Controlled landing requires preparation. Mammals and bipedal birds vary how they prepare for landing by predicting the timing and magnitude of impact from the integration of visual and non-visual information. Here we explore how an animal that moves primarily through hopping, Rhinella marina, the cane toad, integrates sensory information to modulate landing preparation. Earlier work suggests toads may modulate landing preparation using predictions of impact timing and/or magnitude based on non-visual sensory feedback during takeoff rather than visual cues about the landing itself. Here we disentangled takeoff and landing conditions by hopping toads off platforms of different heights and measured electromyographic (EMG) activity of an elbow extensor, m. anconeus, and used high-speed motion capture to quantify whole body and forelimb kinematics to test how toads integrate visual and non-visual information in landing preparation. We asked two questions: 1) when they conflict, do toads correlate landing preparation with takeoff or landing conditions?  And 2) for hops with the same takeoff conditions, does visual information alter the timing of landing preparation?  We found that takeoff conditions are a better predictor of the onset of landing preparation than landing conditions, but that visual information is not ignored. When hopping off higher platforms, toads start to prepare for landing later when takeoff conditions are invariant. This suggests that, unlike mammals, toads prioritize non-visual sensory feedback about takeoff conditions to coordinate landing, but that they do integrate visual information to fine-tune landing preparation.

Animals

Six female cane toads, R. marina, ranging in mass from 63 to 170 g were used for kinematic analysis and electromyography (EMG) recordings. All animals were housed in groups of three to four in large aquaria on a 12 hr. light dark cycle and fed crickets several times weekly.  All experimental work was approved by Mount Holyoke College's IACUC.

Electromyography and kinematic data collection

EMG data were collected from the m. anconeus, an elbow extensor known to show distance-dependent pre-landing onset timing (Gillis et al., 2010). To implant electrodes, toads were anesthetized in a solution of MS-222 (1.5 g l-1). Once anesthetized, 1-2 cm skin incisions were made along each humerus to expose the muscle. Bilateral implants were used to increase the likelihood of successful data collection. Bipolar electrodes were made and implanted as described in detail in previous work (Gillis et al., 2010). EMG signals were amplified 1000X with Grass P511 preamplifiers using a notch filter at 60 Hz. Signals were digitized at 5000 Hz using a NIDAQ 16-bit A/D converter and a custom LABVIEW program and saved onto a personal computer.

Toads alter both the timing of pre-landing EMG activity and the beginning of elbow extension with hop distance.  For completeness, we collected both EMG data and forelimb kinematics. For kinematic data collection, small squares of white cardboard (~3X3 mm) were glued to the skin bilaterally at the wrist and elbow joints as well as at mid-point of the humerus (markers at the shoulder joint are readily obscured). In addition, four markers were also used to form a T along the back of the animal as described in detail in previous work (Cox and Gillis, 2015). Once recovered from anesthesia, animals were placed in a well-lit enclosure (64X107 cm) and, in a random order, hopped off three different height platforms (0 cm (41 hops), 6 cm (36 hops) and 10 cm (31 hops)) lined with felt to ensure purchase. Between 3 and 12 hops per condition per toad were included in the analysis (mean:6.8, SD:3.8 hops/treatment/toad) for a total of 102 hops. Videos of hops were taken with two Fastec HiSpec1 high-speed video cameras at 500 fps. For three-dimensional spatial calibration, a 64-point calibration cube (21X21 cm) was digitized in MATLAB using DLTdv5 (Hedrick, 2008). Videos were synchronized with EMG signals using a 5V trigger pulse that stopped video and EMG recording and was included on its own channel with EMG data. After hopping trials, toads were euthanized by overnight submersion in MS-222 (1.5 gl-1). Post-mortem dissections were used to confirm electrode placements.

Data analysis

Landing preparation in toads is characterized by both distance-dependent amounts of elbow extension and activation of the underlying forelimb musculature at touchdown. But landing preparation at touchdown is not sufficient to determine whether the motor control strategy is altered. Modulation of pre-landing EMG activity and elbow configuration at touchdown can be achieved without varying the timing or intensity of muscle activation. A toad could start elbow extension and muscle activation at the same time relative to liftoff and maintain the same rate of elbow extension and increase of activation intensity throughout the hop. With this strategy, long hops provide more time to generate greater levels of activation or elbow extension without modulating the motor control strategy. Thus, to discriminate changes in motor control, we chose to primarily focus our analysis on how toads changed the timing of landing preparation (the onset and duration of pre-landing EMG activity or elbow extension) since the timing of prelanding EMG activity for the muscle instrumented varied more consistently with hop duration than amplitude. For completeness, we also evaluated metrics of the intensity of both pre-landing EMG activity and the rate of elbow extension 60 ms after liftoff.  To quantify how variations in motor control influence landing preparation at touchdown, we also measured EMG intensity over a 60 ms window before landing as well as the elbow’s configuration (degree of extension) at touchdown.

Video Analysis: Videos were analyzed to identify the timing of the onset of movement (T0), time of takeoff (takeoffs) and landing for each limb (ltouchs: touchdown of left limb, rtouchs: touchdown of right limb, touchs: time of first limb to touchdown). The onset of movement was defined as the moment the toad's vertical velocity first topped 5 cm/s. Three-dimensional coordinates of the forelimbs were calculated with Matlab software (Hedrick, 2008). Data were smoothed with a quintic spline interpolation, and elbow angle, the timing of extension onset (E2) and extension velocity (EEvel60), as well as the trajectory and velocity of the animal during the hop, were calculated as described elsewhere from the vertical velocity of the animal at takeoff (maxT0velVBB), the takeoff height of the snout of the animal (maxT0velHeight) and the height of the animal at reast (startHeight).

Predictions of touchdown times: The time touchdown would have occurred if animals were hopping on flat ground was calculated with equations of motion from the vertical velocity of the center of mass and height of the marker on the tip of the animal’s snout at takeoff. Time of touchdown, t_TD was the sum of the time of liftoff, t_LO, the time to rise to peak height, t_R, and the time to fall back to starting height, t_F.

t_TD= t_LO+t_R+t_F.

Starting height was the height of the tip of snout before hop initiation. The time to peak height, t_R, was found from the vertical component of the velocity of the center of mass at liftoff, Vz_LO, and acceleration due to gravity.  Given that the vertical velocity at peak height is zero,

t_R = Vz_LO/g.

The time to fall back to start height, t_F, was calculated from the total height to fall from peak height, h_P, back to starting height, h_S. Given that rise height can be found from  12at² , the peak height was the sum of the takeoff height, h_LO,

h_P= h_LO+ gt_R²2.

The time to fall, then, is

t_F = 2h_pg.

While several approaches to calculating time of flight were tested, including limb angle at touchdown and equations to estimate anuran jumps (Marsh, 1994), this approach best fit our data for flat hops (Fig. 2B). We evaluated how well our predictions lined up with impact time by comparing predicted to actual impact times for flat hops (Flat hops from Group 1) by fitting two mixed models, a full model with predicted touchdown time as the response variable and actual touchdown time as the fixed effect and a null model with no fixed effect. In both models, individuals were a random factor. The R² value for the full model was calculated from the relative likelihood of each model determined by their AIC values (Nakagawa).

Electromyography and kinematics: EMG activity was analyzed using customized MATLAB scripts in which the onset timing of pre-landing muscle activity (onanc) was identified visually for each hop and the rectified, integrated area, or intensity, of the EMG signals for the first 60 ms after onset (int60Onset)  and the last 60 ms before landing (intAL60touch) was calculated. To control for differences in electrode construction and placement between individuals, each muscle's EMG intensities were normalized to the largest intensity value observed for each animal.  The onset of elbow extension was determined using methods described elsewhere and elbow extension was calculated at touchdown (TDext). The rate of elbow extension (EE_Vel60) was the average rate of change of elbow extension during the 60 ms after the onset of elbow extension. Data was filtered to only include signals from the limb that touched down first.

ReadMe Notes for ‘DataFileCoxGillis2019’.

This data accompanies the paper “The integration of sensory feedback in the modulation of anuran landing preparation”  By SM Cox and GB Gillis published in Journal of Experimental Biology 2019

The file is a csv file with the following columns:

toad                      : animal identifier

hop                       : hop number f: hop on flat ground, l: hop  off 6 cm platform, h: hop of 10 cm platform

takeoffs               : time vertical velocity reaches 5 cm/s (s)

ltouchs                 : time left forelimb touchdown (s)

rtouchs                : time right forelimb touched down (s)

mass                     : animal mass (mg)

T0                          : start of hop (s)

E2t                        : time of start of elbow extension in preparation for landing

onLanc                 : time of start of prelanding EMG activity left forelimb

onRanc                : time of start of prelanding EMG activity left forelimb

dist                        : hop distance (cm)

height                   : vertical height difference from resting to touchdown (cm)

TDext                   : elbow extension of forelimbs at touchdown (180 = straight limb)

treat                     : treatment: f: hop on flat ground, l: hop  off 6 cm platform, h: hop of 10 cm platform

touchs                  : time first limb touchdown

intAL60touch      : integrated EMG 60 ms before touchdown

maxTOvelAngleBB            : angle of torso from horizontal at maximum vertical velocity (deg)

maxTOvelVBB    : maximum vertical velocity of snout at takeoff (cm/s)

maxTOvelHeight: height of snout of the animal at time of maximum vertical velocity during takeoff (cm)

maxTOvelBBT     : time of maximum vertical velocity during takeoff (s)

dropHeight          : difference between maximum vertical position during hop and landing height (cm)

startHeight          : height of the animal at rest from reference zero

LR                          : which limb touched down first L: left, R: right

onanc                   : timing of onset of EMG for limb that touched down first

predictTD            : timing of predicted touchdown

TD60Vel               : elbow extension velocity 60 ms before touchdown

int60Onset          :Integrated EMG activity 60 ms after beginning of elbow extension

EEvel60                : Average elbow extension 60 ms before touchdown (deg/msec)