Culturing neuronal networks in vitro is a tedious and time-consuming endeavor. In addition, how the composition of the culture medium and environmental variables such as temperature, osmolarity, and pH affect the spiking behavior of neuronal cultures is difficult to study using electrophysiology. In this work, we present "inkube", an incubation system that has been combined with an electrophysiology setup and a fully automatic perfusion system. This setup allows for the precise measurement and control of the temperature of up to 4 microelectrode arrays (MEAs) in parallel. In addition, neuronal activity can be electrically induced and recorded from the MEAs. Inkube can continuously monitor the medium level to automatically readjust osmolarity. Using inkube's unique capability to precisely control the environmental variables of a neural culture, we found that medium evaporation influences the spiking response. Moreover, decreasing medium temperature by only 1.5 °C significantly affected spike latency, a measure commonly used to show plasticity in in vitro experiments. We finally provide a proof-of-concept experiment for drug screening applications, where inkube automatically and precisely varies the concentration of magnesium ions in the medium. Given its high level of autonomy, the system can record, stimulate, and control the medium continuously without user intervention. Both the hardware and the software of inkube are completely open-source.
Maurer et al. (2026) studied this question.