(A) Representative traces of ∫preBötC and ∫XII population activity in 3/1 ACSF control, 8 µM Cd2+, and 8 µM Cd2+ + 10 nM DAMGO. In 3/1 ACSF (top), rhythmic activity consisted of both burstlets ( ) and bursts. After Cd2+ application (middle), preBötC bursts were no longer observed, and XII activity was abolished, leaving only a preBötC burstlet rhythm with a f similar to control. With addition of DAMGO (bottom), burstlet f and amplitude decreased. Note: the two large deflections in ∫XII (bottom) are not synchronized with preBötC activity, and are likely non-inspiratory or electrical artifact. Scale bar, 5 s. (B) Average burstlet fraction, preBötC f, and burstlet amplitude (amp.) in 3/1 ACSF control, 6–25 µM Cd2+, and 6–25 µM Cd2+ + 0.1–10 nM DAMGO. Addition of Cd2+ significantly increased the burstlet fraction while preBötC f and burstlet amplitudes were unchanged. Bath application of DAMGO significantly decreased preBötC f (consisting almost solely of burstlets) and burstlet amplitude. *, p<0.05, one-way ANOVA, post-hoc Tukey test, n = 8. (C) Overlays of representative traces of ∫preBötC from successive photoexcitation of 8 Dbx1+ neurons in control 9/1.5 ACSF, 30 nM DAMGO, and 100 nM DAMGO. The illumination pattern consisted of 10 µm soma-centered spots over the targeted neurons. Photoexcitation was provided by 405 nm holographic illumination of 500 µM MNI-glutamate. Arrow represents time of laser stimulation. In 9/1.5 ACSF, a failure was seen, but burst initiation was otherwise reliably successful. Bursts were consistently triggered after a ~ 100 ms latency. In 30 nM DAMGO, failures were more frequent when the same stimulation parameters were used, and the latency between laser stimulation and burst initiation was longer and more variable. In 100 nM DAMGO, bursts in this experiment were no longer triggered with the same stimulation parameters. Scale bar, 200 ms. (D) Average success rate (stim→burst) for burst initiation in control 9/1.5 ACSF conditions and in increasing concentrations of DAMGO using entraining stimuli that elicits >80% success in 9/1.5 ACSF. To entrain the rhythm, 5–10 Dbx1+ neurons were stimulated 8–20 times every 3–6 s. DAMGO produced a dose-dependent decrease in the success rate for triggering bursts with a significant reduction in success between control and at 100 nM and 200 nM DAMGO. *, p<0.05, one-way ANOVA, post-hoc Tukey test, n = 6. (E) Average latency between laser stimulation and burst initiation when bursts were successfully triggered during entraining stimuli was increased in 30 nM DAMGO compared to control 9/1.5 ACSF. *, p<0.05, Student’s t-test, n = 5.