revealed that despite considerable variations in the ensemble of active muscles during RLMs, burst patterns adhered to a common template. The template consisted of alternating flexion and extensor synergies, but also included burst patterns that distin- guished knee extensor activity from ankle extensor activity. These findings are consistent with essential features of locomotor behaviors in hatchlings, and appear most similar to airstepping. Thus we conclude that RLMs in the late stage embryo are locomotor-related and produced by the developing neural circuits for locomotion. Our findings also suggest that RLM patterns are only modestly impacted by small modifications in physical constraint. Whether more dramatic changes in environmental conditions and postural context can significantly impact late stage embryonic behavior remains to be explored. Finally, our results lead us to conclude that RLM rhythm and pattern have different developmental time frames and that further study of their development may advance our understanding of locomotor control. Materials and Methods Fertile Leghorn chicken eggs were incubated under standard conditions with a temperature-controlled humidified incubator (37.5u). Eggs were maintained under similar conditions in a temperature-controlled chamber during preparation and record- ing. The first day of incubation was E0 and the present studies were conducted at E15, E18 or E20. Age was verified at the end of experiments using staging criteria by Hamburger and Hamilton [41]. The viability of the embryo was assessed throughout the experiment by visually monitoring pulse rate and tracking body displacements with a force transducer (FT03C, Grass Instruments) placed in contact with the thigh [42]. If either indicator suggested the embryo was deteriorating, the experiment was terminated. All procedures were approved by the University Institutional Animal Care and Use Committee. EMG, video and force recordings Embryos were prepared for synchronized electromyographic (EMG) and video recording of leg movements in the egg. A sagittal Development of Stepping view of the leg was obtained by creating a window in the shell and dissecting egg membranes. Leg muscles were implanted with silver bipolar electrodes (o.d. 50 mm), including the sartorius (SA), a hip flexor; femorotibialis (FT), a knee extensor; tibialis anterior (TA), an ankle dorsiflexor; and lateral gastrocnemius (LG), an ankle extensor. The location of EMG electrode tips was later verified by dissection after euthanizing the embryo. In E18 and E20 embryos, modified minutin pins were inserted along the leg for digitizing hip, knee and ankle movements. In some experiments additional shell was removed anterior to the foot to reduce mechanical constraints on leg posture and movement (foot-free). Synchronized EMG, force and video were recorded continu- ously for $3–4 hours (Datapac 2K2, Run Technologies) on a grounded anti-vibration table. EMG was band pass filtered (100– 1,000 Hz), amplified (62000), and digitally sampled (4 kHz). Force signals were low pass filtered (30 Hz), amplified (620,000) and digitized (500 Hz). RLM Analyses Sequences of repetitive muscle bursting that were accompanied by leg movement were selected for analyses. The synchronized video and/or force recordings were used to confirm the presence of leg movements. The force recording was particularly useful for detecting RLM when joint motions were only a few degrees in magnitude due to spatial constraint but of sufficient force to produce rhythmic displacements of the body. EMG signals were rectified to detect burst onsets and offsets based on 3 criteria: burst threshold (2–4 times baseline amplitude), burst duration (20– 400 ms), and inter-burst interrupt (20 ms) (Datapac 2K2). Baseline amplitude for each EMG channel was estimated from a quiescent 100 ms segment of recording at the start of the experiment. The threshold for each EMG channel was determined by pilot analyses of several RLMs and based upon the combined parameter set that captured the greatest number of EMG bursts. An example of the burst detection method is shown in Figure 7. EMG burst and cycle measures were referenced to TA burst onset, because TA was the most reliably rhythmic of the muscles recorded [23]. Thus, TA cycle duration was defined as the time between consecutive TA burst onsets. Burst onset latencies for all Figure 7. Burst detection methods. After signal rectification and baseline offset adjustment to 0 mV, average baseline channel noise was estimated during a quiescent interval of 100 ms. In the above example, a threshold of 15 mV (3 times baseline) for TA and LG burst detection is shown. EMG activity remaining above threshold for 20–400 ms and surrounded by subthreshold signal for 20 ms or longer was defined as a burst. Examples of burst detection are indicated by the upward square wave defections in the threshold trace. doi:10.1371/journal.pone.0006111.g007 PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6111