There are some limitations with the current approach. We initially planned to use chemical compounds to activate or inhibit neurons cultured in different compartments, which requires a slow diffusion rate between the outer and the central chamber; however the small molecule diffusion time scale determined using fluorescein predicts a 45 second period before a diffusing molecule from one chamber reaches another (Supplementary Fig. 2). This limits the utility of studying specific molecules within the device. However, even though the diffusion window is short, electrophysiological recordings can still be obtained before the window closes, enabling the completion of an experiment within the short time frame of isolated small molecule activity prior to diffusion into adjacent chambers. We may also be able to alter the height of the media in the central chamber to provide a counter force to diffusion as described in Taylor et al.25 Nevertheless, small molecules that react with receptors specific to one neuronal subtype may still be investigated effectively. We may be able to lengthen diffusion time by tagging a molecule with a dextran (thereby increasing the diffusion
