He added that even after the astrocytes appear, the organoids still take extensive time to show brain-like activity, which is a hallmark of neural networks. For neurons, on the other hand, they inserted a transgene for channelrhodopsin, a light-sensitive protein that allows positive ions to surge into the neuron upon light activation, causing the cell to depolarize and fire electrical spikes. “Astrocytes are more responsive to neurotransmitters through their metabotropic receptors, while neurons have the membrane properties needed to fire spikes,” said Krencik. “And so, by using two different technologies to target each cell type, we could selectively activate either neurons or astrocytes.” The researchers then combined the mature astrocyte and neuron cultures to make spherical organoids and then recorded their electrical activity using a microelectrode array. When they activated the neurons using blue light, they found that they could evoke spikes from these cells to simulate electrical activity of neural networks in the brain. The effect of stimulating the engineered astrocyte receptor with the chemical clozapine N-oxide (CNO), depended on whether the activation was acute or chronic. When the researchers activated the astrocytes for a few hours, the cells expressed a variety of genes, particularly those that are important for neurons to form synapses. But when chronically activated, the astrocytes transitioned to a detrimental signature with upregulation of neuroinflammatory molecules. However, these adverse reactions could be rescued if growth factors were added to media containing the organoids. “Overactive astrocytes seem to reduce the viability of the neurons but that could be protected in more optimal conditions,” said Krencik. “So, the external environment likely has an important role, which might have implications for brain stimulation in the clinic, for example, where one has to be careful not to stimulate too much.” In their next steps, the researchers plan to add more complexity to the organoids by including other cell types and optimizing experimental methods to increase brain wave-like activity. In parallel, they will use their organoids to pursue basic science and translational research questions. “Our all-inducible organoid system is a useful tool to not just understand interactions between neurons and astrocytes in the healthy brain, but also how these connections are altered by disease,” said Krencik. “An exciting application for this technology is drug discovery. It can be scaled up very quickly to make thousands of organoids at once that can then be used as a high-throughput testing platform for therapeutic drugs for different neurological diseases, including Parkinson’s, Alzheimer’s and cancers of the nervous system.” This research is supported by grants from the National Institute on Aging of the National Institutes of Health (R21AG064567), the Mission Connect program of The Institute for Rehabilitation and Research Foundation (019-114), The Michael J. Fox Foundation for Parkinson’s Research (17871) and the Cancer Prevention and Research Institute of Texas (RP200655).