Organization of the core respiratory network: Insights from optogenetic and modeling studies

The circuit organization within the mammalian brainstem respiratory network, specifically within and between the pre-Botzinger (pre-BotC) and Botzinger (BotC) complexes, and the roles of these circuits in respiratory pattern generation are continuously debated. We address these issues with a combination of optogenetic experiments and modeling studies. We used transgenic mice expressing channelrhodopsin-2 under the VGAT-promoter to investigate perturbations of respiratory circuit activity by site-specific photostimulation of inhibitory neurons within the pre-BotC or BotC. The stimulation effects were dependent on the intensity and phase of the photostimulation. Specifically: (1) Low intensity (≤ 1.0 mW) pulses delivered to the pre-BotC during inspiration did not terminate activity, whereas stronger stimulations (≥ 2.0 mW) terminated inspiration. (2) When the pre-BotC stimulation ended in or was applied during expiration, rebound activation of inspiration occurred after a fixed latency. (3) Relatively weak sustained stimulation (20 Hz, 0.5–2.0 mW) of pre-BotC inhibitory neurons increased respiratory frequency, while a further increase of stimulus intensity (> 3.0 mW) reduced frequency and finally (≥ 5.0 mW) terminated respiratory oscillations. (4) Single pulses (0.2–5.0 s) applied to the BotC inhibited rhythmic activity for the duration of the stimulation. (5) Sustained stimulation (20 Hz, 0.5–3.0 mW) of the BotC reduced respiratory frequency and finally led to apnea. We have revised our computational model of pre-BotC and BotC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BotC that interacts with other neurons in the network. This model was able to reproduce the above experimental findings as well as previously published results of optogenetic activation of pre-BotC or BotC neurons obtained by other laboratories. The proposed organization of pre-BotC and BotC circuits leads to testable predictions about their specific roles in respiratory pattern generation and provides important insights into key circuit interactions operating within brainstem respiratory networks.