Sensory-evoked turning locomotion in red-eared turtles: kinematic analysis and electromyography
We examined the limb kinematics and motor patterns that underlie sensory-evoked turning locomotion in red-eared turtles. Intact animals were held by a band-clamp in a water-filled tank. Turn-swimming was evoked by slowly rotating turtles to the right or left via a motor connected to the shaft of the band-clamp. Animals executed sustained forward turn-swimming against the direction of the imposed rotation. We recorded video of turn-swimming and computer-analyzed the limb and head movements. In a subset of turtles, we also recorded electromyograms from identified limb muscles. Turning exhibited a stereotyped pattern of (1) coordinated forward swimming in the hindlimb and forelimb on the outer side of the turn, (2) back-paddling in the hindlimb on the inner side, (3) a nearly stationary, “braking” forelimb on the inner side, and (4) neck bending toward the direction of the turn. Reversing the rotation caused animals to switch the direction of their turns and the asymmetric pattern of right and left limb activities. Preliminary evidence suggested that vestibular inputs were sufficient to drive the behavior. Sensory-evoked turning may provide a useful experimental platform to examine the brainstem commands and spinal neural networks that underlie the activation and switching of different locomotor forms.
Animal locomotion in water, air, or terrestrial environments exhibits a variety of continual fine-steering adjustments along with more abrupt turns and reversals. During swimming or flight, in which animals are free to move in three dimensions, directional adjustments can involve changes in yaw, pitch, and roll orientations. In the present paper, we focus on lateral turning (changes in yaw, or horizontal orientation) during swimming locomotion in freshwater turtles. The biomechanical and neuromuscular mechanisms that mediate lateral turns during animal locomotion are much less thoroughly examined than those that underlie forward rectilinear locomotion. However, the inherent asymmetry of lateral turning behavior provides unique opportunities for examining the differential selection and modulation of locomotor patterns on the right and left sides. Among some arthropods, terrestrial turning locomotion, especially during tight turns or in-place rotation, can involve different locomotor forms in legs on the outer and inner sides of the turn with different intralimb timing (e.g., forward stepping on the outer side, backward stepping on the inner side) that rotates the animal around a vertical axis (jumping spiders: Land 1972; honeybee: Zolotov et al. 1975; cockroach: Bell and Schal 1980; fruitfly: Strauß and Heisenberg 1990; Mu and Ritzmann 2005). In other cases, particularly during more gradual curve walking, arthropods may shorten stride length on the inner side of the turn (ants: Zollikofer 1994; crayfish: Cruse and Silva Saavedra 1996; walking sticks: Gruhn et al. 2009). Similar asymmetries occur during aquatic turns. For example, whirligig beetles swim in a tightly curved trajectory via the asymmetric paddling of outer legs or sculling of the outer wing, combined with the extension of a stationary forelimb or elytra (wing cover) on the inner side that acts as a brake or pivot (Fish and Nicastro 2003). Vertebrates that swim via axial undulations (teleost fish: Gray 1933; lamprey: McClellan 1984; McClellan and Hagevik 1997; Saitoh et al. 2007) execute lateral turns via an abnormally large wave of muscular contraction passing down the trunk and tail on the inner side of the turn. In sea turtles, which swim via a lift-based mechanism involving synchronous flapping movements of both wing-like forelimb flippers, turns are executed either by reducing the duration of the forelimb cycle on the inner side of the turn (Renous and Bels 1993; Wyneken 1996) or by a combination of increasing the stroke amplitude in the outer-side forelimb and using the hindlimb flipper on the inner side of the turn as a stationary rudder (Walker 1971b; Lohmann et al. 1995; Avens et al. 2003). Some previous work has also been done on straight swimming vs. turn-swimming in fresh-water turtles. Rivera et al. (2006) described the pattern of limb movements during voluntary forward and backward lateral turns in free-swimming painted turtles (Chrysemys picta) in a study that investigated the dynamics of turning performance rather than the specifics of limb motor patterns and inter- or intralimb coordination. The bilateral movements and motor patterns of the turtle hindlimbs (but not forelimbs) were also described for spontaneous turning episodes in carapace-restrained red-eared turtles (Trachemys scripta elegans) held by a band-clamp (Field and Stein 1997a, b; Earhart and Stein 2000).
Surgically reduced vertebrate preparations have been developed to enable the study of actual and fictive behavior elicited by natural sensory stimulation, permitting the close examination of neural mechanisms. Lamprey tail fin–spinal cord preparations exhibited episodes of tail withdrawal followed by fictive escape swimming in response to cutaneous stimulation of the tail fin (McClellan and Grillner 1983), and head–brainstem–spinal cord preparations from lampreys could be induced to generate fictive turning (aversive withdrawal) and escape swimming bouts by mechanical or electrical stimulation of the trigeminal-innervated skin of the snout (McClellan 1984). There have been no comparable means of eliciting locomotion in restrained or semi-intact turtle preparations via sensory stimulation, and the search for such a method was part of the motivation for our present experiments. Freely behaving freshwater turtles are capable of making both gradual and abrupt voluntary lateral turns while swimming, especially during pursuit of fish prey (Rivera et al. 2006); however, these turns are typically executed by single, bilaterally asymmetric swim cycles.
In the present study, we describe sustained sensory-evoked turn-swimming in red-eared turtles, elicited by horizontal rotation of the animals around their mid-body vertical axes. We assessed the kinematics and electromyogram (EMG) motor patterns of bilateral forelimb and hindlimb movements during prolonged episodes of turn-swimming and during switches between right and left turn-swimming in response to switched rotational direction. Our results enabled us to identify a highly stereotyped set of bilaterally asymmetric movements and EMG motor patterns that underlie sensory-evoked turning behavior and provide a potential basis for the development of reduced preparations that express fictive turning motor patterns in response to rotational stimulation. Most of the data presented in this paper were submitted by the first author as part of his dissertation in partial fulfillment of the requirements of the doctoral degree (Welch 2011).
Rotation of carapace-restrained turtles
We recorded turtle locomotor behaviors from below with a digital video camera (Canon Optura 20 mini-DV), aimed at a 45° mirror beneath the clear Plexiglas bottom of the swim tank (Fig. 1a). Brightly colored plastic beads (3 mm diameter) were attached to the ventral-medial side of the wrists (h and i in Fig. 1b) and the ventral-anterior side of the knees (f and g) and ankles (k and l) to track movement of the limbs relative to fixed reference markers painted on the ventral plastron with white correction fluid (a–e). Reference points a and c marked the rostral and caudal ends of the ventral midline. Reference point b marked the rostro-caudal level of pectoral-girdle pivot points along the ventral midline, where the scapulae meet the underside of the dorsal carapace. Our examination of red-eared turtle skeletons showed that this point could be closely estimated from external anatomy, as it occurs at nearly the same rostro-caudal level as the anterior border of the lateral shell bridge (the “axillary notch”; see Carr 1952, pp. 37–38). Reference points d and e marked the positions of the right and left hip joints (note that images are inverted in the mirror), while j and m marked spots painted on the chin and near the tip of the tail, respectively. Video recordings were obtained with a 30-Hz frame rate and 1/250-s shutter speed and recorded on a DVC tape. Selected video sequences were de-interlaced and digitized later on a lab PC at a resolution of 60 fields per second. We tracked marker locations on the limbs and plastron field-by-field, using Datapac 5 software (Run Technologies, Laguna Hills, CA, USA). A detailed description of limb angle measurements was given previously (Samara and Currie 2007). Briefly, forelimb angle was defined as ∠abh on the right and ∠abi on the left, and was measured relative to a stationary reference point on the plastron midline (b, see above) (Walker 1971a; Samara and Currie 2007). “Forelimb angle” was used instead of “shoulder angle” because the shoulder joint is not stationary during locomotion, but moves back and forth through an arc as the pectoral girdle rotates (Walker 1971a; Samara and Currie 2007). Hip angle was defined as the angle between the thigh line (d–f on the right or e–g on the left in Fig. 1b) and a line parallel to the ventral midline (a–c) with its origin at the stationary hip joint. Knee angle was defined as ∠dfk on the right and ∠egl on the left. Head angle was defined as ∠abj, where an angle of 0° meant that the head was pointed straight forward, and movements of the head to the right and left were indicated as positive and negative angles, respectively. Tail angle was not measured, but the position of the tail marker was tracked in Fig. 2.
To visualize the trajectories of digitized limb, head and tail markers during turning behavior in rotating turtles (Fig. 2a, c), we used a customized feature in the two-dimensional motion analysis module of our Datapac 5 software, developed by Youan Chang at Run Technologies. This allowed us to stabilize the animal’s ventral mid-line (line segment b–c in Fig. 1b) in digitized stick-figures and observe the overlapping movement trajectories of each limb marker relative to a stationary body axis.
EMGs were recorded with bipolar electrodes consisting of two H-ML-insulated 0.004-in. silver wires, approximately 24 in. in length and glued together with Permabond 910 adhesive (National Starch and Chemical, Bridgewater, NJ, USA). Electrodes were implanted into selected hindlimb muscles (n = 7), forelimb muscles (n = 3), or both (n = 1) on the right side, using previously described surgical techniques (Stein 1978; Earhart and Stein 2000; Samara and Currie 2007). The muscle-end of the paired EMG wires were separated over the last 1–2 cm and about 0.5 mm of insulation was scraped off at the tips; these were then hooked with fine forceps and inserted 4–8 mm apart in the thickest part of each muscle with a hypodermic needle. Recorded hindlimb muscles included (1) a hip retractor–knee flexor (HR: flexor cruris pars flexor tibialis internus, n = 5), (2) a hip protractor (HP: puboischiofemoralis internus pars anteroventralis, n = 4), and (3) a knee extensor (KE: triceps femoris pars femorotibialis, n = 4). Note that the hip retractor and protractor muscles (HR and HP) referred to here were called hip extensor and hip flexor muscles (HE and HF) in previous papers (Samara and Currie 2007, 2008a, b). Recorded forelimb muscles included (1) a shoulder retractor–adductor (SR: pectoralis, n = 3) that also rotates the pectoral girdle backward, (2) a shoulder protractor–abductor (SP: deltoideus, n = 1) that also rotates the pectoral girdle forward, and (3) an elbow extensor (EE: triceps brachii, n = 2) (Walker 1973; Stein 1978). Electrodes from different muscles were loosely bundled together to minimize tangling and movement artifacts, and were sufficiently long to wind around the band-clamp post for several complete rotations without breaking or becoming taut. We conducted a post mortem analysis to verify correct electrode placements. Recordings from electrodes that were not properly placed in the correct muscle were excluded from our counts. EMGs were amplified and filtered (30–1,000 Hz) with Grass P511 AC amplifiers (Astro-Med., West Warwick, RI, USA) and digitized on a lab PC, using Datapac 5 software (Run Technologies, Laguna Hills, CA, USA). A remote video synchronization unit (Peak Performance Technologies, Los Angeles, CA, USA) was used to synchronize analog recordings (EMGs and the output of the rotational position sensor) with the video data. Episodes chosen for figures were formatted with Datapac 5 and CorelDraw.
We used digitized kinematic data to calculate average minimum and maximum joint angles, cycle frequency, and intra- and interlimb phase values for turning responses, using the event selection and advanced spreadsheet modules of Datapac 5. The cycle frequency of turn-swimming was calculated for leftside forward swimming hip movements on the outer side of turns, using Datapac 5 software. The time between consecutive hip protraction onsets was measured in kinematic recordings and converted to cycle frequency. The data were imported into SYSTAT 11 (Systat Software, Inc. Chicago, IL, USA) for analysis. Repeated-measures ANOVA (Zar 1999) was used to assess the effects of rotation speed and duration on the cycle frequency of turn-swimming responses. We used t tests to isolate the differences between groups in our ANOVA analyses. Post hoc pair-wise comparisons (with Bonferoni corrections) were made to assess the differences between mean cycle frequency values for different rotation velocities within the same time-bin and for different time-bins at the same rotation velocity.
Dual-referent phase analysis was used to assess intralimb and interlimb coordination (Berkowitz and Stein 1994; Field and Stein 1997a, b); phase values were calculated with Datapac 5. One joint or limb angle was selected as “referent” and the other as “target”. The onsets of referent-limb protraction were defined by phase values of 0.0 and 1.0. The onsets of referent retraction were defined by a phase value of 0.5 (see Field and Stein 1997a, b). Phase data were imported into Oriana 3.0 (Kovach Computing Services, Anglesey, Wales, UK) to obtain circular statistics, which are appropriate for cyclical events (Batschelet 1981; Zar 1999; Mardia and Jupp 2000). The angle and length of the mean vector were calculated using standard trigonometric functions. The angle of the mean vector (μ) represents the average phase value on a circular scale ranging between 0.0 and 1.0. The length of the mean vector (r) indicates the directional concentration of data points around the mean vector angle. We used the Rayleigh test to discriminate between uniform and unimodal-clustered phase distributions and the Watson U2 test (Batschelet 1981; Zar 1999) to determine if phase values were significantly different.
In response to slow horizontal rotation around their central vertical axes (Fig. 1), turtles that were held by a band-clamp and immersed in water displayed stereotyped forward turn-swimming against the direction of imposed rotations. In the absence of rotation, the same animals could be induced to execute episodes of rectilinear forward swimming by visual stimulation (e.g., movement of the experimenter) and/or tactile stimulation of the animal’s body surface. We recorded digital video of rotation-evoked turn-swimming and rectilinear forward swimming from below the tank and computer-analyzed forelimb and hindlimb movements as well as, in some cases, head and tail movements. In Fig. 2, we compared foot trajectories and head and tail positions in the same turtle during 2-s sequences of right and left turn-swimming evoked by counter-clockwise and clockwise rotation (Fig. 2a, c) and rectilinear forward swimming in the absence of rotation (Fig. 2b). During rectilinear swimming (Fig. 2b), the turtle displayed the typical bilaterally symmetric pattern of forward swim cycles in all four limbs, including 1:1 out-of-phase movement of contralateral limbs (right–left forelimbs and right–left hindlimbs) and ipsilateral limbs (right forelimb–hindlimb and left forelimb–hindlimb), and 1:1 nearly in-phase movement of diagonal limbs (right forelimb–left hindlimb and left forelimb–right hindlimb). During rotation-evoked turning (Fig. 2a, c), the same animal exhibited a highly stereotyped pattern of (1) tightly coordinated forward swimming motor patterns in the hindlimb and forelimb on the outer side of the turn, (2) back-paddling motor patterns in the hindlimb on the inner side of the turn, (3) a stationary and protracted forelimb on the inner side of the turn that appeared to act as a brake, and (4) neck and tail bending toward the direction of the animal’s turn. The same movement pattern was observed in all the turtles we examined (n = 17).
We present the average minimum and maximum limb angles for all four limbs during left and right turn-swimming in Fig. 3 (n = 5 turtles). Statistical comparisons were made between corresponding minimum angles and between corresponding maximum angles for a given limb during left and right turns, using the Mann–Whitney U test (Siegel 1956). The most obvious difference in angular range (average minimum to maximum angle) was between forelimb forward swimming, with very large-amplitude movements, and forelimb braking, with little movement at all. The next most striking difference was in the angular ranges of forward swimming vs. back-paddling movements at the hip. Hip movements during back-paddling occurred over a relatively narrow range and were protraction biased (biased toward smaller minimum and maximum hip angles) compared to forward swimming. These differences in minimum and maximum hip angles during back-paddling and forward swimming may only occur during rotation-evoked turning behavior, since no such difference was noted in previous work for spontaneous turning in non-rotated turtles (see Fig. 2 in Earhart and Stein 2000).
The lateral head movements (neck bending) that accompanied changes in rotational direction could be explained as passive responses to rotational inertia and water-associated drag forces acting on the head; however, our evidence suggests that the head movements were largely driven by active muscular control of the neck. First, head movements in water were often not smooth, as would be expected if they were passively driven, but jerky and discontinuous (see downward arrow at left side of head position trace in Fig. 7), suggesting muscular control. Second, identical head movements were observed when turtles were slowly rotated in the air, where water-induced drag on the head could not contribute to passive neck bending (see “Sensory activation”). Third, we observed similar head movement toward the direction of a turn even during spontaneous right and left turning behavior in non-rotating animals that were held stationary by a band-clamp (Welch and Currie, unpublished observations).
We evoked robust, sustained turning behavior (forward turn-swimming) in carapace-restrained turtles by slowly rotating animals to the right or left via a motorized band-clamp encircling middle of the shell. Turtles reliably exhibited forward turning in the opposite direction of the imposed rotation, in an apparent effort to counter the rotation and stabilize their yaw position. They also switched rapidly back-and-forth between right and left turns when the direction of rotation was reversed. We examined the hindlimb and forelimb contributions to turning behavior via video kinematics and synchronized EMG recordings of limb motor patterns. Our data confirmed and extended previous studies (Field and Stein 1997a, b; Rivera et al. 2006) that characterized some elements of turning behavior in freshwater turtles. During a turn, the “outer” forelimb and hindlimb produced tightly coordinated, out-of-phase forward swimming, while the “inner” hindlimb exhibited back-paddling, and the inner forelimb was held in a tonic protracted position, apparently acting as a brake, or “pivot” as earlier described by Rivera et al. (2006). Rotation-evoked turning was always accompanied by neck and tail bending toward the direction of the turn. In addition, the characteristic asymmetric pattern of limb and head movements during turn-swimming still occurred when animals were rotated in the air instead of in water (to prevent drag-associated movement of the limbs and head and the associated proprioceptive stimulation) and blindfolded (to abolish visual inputs), suggesting that turn-swimming was primarily triggered by rotation-evoked vestibular activity.
Sensory-evoked turning behavior was previously described in hatchling sea turtles (Lohmann et al. 1995; Avens et al. 2003). Lohmann et al. (1995) constructed a wave simulator that reproduced the circular motion of hatchling turtles swimming beneath ocean waves. Animals suspended in the air and subjected to this wave action consistently attempted to turn-swim into the simulated wave (against the wave direction), just as our adult freshwater turtles swam against the direction of horizontal rotation. In both cases, the animals were attempting to maintain a consistent heading in response to externally imposed accelerations. Hatchling sea turtles (green and loggerhead: Lohmann et al. 1995; Avens et al. 2003) swim in the same manner as their adult forms (green, loggerhead, Ridley, hawksbill, leatherback: Walker 1971b; Renous and Bels 1993; Wyneken 1996), via the synchronized strokes of powerful front flippers. Turns are initiated in sea turtle hatchlings by extending a rear flipper as a stationary rudder on the inner side of the turn and continued during sustained turns by increasing the amplitude of the stroke cycle in the outer-side front flipper (Lohmann et al. 1995; Avens et al. 2003). Similar movements, along with a shorter stroke cycles on the inner side of the turn compared to the outer side, have been observed in adult sea turtles during lateral turns (Walker 1971b; Renous and Bels 1993). However, in freshwater turtles like those in our present study, forward propulsion is achieved via the alternating forward swim strokes of large, webbed hind-feet, while the smaller, non-webbed forelimbs may serve mainly to counter the horizontal rotational torque produced by the alternating hindlimb power-strokes and regulate orientation, keeping the animal on a straight heading (Pace et al. 2001; Rivera and Blob 2010). During rotation-evoked turns, our red-eared turtles continued producing strongly coordinated, out-of-phase forward swim cycles in the outer forelimb and hindlimb, while protracting the inner forelimb as a brake to increase drag and switching the inner hindlimb from forward swimming to back-paddling. Turn-swimming motor patterns sped up in response to increased rotational velocity and were sustained for as long as the rotation continued (up to 24 s), albeit with gradually diminishing cycle frequencies (Fig. 3). There were both similarities and differences between the kinematics of our sustained, sensory-evoked turning episodes in red-eared turtles and those of the brief, voluntary forward turns in freshwater painted turtles, as described by Rivera et al. (2006). In both cases, turtles held the inner (or, “inboard”) forelimb in a stationary, protracted position during forward turns, which would be expected to increase drag on the inner side and contribute to rotational torque. However, Rivera et al. (2006) described the outer (“outboard”) forelimb and both hindlimbs as continuing with normal “rectilinear” forward swimming” during turns. Thus, they suggested that painted turtles produced forward turns primarily by increasing drag on the inner side of the turn via the braking forelimb, while the remaining limbs continued to execute rectilinear forward locomotion. In contrast, we found that while the outer forelimb and hindlimb did both exhibit robust forward swimming during a rotation-evoked turn, the inner hindlimb switched to a back-paddling motor pattern (see: Field and Stein 1997a; Earhart and Stein 2000) in which the power-strokes occurred during hip protraction. Also, we saw head and tail movements toward the direction of the turn in every animal, but these were not mentioned in the Rivera et al. (2006) article, and no neck or tail bending was apparent from their photographs of an animal during a right forward turn (see their Fig. 2b). It may be that the neck and tail bending that we always observed in our experiments (Figs. 2, 7, 11) occurs only in response to rotational stimulation, as vestibular reflexes, and that these do not normally accompany brief voluntary turns.
Forward swimming, back-paddling and braking motor patterns contribute to turn-swimming
Red-eared turtles exhibit two distinctly different hindlimb motor patterns during aquatic locomotion: forward swimming and back-paddling (Fig. 5; see also Field and Stein 1997a; Earhart and Stein 2000). During hindlimb forward swimming, the onsets of knee extension occur during the middle of the hip protraction phase of each swim cycle, and the power-stroke occurs during hip retraction. Knee extensor (KE) EMG activity begins near the middle of each hip protractor (HP) EMG burst and continues until shortly after HP offset (Fig. 8b; see also Earhart and Stein 2000). During hindlimb back-paddling, the onsets of knee extension occur before the onset of the hip protraction phase of each swim cycle, and the power-stroke occurs during hip protraction. Knee extensor (KE) EMG activity begins before the onset of each hip protractor (HP) EMG burst and continues until HP offset (Fig. 8a; see also Earhart and Stein 2000). In the present experiments, we always observed forward swimming in the outer hindlimb and back-paddling in the inner hindlimb during rotation-evoked turn-swimming (n = 17 turtles). This was previously noted in the spontaneous, voluntary turn-swimming of turtles held by a stationary band-clamp (Field and Stein 1997a, b; Earhart and Stein 2000); however, further kinematic measurements and EMG recording will be required to determine if a similar pattern of outer and inner hindlimb motor patterns, perhaps lasting only a single cycle, underlies some voluntary turns in free-swimming turtles.
We also recorded EMGs from three different forelimb muscles during turns, including SP (deltoid, shoulder protractor), SR (pectoralis, shoulder retractor) and EE (triceps brachii, elbow extensor). We observed only forelimb forward swimming and braking patterns, but never saw forelimb back-paddling like that of the inner hindlimb. Surprisingly, forelimb braking was not accompanied by significant EMG activity in any of the three recorded muscles, except for some very low amplitude, rhythmic discharge in the SP muscle (Fig. 10a). We expected to see higher levels of tonic or modulated discharge in both SP and EE, given the elbow-extended appearance of the braking forelimb. Forelimb forward swimming motor patterns, however, were robust, with intralimb EMG timing consistent with what Rivera and Blob (2010; see their Fig. 4; Table 2) described in great detail for intact, free-swimming animals, and what Stein (1978) observed during forward swimming elicited by spinal cord stimulation in high-spinal turtles.
We have not yet recorded EMGs from neck or tail muscles during turn-swimming, so we are unsure which muscles control the lateral neck and tail bending in the direction of forward turns. However, it appears likely that lateral neck bending is produced, at least in part, by the actions of cervico-capitis and sternomastoid muscles (Ashley 1955; Herrel et al. 2007).
Interlimb coordination during turn-swimming
During rectilinear swimming, freshwater turtles exhibit forward swim motor patterns in all four limbs, with 1:1 alternating coordination of contralateral limbs (right–left forelimbs and right–left hindlimbs, mean phase ≈ 0.5), 1:1 out-of-phase coordination of ipsilateral limbs (right forelimb–hindlimb and left forelimb–hindlimb, mean phase ≈0.4), and 1:1 nearly in-phase coordination of diagonal limbs (right forelimb–left hindlimb and left forelimb–right hindlimb, mean phase ≈ 0.9) (Stein 1978; Davenport et al. 1984; Field and Stein 1997b; Gillis and Blob 2001; Samara and Currie 2007, 2008b). Field and Stein (1997b) also showed that the hindlimbs continued to alternate 1:1 (mean phase = 0.48) during voluntary turning behavior in turtles held by a stationary band-clamp, in which one hindlimb executed forward swimming and the other executed back-paddling. During rotation-evoked turning in the current study, we found that contralateral back-paddling and forward swimming hips were still out of phase with each other (mean phase = 0.29), but not clearly alternating as they were in Field and Stein (1997b).
We demonstrated in previous work that the alternating right–left hindlimb movements that occur during voluntary rectilinear turtle swimming were not dependent on crossed commissural pathways in the spinal hindlimb enlargement (Samara and Currie 2007). By surgically bisecting the posterior spinal cord down the longitudinal midline, including the 5-segment hindlimb enlargement (segments D8–S2) and the pre-enlargement segment (D7), we showed that the phase-coupling of contralateral hindlimb forward swimming movements was unchanged after destruction of all crossed commissural axons in those segments, although hindlimb movement amplitudes were reduced. These results are consistent with a model involving alternating descending propriospinal drive from the left and right sides of the forelimb enlargement, sufficient to maintain the out-of-phase coordination of right and left hindlimbs in the bisected-cord preparation. Thus, descending propriospinal and crossed commissural spinal cord pathways appear to function together during rectilinear swimming as redundant mechanisms to sustain right–left hindlimb alternation during turtle locomotion. The fact that the braking inner forelimb is virtually motionless during rotation-evoked turn-swimming suggests that there is little or no propriospinal rhythmicity arising from inner forelimb circuitry to contribute to interlimb coordination, in contrast to the outer side, which is strongly rhythmic. In this regard, note the strong uncrossed interlimb coordination between the outer forelimb and hindlimb in Fig. 6b (r = 0.94), and the weaker crossed coordination between outer and inner limbs in Fig. 6a (r = 0.67) and c (r = 0.60). This is in contrast to bilaterally symmetrical, rectilinear forward swimming in which all four limbs are rhythmically active and strongly phase-coupled (Samara and Currie 2007, 2008b). We suggest that the weakened crossed coordination among the three rhythmically active limbs during rotation-evoked turning is due to the absence of significant descending propriospinal drive on the side of the braking (inner) forelimb that would normally augment the crossed coupling produced by commissural pathways.
Descending activation of turn-swimming by brainstem command systems
What is the nature of descending brainstem command systems that activate forward swimming in outer limbs versus braking and back-paddling in the inner limbs of turtles during sensory-evoked turning behavior? How do those commands switch between locomotor forms when the direction of imposed rotation is reversed? In vitro head–brain–spinal cord preparations from the lamprey made it possible to activate fictive forward swimming via mechanical or electrical stimulation of trigeminal afferents innervating the skin of the snout (McClellan 1984; Fagerstedt et al. 2001) and permitted intracellular characterization of identifiable reticulospinal neurons. Another approach in lamprey has been to record and sort extracellular spikes from the larger reticulospinal axons in the spinal cords of freely behaving animals and restrained animals subjected to rotational stimulation (Zelenin 2005, 2011); this enabled the activity of identified classes of cell to be correlated with specific rotational stimuli and different forms of locomotion (forward vs. backward swimming). In our own earlier work, we evoked fictive forward swim motor patterns in contralateral hindlimb nerves via electrical stimulation in the dorsolateral funiculus of the mid-body spinal cord in low-spinal immobilized animals (Juranek and Currie 2000) and by stimulation in the anterior-lateral brainstem of decerebrate-immobilized preparations (Currie 2003). Future studies should investigate the origin and identity of descending reticulospinal drive that selects and activates asymmetric locomotor forms on the right and left sides during sensory-evoked turtle turning behavior. We note that an in vitro turtle brain/ear-capsules preparation has been in use for years to examine the neuronal basis of rotation-evoked vestibulo-ocular reflexes (Fan et al. 1997; Jones and Ariel 2008). A similar preparation, with cervical-brachial spinal cord and selected forelimb nerves attached, might permit a cellular investigation of sensory-activated fictive turn-swimming and a better understanding of vertebrate locomotor pattern selection.
We thank Basilio Haro and Clifford Jung Hun Kye for assistance with video digitization. All procedures were performed according to protocols approved by the UC Riverside Institutional Animal Care and Use Committee in accordance with federal guidelines. This research was supported by U.C.R. Academic Senate grants to S.N.C.