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.