to control (15615%, p,0.011), but burst durations (TA 65 ms, FT 55 ms) and duration of overlap (< 20 ms) were similar between conditions. Interlimb alternation during RLMs Given some RLM features appeared to be similar to locomotor patterns while others were more ambiguous, we implanted leg muscles bilaterally at E20 in 4 experiments to determine if both legs were active during RLMs. We found that all experiments exhibited evidence of interlimb coordination. For example, in Figure 6A, the right TA and LG were alternately active, as were the right and left TA, and the right LG alternated with and the left flexor digitorum profundus (toe flexor/ankle extensor, FDP). The plot of ankle markers tracking anterior-posterior displacements indicated that the legs were moving in opposite directions, as during fore-aft foot trajectories while walking. Burst analyses for this experiment indicated that 56% of all cycles for the right LG (N = 211) were accompanied by bursts in the left LG or FDP. Results indicated that 50% of left FDP bursts began mid cycle (0.4–0.6). In the 4 experiments combined, EMG bursts were detected in the left leg during 49% to 62% of rhythmic sequences in the right leg, and the majority of left bursts began mid cycle (Figure 6B). A plot of burst frequencies for each leg (4 experiments) verified that these interlimb events fell within the RLM frequency range (Figure 6C). Discussion RLM patterns resemble locomotor patterns Our first aim was to determine if EMG patterns for RLMs resemble any of the three locomotor patterns in hatchings. During walking, swimming and airstepping, several features are charac- teristic of leg muscle activity [1,2]. Nearly all leg muscles exclusively participate in either the flexor synergy for limb protraction or extensor synergy for limb retraction. Burst onsets are close in time for flexor synergists (i.e., TA, SA). The ankle extensor (LG) alternates with flexors and the overlap between the bursts is little or none. In addition, activity of the knee extensor (FT) is distinctly different from other leg extensors. FT bursts twice per step cycle in walking and swimming, and once during airstepping; one FT burst begins during flexor activity in all 3 forms of locomotion. Our results indicated that EMG patterns for RLMs shared these features. SA burst onsets were closely associated with TA onset, regardless of TA burst frequency (Figure 3B). The ankle extensor, LG, was consistently recruited in the interval between TA bursts (Figures 1, 3). Recruitment of the knee extensor, FT, was distinct from LG; it most often began early in the cycle, but it could also burst late or twice per cycle (Figures 3–5). Also, like locomotion, RLMs could include alternating interlimb coordination (Figure 6). This array of similarities leads us to propose that RLMs in late-stage embryos are locomotor-related behavior. Was there a match between the RLM burst pattern and any of the 3 locomotor forms at hatching? The RLM frequency range (1– 10 Hz) embraced the combined cycle frequencies for the 3 locomotor behaviors at hatching [2]. On average, RLM frequencies at E18 (4 Hz) and E20 (5.4 Hz) were faster than the approximate averages for walking (2.8 Hz) and swimming (3.3 Hz), but airstepping (5.1 Hz) includes the higher frequencies common to RLMs. Though studies of locomotor patterns for hatchlings used the LG as reference for analyses, it also appears that the relative timing of burst onsets during RLMs closely resembled airstepping EMG [1,2]. During RLMs, LG began in the latter half of the TA cycle and was immediately followed by the Development of Stepping Figure 6. Interlimb coordinated during RLM at E20. Interlimb data are shown for 4 experiments at E20. A: A 3-burst sequence in the right (R) TA (3.461.2 Hz) alternated with a 4-burst sequence in the left (L) TA (4.060.7 Hz). R TA bursts also alternated with R LG bursts; L TA bursts alternated with left flexor digitorum profundus (L FDP) bursts. Anterior (upward)/posterior (downward) displacements of the 2 ankles were out of phase with one another. B: Left ankle extensor burst onsets were normalized to right extensor burst cycles (relative phase) and phase values were binned in 0.05 increments before plotting the distribution of interlimb phase patterns. The majority of left leg bursts fell between relative phases of 0.4–0.6 in all 4 experiments. C: The burst frequency distributions for the cycles plotted in B are shown for each leg (L TA and R TA). TA burst frequencies ranged from 1 to 10 Hz for both legs in each experiment. doi:10.1371/journal.pone.0006111.g006 next TA burst (Figures 1, 3). FT began near TA offset and before onset of the next LG burst (Figures 1, 4–5). Extrapolating from published averages for airstepping (see [2] Figure 5), LG appears to begin in the later 40% of the TA cycle. A single FT burst begins PLoS ONE | www.plosone.org 7 July 2009 | Volume 4 | Issue 7 | e6111