extensor activity also distinguish the three locomotor patterns from one another. During walking there are two knee extensor bursts; one in the latter part of swing to extend the knee and advance the foot, and one in late stance to propel the body forward [1]. Only the duration of the knee extensor burst in the stance phase co- varies with step cycle duration. We reasoned that locomotor pattern generation must be established prior to hatching, given that chicks walk within hours afterwards. We also reasoned that RLMs might employ locomotor patterns given the similarities in cycle frequency range. However, in our previous study we observed that RLMs exhibited considerable variability in EMG activity [23]. This variability obscured ready identification of a fundamental pattern resembling any of the locomotor patterns observed in hatchlings. Thus, in this study we extend earlier findings by reporting quantitative analyses for the burst patterns at 3 ages. One aim was to determine if the EMG patterns for RLMs resemble any of the three locomotor patterns in hatchings. In addition, our earlier kinematic study of motility between E9 and E18 suggested that mechanical constraints due to growth of the body within a fixed egg volume increasingly alters joint kinematics between E15 and E18 [22]. Thus, differences we might find between locomotor and RLM muscle patterns could be attributable to constraints that include placement of flexor muscles at their shortest length and extensors at their greatest length. Therefore, another aim was to determine if EMG patterns would appear more similar to locomotor patterns when the shell constraint was removed. We provide evidence that EMG patterns for RLMs share some features common to locomotor behaviors at hatching, and the first evidence of alternating interlimb stepping in the embryo. We also provide evidence that the effects of shell removal were limited to the primary knee extensor, the only muscle that is known to distinctly vary its participation across the three locomotor behaviors in hatchlings [2]. Results We report EMG analyses for intralimb coordination based on 1206 RLM sequences. These data represent control conditions at E18 (386 RLM, 11 embryos) and E20 (353 RLM, 12 embryos), and the experimental condition, foot-free, at E20 (467 RLM, 9 embryos). The sample was drawn from a larger sample of 1569 RLMs whose rhythm properties were previously reported [23]. For these analyses, we excluded RLMs in which the tibialis anterior (TA), an ankle flexor and the reference muscle for our analyses, was the only rhythmically active muscle or if there were fewer than 10 rhythmically stable RLMs representing an experiment. EMG patterns at E15 are also reported (10 embryos). Leg muscle participation varied within and between experiments The wide array of RLM rhythm frequencies and combinations of active muscles reported in our previous study raised the possibility that RLMs are a collection of rhythmic behaviors. Kinematic differences between RLMs, such as ankle motions that were in phase with proximal joints (Figure 1A) or out phase (Figures 1B, 3A) might be evidence of different limb behaviors. Quantitative analyses for this study extended those results revealing that the combination of active muscles varied markedly throughout every experiment, even in RLMs only seconds to minutes apart, though conditions were seemingly unchanged. In one E20 experiment for example, 4 leg muscles participated during one RLM (Figure 1A). Approximately 20 min later, a rhythmic sequence of TA bursts was spontaneously initiated that Development of Stepping lacked rhythmic activity in the other EMG channels. It was followed immediately by a sequence of 6 TA cycles that included bursting in the sartorius (SA), a hip flexor (Figure 1B). In other RLMs during this experiment, TA sequences were accompanied by bursts in the femorotibialis (FT), a knee extensor (Figure 1C). To determine if there were any consistencies in recruitment, we determined the participation rate for each muscle across RLM cycles for each experiment. Based on within-subject averages (Figure 1D), the SA participated in nearly half of all RLM cycles at E18 (48%) and E20 (52%), FT participated in more than a third (38–40%), and the lateral gastrocnemius (LG), an ankle extensor, was least likely to participate at both E18 (26%) and E20 (13%). However, rates of participation for each muscle varied widely across experiments at both ages (Figure 1D). RLM sequences were difficult to detect at E15 due to the low amplitude and irregular bursting that dominated recordings. Based on a stable burst rhythm in one muscle, though not necessarily TA, 160 EMG sequences (N = 10 embryos) were examined for muscle patterns. Participation of a second muscle was detected in 24 RLMs (15%). In these instances FT bursts occurred synchronously with repetitive LG bursting (Figure 2A); TA bursts alternated with LG (Figure 2B); or FT bursts occurred synchronously with TA (Figure 2C). Participation of a third muscle was observed only twice (Figure 2C; see also [23] Figure 1F). Thus at E15, rhythmic RLM bursting was more readily apparent than any RLM muscle pattern. RLM muscle patterns also exhibited reliable features Though participation rates varied substantially across RLMs in an experiment, burst onsets for participating muscles were distinct. SA bursts began nearly synchronous with TA burst onset at both slower and faster RLM frequencies at both E18 and E20, with a mean onset latency of 630 ms. An exemplary RLM and analyses for one E18 experiment are shown in Figure 3. The regression plot for SA burst onsets indicated SA onset latency clustered around 0 ms, the slope near 0.0 (Figure 3B1). Relative onsets fell mostly between 0–0.2 across a TA frequency range of 1–7 Hz (Figure 3B2). Slopes approximating 0.0 were found in 13 of 16 experiments at E18 and E20 combined. (See Table S1 in supplementary material for a detailed summary.) When LG activity was well-formed, it alternated with TA bursts (Figure 3A). For example, regression results for the same E18 experiment indicated LG burst onsets co-varied closely with TA cycle duration (Figure 3C1). Relative onsets fell mostly in the latter half of the TA cycle at all RLM frequencies (Figure 3C2). These results were typical at both E18 and E20. LG onset closely varied with cycle duration in 6 of 9 experiments (R2.0.6), and was weak in only 2 (R2,0.4). Also, more than 60% of LG bursts began in the latter half of the TA cycle in 5 experiments (e.g., relative onset of 0.5–1.0), and no bias was found in 4 experiments. (See Tables S2 and S3 in supplementary materials for detailed summaries.) The knee extensor, FT, also alternated with TA, but onset trends were distinct from LG. FT bursts exhibited 3 onset patterns relative to the TA cycle: early (relative onset ,0.5), late (relative onset .0.5), or double bursting (early and late) at both E18 and E20. All 3 patterns were found within single experiments, as illustrated by 2 RLMs in Figure 4A. During the 1st RLM (Figure 4A1) an FT burst began in the first half of each cycle, and included a double burst in cycle 3 (q). During the 2nd RLM approximately 30 s later (Figure 4A2), FT bursts began in the latter half of cycles 2–4. FT burst onsets for this experiment modestly co-varied with TA cycle duration, and 2 clusters, early and late, were observed in the relative onset plot (Figure 4A3, bottom plot). FT burst onset varied closely with cycle duration in PLoS ONE | www.plosone.org 2 July 2009 | Volume 4 | Issue 7 | e6111