Figure 3:

Injection sequence for intravenous remifentanil and local naloxone microinjections. The initial intravenous remifentanil bolus (red arrow) was chosen to cause apnea for more than 60s. Once respiratory activity returned, the remifentanil infusion was started (red line) until steady-state respiratory rate depression of 50%. This dose-rate was continued unchanged throughout the entire injection sequence. For Cohort A (n=11), naloxone (dark blue arrows) was microinjected into the bilateral Kölliker-Fuse Nucleus, Parabrachial Nucleus, preBötzinger Complex (including premotor neurons), and Caudal Medullary Raphe. Apneic remifentanil boluses (red arrows) were given after naloxone injections into the pons and preBötzinger Complex, and after subsequent naloxone injection into the raphe. After each “apneic” remifentanil bolus, we awaited complete recovery of the respiratory rate to prebolus levels before continuing the protocol. The “very high” remifentanil bolus was administered shortly after the last “apneic” bolus to maximize effect (light blue arrow). In Cohort B (n=10), naloxone was microinjected in the reverse order. In Cohort C, naloxone was microinjected only into the preBötzinger Complex including the premotor neurons and caudal medullary raphe. This protocol was truncated after five animals.