Common onset of predictive spino-ocular, visual motion and otolith organ-evoked eye movements
Free swimming in Xenopus laevis larvae commences around stage 37/38 followed by a rapid acquisition of the typical kinematic profile that remains essentially unaltered between stage 38-4029 and stage 509. The early developmental occurrence of undulatory swimming, however, is at variance with the lack of locomotor efference copy-driven gaze-stabilizing spino-ocular motor behavior8 at stage 40 (Fig. 1a1; Supplemental fig. 1b, N = 3). In fact, the earliest activation of reliable and robust compensatory eye movements triggered by ascending copies of spinal locomotor signals was observed at stage 42 and had a gain of 0.34 ± 0.08 (N = 3; Fig. 1a2; Supplemental fig. 1b). This onset coincided with the developmental acquisition of sensory stimulus-driven visuo- and gravitoinertial (otolith organ) ocular motor behaviors. In fact, stage 40 larvae were incapable of expressing a horizontal optokinetic (OKR; Fig. 1b1; Supplemental fig. 1b, N = 5) or a gravitoinertial vestibulo-ocular reflex (gVOR; Fig. 1c1; Supplemental fig. 1b, N = 5). First evidence for the presence of these sensory-motor-driven gaze-stabilizing eye movements was encountered at stage 42 (Fig 1b2,c2). At that stage, the OKR and gVOR consisted of small, yet reliable eye movements with gains that exceeded 0.15 (0.16 ± 0.02 for OKR, N = 5 and 0.18 ± 0.05 for gVOR, N = 7; Supplemental fig. 1b). Thereafter, all gaze-stabilizing eye movement components increased further in robustness to reach gains at stage 45 (N = 4) of 0.65 ± 0.02 for the spino-ocular motor coupling, 0.33 ± 0.08 for the OKR and 0.47 ± 0.21 for the gVOR (Fig. 1a3-c3, Supplemental fig. 1b). The performance continued to improve until maximal gain values were reached at stage 48 with a generally better performance of the gVOR compared to the OKR (Supplemental fig. 1c-e). The common developmental onset of all three gaze-stabilizing eye movement components coincided with a major step in the maturation of the rhythmically alternating spinal locomotor output of the Vrs, which changes from single-spikes at stage 37/38 to spike bursts at stage 42 (Supplemental fig. 1a)30.
Interactive maturation of locomotor-induced ocular motor behavior and angular vestibulo-ocular reflexes
Spino-ocular motor coupling experiences extensive spatio-temporal plasticity during the amphibian metamorphosis, culminating during the emergence of appendicular and concurrent loss of axial muscle-based locomotor strategy in post-metamorphic froglets10, 24. To avoid interference with plasticity processes related to this latter final transition at stage 59, evaluation of the developmental plasticity in spino-ocular motor coupling was therefore restricted to larvae prior to this stage (Fig. 2a). At stage 49, ascending spinal locomotor efference copies, in the absence of motion-related visuo-vestibular sensory inputs, elicited phase-coupled oscillatory eye movements with relatively invariable magnitudes of 5-7° (Fig. 2a1-b1) independent of the tail bending amplitudes (R = 0.05 ± 0.05, r² = 0.02 ± 0.02, N = 8; Fig. 2b1-b2). Consequently, the gain for locomotor efference copy-induced eye movements was relatively high for small tail excursions (0.74 ± 0.10 at left-right tail deflections of ~10°, N = 8; Fig. 2b3, left panel) but decreased gradually for larger tail oscillations (Fig. 2b3; Supplemental fig. 2a). In contrast, at stage 58, eye and tail movement amplitudes were more closely correlated (R = 0.56 ± 0.11; r² = 0.46 ± 0.07, N = 8; Fig. 2b1-b2) with a rather invariant gain of ~0.4 independent of tail excursion magnitudes (Fig. 2b3, left panel; Supplemental fig. 2a). The spino-ocular motor coupling revealed an average phase lag re deflection of the rostral tail region of 0-30° (Fig. 2b3, right panel) that was unrelated to tail excursion magnitude as well as age (Watson-Williams F-test, F = 0.952, p = 0.337). The relative timing of the responses was rather variable at stage 49 but became considerably more homogeneous in older larvae (Supplemental fig. 2d,e, compare vector length of the mean phase relation).
The developmental period between stage 49 and 58 is generally characterized by a considerable improvement of the performance of the horizontal angular vestibulo-ocular reflex (aVOR; Fig. 2c-d). First noticeable compensatory eye movements during horizontal head/body rotation were observed as early as stage 49-50 (Fig. 2c1; N = 10), although with a gain below 0.2, independent of stimulus strength (Fig. 2d1,2). This relatively late aVOR onset, compared to gVOR and OKR onsets is related to the necessity of semicircular canals to acquire sufficiently large duct diameters to become operational, which in Xenopus occurs shortly before stage 49-5020. Thereafter, aVOR gains became significantly increased by stage 52 (N = 6), especially during large amplitude head rotations (1 Hz, ± 15-25°; Fig. 2d,e) with further enhancement, although at a slower rate, until stage 58 (Fig. 2d,e; N = 5). The phase relation between semicircular canal-induced eye movements and stimulus waveform was relatively stable between stage 49 and stage 58, with small phase leads of the response re stimulus (~0-30°) at 1Hz (Fig. 2d1-2).
Collectively, these findings suggest that implementation and refinement of locomotor efference copy-driven eye movements are closely intertwined with the developmental maturation of visuo- and vestibulo-ocular reflexes. Thus, spino-ocular motor coupling co-emerges together with visuo- and otolith organ-driven ocular motor responses at stage 42 followed by progressive improvement in performance with a common time course until stage 49 (Fig. 1; Supplemental fig. 1). Thereafter, further enhancement of locomotor efference copy-driven eye movements coincides with the maturation of the horizontal aVOR, with a potentially cross-functional entanglement of the two ocular motor behaviors (Fig. 2e).
The potential impact of gradually improving aVOR performance on the maturation of locomotor efference copy-driven eye movements was elucidated in semicircular canal-deficient stage 52 larvae. These animals were obtained following bilateral injection of hyaluronidase into both otic capsules at stage 44 prior to inner ear duct completion26. This experimental perturbation at early larval stages caused a failure to form semicircular canals on both sides and consequently disabled the aVOR25. In compliance with the absence of functional semicircular canals, imposed horizontal head rotation at stage 52 failed to elicit compensatory eye movements (Fig. 3a), while in contrast locomotor efference copy-driven eye movements during tail undulations persisted (Fig. 3b). However, the performance of these predictive eye movements was inferior to that of age-matched controls and more comparable to that of younger larvae (Fig. 3c,d). The apparent retardation in spino-ocular motor performance might have derived from altered tail movements in semicircular canal-deficient stage 52 larvae, which in fact had rather small amplitudes and high oscillation frequencies (Fig. 3e,f) reminiscent of those, predominating in younger, stage 48 controls prior to aVOR onset (Fig. 3e,f)31. This suggests that semicircular canal signals are not only required for a functional aVOR, but also for the maturation of locomotor efference copy-driven predictive eye movements with stage-specific dynamic profiles, likely through an impact on locomotor pattern formation.
Progressive developmental influence of semicircular canals on locomotion-induced eye movements
The interaction between semicircular canal- and locomotor efference copy-driven eye movements was further evaluated by calculating the impact of horizontal head rotations on concurrent spino-ocular motor performance during undulatory swimming between developmental stages 45 and 58. Discrimination of vestibular and locomotor efference copy-driven response components in resultant eye movements was achieved by spectral analyses, which required activation of the two components at different frequencies, respectively. Accordingly, passive head rotation was applied at 1 Hz (± 10° positional excursions), known to elicit an aVOR in larvae older than stage 4920. This stimulus frequency was sufficiently distant from swim-related tail beat oscillations that occurred within a range of 6-15 Hz31.
At stage 48, oscillatory eye movements during swim-related tail undulations and concurrent horizontal sinusoidal head rotation were robust (Fig. 4a1). However, noticeable 1 Hz vestibular motion stimulus-related eye movement components were absent as indicated by the lack of the respective spectral component (see absence of red band at 1 Hz in the spectrogram in Fig. 4b1 and of peak at 1Hz in the periodogram in Fig. 4c1). This lack complies with the known failure to elicit an aVOR at this developmental stage20. The eye movement frequency component at stage 48 therefore corresponds exclusively to the swim frequency (~12 Hz), indicated by the red band in the spectrogram and by the respective peak in the periodogram (Fig. 4b1,c1). As expected, at stage 52, eye movements during tail undulations and sinusoidal horizontal head rotation were also rather robust (Fig. 4a2). Spectral analysis demonstrated, however, that eye movements contained two clearly discernable components that coincided with the vestibular stimulus frequency (1 Hz) and the tail beat oscillations (~6 Hz), indicated in the typical example by red bands in the spectrogram (Fig. 4b2) and the corresponding peaks in the periodogram (Fig. 4c2).
Respective spectral analyses of eye movements during swimming and concurrent head rotation in larvae between stage 45 and 58 revealed a progressive reconfiguration of sensory-motor and predictive spino-ocular response components (Fig. 4d,e). Horizontal rotation-induced eye motion components experienced a gradual, yet significantly increasing contribution (Kruskal-Wallis test, p < 0.001, N = 17) after the onset of semicircular canal function at stage 49, culminating at stage 58, just prior to the metamorphic switch from tail- to limb-based locomotion (Fig. 4d,e). Possible underlying plasticity mechanisms include an increasing efficacy of peripheral and central processing of semicircular canal signals. This obviously has a direct impact on extraocular motoneuronal activity as well as on spinal central pattern generator performance. A possible mechanism likely includes fusion of vestibular and locomotor efference copy signals at the spinal level before being transmitted by ascending pathways as joint premotor signal to extraocular motoneurons.
The superposition of vestibular and spino-ocular response components for the generation of eye movements during episodes of tail undulations and passive head motion was further specified in extraocular motoneurons as a more sensitive indicator for signal integration (Fig. 5). The spike discharge of the left lateral rectus (LLR) nerve along with the motion of the right eye was recorded in semi-intact preparations at stage 52 (Fig. 5a-c). Fictive swimming, identified by episodes of rhythmic spike bursts in the right spinal ventral root (RVr), caused a phase-coupled spike discharge in the LLR nerve (Fig. 5a,b). Following integration at the neuromuscular interface of the eye muscles, the dynamics of this extraocular motor command corresponded to the horizontal oscillations of the intact, right eye (Reye in Fig. 5b). Spectral analyses of the eye movements and LLR nerve discharge revealed a synchronous oscillation frequency at ~8 Hz, phase-coupled to the rhythm of the fictive swimming (Fig. 5c-e).
Swim-related LLR nerve spiking and eye oscillations were superimposed, however, on a second, slower rhythm with a frequency of 1 Hz (Fig. 5c-e). This eye motion component corresponded to the head rotation-induced generation of ocular motor commands for an aVOR. Integration of signals related to swimming and concurrent sinusoidal head rotation, each with a different frequency, produced broadly tuned ocular motor oscillations with an amplitude modulation that matched the vestibular stimulus rhythm (Fig. 5c1). The presence of two distinct response components (Fig. 5c1 and 5c2, red bands in Fig. 5d and peaks in Fig. 5e) are thus of semicircular canal and spinal locomotor efference copy origin, respectively (Fig. 5e) and after fusion, jointly produce compensatory eye movements. Most interestingly, however, the cyclic spinal Vr discharge was also broadly tuned, producing a spindle-shaped envelope with the timing of the head rotation frequency. This provides suggestive evidence that horizontal semicircular canal signals modulate the activity of spinal CPG circuits with direct implications for locomotor commands but also ascending copies thereof, designated as ocular premotor signals.
Developmental plasticity of vestibular influences on locomotor efference copy-evoked eye movements
Eye movements during swim episodes and simultaneous horizontal head rotation appeared to be governed by cross-modal interactions that principally fell into two categories (Figs. 6a,b, 7a,b). In the first category, typically found in younger larvae, sinusoidal head rotation during tail undulations were found to modulate the amplitude of individual swim cycle-related eye oscillations (Figs. 6a, 7a) with alternating enhancement and attenuation depending on the head rotation half cycle (Fig. 7e, light blue dots). The variations (Δ amplitude, Fig. 7i) were in the range of ~2 - 8° between the largest and smallest eye excursions (5.14 ± 0.9°, N = 10, Fig. 7i). In contrast, the ocular position around which swim-related eye oscillations were centered remained relatively stable (Figs. 6a, 7a,f, light blue dots) with only small baseline positional fluctuations (Δ position, 2.34 ± 0.28°,N = 9, Fig. 7j). The absence of larger, head rotation-timed eye oscillations suggests cancellation of these vestibular sensory signals, in compliance with earlier findings8. This outcome was further substantiated by recordings of extraocular motor activity from the LLR nerve (Figs. 6c, 7c). Individual efference copy-triggered spike burst amplitudes were modulated in phase-time with the head motion (Fig. 6c, reconstructed 1 Hz wavelet; 7c; light red dots in Fig. 7g; Δ amplitude, 178.7 ± 120.2%, N = 5, Fig. 7k). In contrast, the baseline spike rate remained rather unaffected by the head rotation, illustrated in the typical example by minor vestibular stimulus-timed fluctuations of the discharge across a single motion cycle (Fig. 7c; light red dots shown in Fig. 7h; Δ baseline, 77 ± 20%, N = 5, Fig. 7l).
Cross-modal interactions that fell into the second category were typically found in older larvae (Fig. 6b,d). Amplitudes of individual swim cycle-related eye oscillations and corresponding LLR nerve motor commands were modulated by the head rotation. The overall pattern was comparable to that of the first category (Fig. 7b,d), although with somewhat smaller amplitude variations between the largest and smallest eye oscillations (Fig. 7b; dark blue squares in Fig. 7e; Δ amplitude, 4.5 ± 0.7°, N = 8, Fig. 7i). Most noticeable, however, the baseline ocular position around which swim-related eye movements oscillated was considerably modulated with a waveform that corresponded to the head rotation, causing considerable left-right eye movements (Fig. 7b; dark blue squares in Fig. 7f; Δ position, 5.05 ± 0.62°, N = 8, Fig. 7k). This eye movement profile complies with a summation of VOR components and locomotor efference copy-triggered eye oscillations during joint swimming and passive head rotation. This eye motion pattern matched corresponding variations of the spike discharge of the LLR nerve (Figs. 6d, 7d). Locomotor efference copy-triggered spike burst amplitudes as well as the baseline spike activity were modulated during the rotation with a profile that was phase-timed to the head motion (Fig. 7g,h, dark red squares; Δ amplitude, 102.3 ± 29.7%, N = 5; Δ position, 382.6 ± 103.4%, N = 6), compatible with the eye oscillation dynamics (Fig. 7e,f, dark blue squares).
Quantification of the two patterns of signal integration principles across a larger population of animals revealed that the vestibular influence on efference copy-triggered eye oscillations and associated spike bursts appeared to be generally independent of larval age (Fig. 7i,k; Mann Whitney U-test, p = 0.910 and p = 0.690 respectively). Although, age was not indicative for either one of the two integrative principles, the vestibular influence on baseline eye position and extraocular motor spiking was significantly larger in older tadpoles (Fig. 7j,l; Mann Whitney U-test, p < 0.01). In fact, extraocular motor signal cancellation versus summation was typically related to younger versus older tadpoles, respectively (Fig. 7m). Although, either one of the two modes could be found in tadpoles of any age (Sup. Fig. 6), signal cancellation (light green in Fig. 7m) predominated in younger tadpoles (~66% of stage 49 larvae, Fig. 7m) but was rarely observed in older larvae (~10% of stage 58 larvae, Fig. 7m). Conversely, additive signal interactions (summation; dark green in Fig. 7m) of vestibular and intrinsic locomotor-induced extraocular motor signals correspondingly predominated in older larvae (90% at stage 58, Fig. 7m).
Despite the clear evidence for an age-related shift in signal processing (light and dark green bars in Fig. 7m), larval age might only be an indirect classifier. As demonstrated earlier9, 31, the frequency of swim-related tail oscillations decreases progressively during larval development (black dots and bowls in Fig. 7m). Thus, using swim frequency as decisive parameter, cancellation of motion-related ocular motor response components by locomotor efference copies occurred mainly at higher tail undulatory frequencies (~10 Hz, black dots and bowls in Fig. 7m), while summation of signals was found, whenever swimming occurred at a lower frequency such as it is usually the case in stage 58 larvae (~6 Hz, black dots and bowls in Fig. 7m). Accordingly, swim episodes in which vestibular signals were additively combined with locomotor efference copies had significantly lower frequencies (Mann Whitney U-test, p < 0.001; 6.77 ± 0.24 Hz, Fig. 7n) than those where concurrent vestibulo-ocular signals were canceled (9.16 ± 0.60 Hz, Fig. 7n), independent of developmental stage (see color-coded dots in Fig. 7n).
This differential interaction therefore suggests that the interaction between locomotor efference copies and vestibular sensory signals becomes gradually altered during larval development. In young tadpoles at stage 49-50, horizontal semicircular canal signals modify spinal CPG activity, while the VOR-related motor output is suppressed. In older animals, at stage 58, VOR cancelation by locomotor efference copies disappears and instead is replaced by a summation of intrinsic and sensory-motor components in the process of generating extraocular motor commands. Key to this developmental switch is a gradually reduced efficacy or rhythmicity of the locomotor program, visualized by the slower tail undulation during metamorphic progression with concurrent vanishing influences of locomotor efference copies on the ocular motor output.