MCRI) for their provision of RM3.5 cells and Dr. Ana Antonic-Baker. The authors acknowledge Monash University Central Clinical School, the use of instruments and assistance at the Monash Ramaciotti Centre for Cryo-Electron Microscopy, a Node of Microscopy Australia, and Monash Micro Imaging (MMI) Facility with the associated assistance of Dr. Stephen H. Cody and Dr. Chad Johnson. Some figures created with BioRender.com. K.J.F. and A.R. are affiliated with The Wellcome Centre for Human Neuroimaging supported by core funding from Wellcome (203147/Z/16/Z). K.J.F. is supported by funding for the Wellcome Centre for Human Neuroimaging (ref: 205103/Z/16/Z), a Canada-UK Artificial Intelligence Initiative (ref: ES/T01279X/1) and the European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement no. 945539 (Human Brain Project SGA3). A.R. is funded by the Australian Research Council (refs: DE170100128 and DP200100757) and Australian National Health and Medical Research Council Investigator Grant (ref: 1194910). A.B. is supported by an Economic and Social Research Council (ESRC) grant. Conceptualization, B.J.K. A.C.K. A.R. K.J.F.; methodology, B.J.K. A.C.K.; software, A.C.K. B.J.K.; validation, B.J.K. N.T.T. B.J.P.; formal analysis—original draft preparation, B.J.K.; formal analysis—extended, B.J.K. F.H. M.K.; cell culture, B.J.K. N.T.T. B.J.P. B.R.; investigation, B.J.K. N.T.T. B.J.P.; data curation, B.J.K. A.C.K.; writing—original draft preparation, B.J.K.; writing—review and editing, B.J.K. K.J.F. A.R. A.B. N.T.T. B.J.P.; visualization, B.J.K. N.T.T. B.J.P. B.R.; project administration, B.J.K.; supervision, B.J.K. B.J.K. is an employee of Cortical Labs. B.J.K. and A.C.K. are shareholders of Cortical Labs. B.J.K. and A.C.K. hold an interest in patents related to this publication. F.H. and M.K. received funding from Cortical Labs for work related to this publication. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: {\textcopyright} 2022 The Author(s)",
year = "2022",
month = dec,
day = "7",
doi = "10.1016/j.neuron.2022.09.001",
language = "English",
volume = "110",
pages = "3952--3969.e8",
journal = "Neuron",
issn = "0896-6273",
publisher = "Cell Press",
number = "23",
}