near the end of the TA burst and terminates during the LG burst [2]. Further, during airstepping extensor burst durations are brief (,100 ms) and vary independent of cycle duration, a feature that was also typical of RLMs. Features that distinguish RLMs from locomotor behaviors RLM EMG patterns also differed from airstepping. RLMs included burst frequencies below 3.5 Hz, a broader spectrum of FT onsets, and short lasting TA bursts that did not co-vary with cycle duration. Whereas during airstepping, TA burst durations are longer and scale with the step cycle [2]. The absence of covariation between extensor burst and cycle duration also distinguished RLMs from locomotion and swimming, for extensor bursts during stance (locomotion) and limb retraction (swimming) closely co-vary with the step cycle [2]. Thus, the absence of scaling between burst and cycle duration distinguished RLMs from all 3 posthatching forms of locomotion. The absence of burst scaling during RLMs could be an indication that proprioception at E18– E20 is not sufficiently mature to code movement dynamics [24]. However, longer extensor bursts are observed during hatching at E20 [17], possibly as embryos extend their legs into the shell wall to rotate their posture [25]. Thus, another possibility is that during most RLMs, the leg was not adequately loaded by foot contact with the shell to modulate extensor activity, for there were a few instances when LG burst durations were modestly lengthened (Figures 5A, 6A). On the other hand, flexor burst durations were probably not modulated by a load because flexor muscles operated in a shortened range due to the postural constraint imposed by the shell. It is also possible that descending pathways are required to modulate burst duration in ovo but that they do not modulate RLM activity. Shell removal did not enhance locomotor features of RLMs We also considered the possibility that the extreme flexed posture imposed by shell constraint could mediate a set of cues that might mask locomotor patterns for airstepping, walking and/or swimming, and match force output to the limited movement space. The extreme flexed posture in ovo during the final days of incubation puts flexors at their most shortened length and all leg extensors at their greatest length. Thus our second aim was to determine if EMG patterns would appear more similar to one or more locomotor patterns when the shell constraint was removed. We reasoned that if leg movements were unconstrained, movements would be larger, plus flexor muscle lengths and loads would increase as extensor lengths decreased. However our results indicated that the temporal structure of RLM EMG activity was preserved, despite substantial increases in joint excursion range after shell removal (Figure 5B) and a significant increase in cycle frequency [23]. The resilience of the pattern during larger excursions was consistent with the observation that TA and SA burst onsets co-varied even when the ankle rotated out of phase with the hip and knee during control RLM (Figures 1B, 3A). FT onset was the only measure that exhibited a modest variation with shell removal. FT onset was more likely to co-vary with TA cycle duration, but relative onset was more ambiguous or shifted from an early to late burst in the TA cycle (Figure 5B3); and the incidence of briefly overlapping TA and FT activity significantly increased. It was somewhat surprising to us that the EMG pattern for RLMs at E20 was not more mutable during the larger unconstrained motions, given the embryo would hatch and walk within 24 hrs. Any of the possible mechanisms discussed above might account for the minimal effect of shell removal. Also, the Development of Stepping sensory cues for larger motions may have been insufficient because the embryo was in a side lying position and the leg flexors experienced less gravitational loading than if held vertically suspended, as during airstepping studies. Further, shell removal did not appreciably alter head/neck/spine flexion, and these inputs might act to reduce descending drive to spinal motor networks during foot-free as well as control experiments. On the other hand, it may be instructive that recruitment of FT, the muscle with the most varied pattern during control RLMs, was also the only muscle responsive to motion-related changes. The early and late onset variability in FT seems to be comparable to the onset shifts also seen in the vastus lateralis, a knee extensor in cat, during the ballistic motions of paw shaking [26,27]. RLM pattern and rhythm have different developmental time frames In our previous study we propose that RLM rhythm generation may be modular because stable rhythms appeared to be isolated to individual muscles [23]. The same array and distribution of rhythms were observed by E15. Our present findings extend these results, suggesting that rhythm is established within a motor pool before it is expressed by an array of motor pools to form the RLM pattern. Participation of two muscles was infrequent at E15 but well established at E18. Studies of fictive locomotion and scratching in adult preparations have also observed that rhythmic activity in motor neuron pools can be sustained or resumed without temporal resetting as some motor pools drop out [28–31]. These observations led to proposals that central pattern generation for rhythmic limb movements involves two or more levels of control, i.e., one for rhythm and another for pattern (see [32] for a recent review). Thus, the differences in developmental time frame found for RLM rhythm and pattern generation in our studies appear to provide support for the proposal that pattern and rhythm generation involve multiple levels of control. The developmental time frames for rhythm and pattern generation extend even more broadly than addressed by our present data. Repetitive limb movements are slow (0.2–2 Hz) at E9 [20], and temporally irregular between E12 and E15 [17,19,22]. The slow rhythms at E9 are likely produced by transient spinal network dynamics resulting from immature membrane properties [33,34], and their progressive maturation may partially account for the temporal irregularities preceding emergence of RLM rhythms at E15. We do not know the mechanisms responsible for RLM rhythms, but descending pathways are well established by E15 [35,36], and might provide excitatory drive as maturing spinal neurons lose their intrinsic excitability. Both slow and fast rhythms are also expressed in adult lamprey locomotion [37]. The slow frequencies are attributed to neural membrane properties, and the fast frequencies are attributed to recurrent excitation within the neural network (see [38] for a recent review). Our results suggest that the RLM pattern appears 1 to 3 days after RLM rhythm, however RLM pattern development may begin with the emergence of alternating flexor and extensor synergies by E9 [18]. Some of the subsequent transformations are likely attributable to maturation of membrane properties such as persistent inward currents [39]. However, the emergence of the FT double burst and its variants at E18 may require development of additional mechanisms such as another control layer in CPG circuitry [32]. Maturation of sensory inputs may also shape FT burst patterns [2]; however FT double bursting is retained in fictive locomotion after hatching, suggesting that it is centrally specified [40]. In sum, our present study of RLM burst patterns completes the first detailed studies of RLMs in the chick embryo. Findings PLoS ONE | www.plosone.org 8 July 2009 | Volume 4 | Issue 7 | e6111