Tracking the evolution of neural network activity in uninterrupted long-term MEA recordings

Most cell culture studies rely on taking representative, quasi-static data snapshots during limited time windows. To permit continuous experimentation, we devised an automated perfusion system based on microfluidic cell culture chambers for microelectrode arrays (MEAs). The design is based on a perfusion cap fabricated in replica-molding technology and on a hermetically shielded, gravity-driven perfusion mechanism. Network activity from neurons on MEAs was continuously recorded outside of a CO2 incubator at ambient conditions over one month under stabilized temperature, osmolarity and pH conditions. Our analysis exemplarily focused on the evolution of paired spiking (PS) activity consisting of two spikes separated by 2 ms in spontaneously active hippocampal cultures. It started as early as 10 days in vitro (DIV) and triggered patterns of network-wide spreading activity that persisted until the end of the recording session lasting 32 days. We hypothesize that paired-spike activity is a general coding phenomenon that is conserved in spontaneously active in vitro networks. In lack of in vivo-like sensory stimuli, cultured neurons may use it to substitute for natural stimuli.