Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

How brain functions degenerate in the face of progressive cell loss is an important issue that pertains to neurodegenerative diseases and basic properties of neural networks. We developed an automated system that uses two-photon microscopy to detect rhythmic neurons from calcium activity, and then individually laser ablates the targets while monitoring network function in real time. We applied this system to the mammalian respiratory oscillator located in the pre-Botzinger Complex (preBotC) of the ventral medulla, which spontaneously generates breathing-related motor activity in vitro. Here, we show that cumulatively deleting preBotC neurons progressively decreases respiratory frequency and the amplitude of motor output. On average, the deletion of 120 +/- 45 neurons stopped spontaneous respiratory rhythm, and our data suggest approximate to 82% of the rhythm-generating neurons remain unlesioned. Cumulative ablations in other medullary respiratory regions did not affect frequency but diminished the amplitude of motor output to a lesser degree. These results suggest that the preBotC can sustain insults that destroy no more than approximate to 18% of its constituent interneurons, which may have implications for the onset of respiratory pathologies in disease states.