(A) Simultaneous intracellular recordings from R-Si1, L-Si2, and R-Si3 show the motor pattern in normal saline (left). Bath application of 0.1 mM curare (gray bar, right) slowed down the rhythm by extending the duration of bursts and causing them to become irregular. Schematics above the traces indicate the neural circuit. The blocked Si3 synapses are shown in gray (right).

(B) Curare (0.1 mM) significantly decreased the burst frequency (inverse burst period). In normal saline, the burst frequency was 0.26 ± 0.09 (n = 16 animals) Hz, which decreased to 0.06 ± 0.02 Hz (n = 16 animals) in curare. The burst frequency recovered to 0.19 ± 0.08 Hz (n = 8 animals) after more than 30 min washout of curare. There was a statistically significant effect of curare on the burst frequency (F (2,22) = 47.9, p < 0.001 by one-way repeated-measures [RM] ANOVA).

(C) Curare (0.1 mM) significantly increased the coefficient of variation (CoV) in the burst period. CoV of the burst period during the swim motor pattern was measured from the right Si1 or Si2 in each individual in the duration of at least 40 s. In normal saline (white), the CoV was 0.04 ± 0.02 (n = 16 animals), which increased to 0.21 ± 0.12 (n = 16 animals) in curare (dark gray). The CoV decreased to 0.08 ± 0.03 (n = 8 animals) after washout of curare (light gray). There was a statistically significant effect of curare on the CoV (F (2,22) = 20.8, p < 0.001 by one-way RM ANOVA).

(D) Curare had no effect on the intraburst spike frequencies of the swim interneurons. The bar graph shows averaged intraburst spike frequencies (Hz) measured from Si1, Si2, and Si3, in the normal saline (white), in curare (dark gray), and after washout (light gray). Curare had no significant effect on intraburst firing (Si1, F (2,14) = 1.44, p = 0.27 by one-way RM ANOVA, n = 14 cells; Si2, F (2,14) = 0.92, p = 0.42 by one-way RM ANOVA, n = 12 cells; Si3, F (2,16) = 2.27, p = 0.14 by one-way ANOVA, n = 13 cells).

In (B)–(D), data are represented as mean ± SD. Bracket with asterisks indicating significant differences by all pairwise multiple comparison procedures (Holm-Sidak method, two asterisks for p < 0.001 and one asterisk for p = 0.011). Individual data points are shown on each bar as open circles.

(E) Suppression of firing in Si3s by hyperpolarizing current injection (−4 nA) had similar effect as curare application by extending the burst duration of Si1 and Si2. Schematic above the traces indicate both Si3s were simultaneously hyperpolarized by injecting a negative current step.

(F) The Si3 pair do not act as a half-center oscillator when not receiving periodic synaptic input from Si2. Suppression of firing in both Si1 and Si2 by hyperpolarizing current injection (−4 nA) made Si3 fire tonically with no LR alternation.