Semicircular canal size determines the developmental onset of angular vestibuloocular reflexes in larval Xenopus

Abstract

Semicircular canals have been sensors of angular acceleration for 450 million years. This vertebrate adaptation enhances survival by implementing postural and visual stabilization during motion in a three-dimensional environment. We used an integrated neuroethological approach in larval Xenopus to demonstrate that semicircular canal dimensions, and not the function of other elements, determines the onset of angular acceleration detection. Before angular vestibuloocular function in either the vertical or horizontal planes, at stages 47 and 48, respectively, each individual component of the vestibuloocular system was shown to be operational: extraocular muscles could be activated, central neural pathways were complete, and canal hair cells were capable of evoking graded responses. For Xenopus, a minimum semicircular canal lumen radius of 60 microm was necessary to permit endolymph displacement sufficient for sensor function at peak accelerations of 400 degrees /s(2). An intra-animal comparison demonstrated that this size is reached in the vertical canals earlier in development than in the horizontal canals, corresponding to the earlier onset of vertical canal-activated ocular motor behavior. Because size constitutes a biophysical threshold for canal-evoked behavior in other vertebrates, such as zebrafish, we suggest that the semicircular canal lumen and canal circuit radius are limiting the onset of vestibular function in all small vertebrates. Given that the onset of gravitoinertial acceleration detection precedes angular acceleration detection by up to 10 d in Xenopus, these results question how the known precise spatial patterning of utricular and canal afferents in adults is achieved during development.

Figure 1.

Schematic depicting locations of nerve recording and electrical and pressure stimulation sites in the labyrinth in larval Xenopus. A, B, The activity of motor nerves innervating extraocular muscles were recorded with suction electrodes (A) after either electrical stimulation (Stim) of an identified semicircular canal nerve or displacement of the canal cupula (Cup) by pressure injection of endolymph Ringer's solution, e.g., into the horizontal (Hor) canal (B). C, Graph of body length (mean ± SD) versus developmental stage according to Nieuwkoop and Faber (1994). Superimposed on the graph is a photomontage of larval Xenopus from stages 42–49 showing the significant change in length and head size with age. The peak growth in larval size occurs between stages 46 and 49. Adjacent to each marker on the graph are the ages of the larvae in days postfertilization. Inf, Inferior; Lat, lateral; Med, medial; Opt, optic; Sup, superior; Utr, utricle.

Figure 2.

VOR behavior and extraocular nerve discharge of larval Xenopus in response to horizontal axis rotation. A, B, Larval Xenopus exhibit a robust gVOR to dynamic rotation (0.125 Hz; ±10°/s) in the horizontal (roll) plane at stage 45 (A) that becomes more refined by stage 47 (B). C, D, Suction electrode recording of the nerve innervating the left medial rectus in a stage 47 animal demonstrates a strong modulation in motor neuron activity during horizontal axis rotation in the roll plane. One cycle of the recording shown in C at an extended time scale is shown in D. E, Averaged response ± SD (red area) to horizontal axis rotation from five stage 47 larvae show discharge modulation correlated with head velocity. Pos, Position; Vel, velocity; Med, medial.

Figure 3.

VOR behavior and extraocular nerve discharge of larval Xenopus in response to vertical axis rotation. A, A stage 47 larvae did not exhibit any behavioral response during angular acceleration stimulation in the vertical axis (1 Hz; ±400°/s²). B, Only by stage 49 did Xenopus larvae demonstrate a reliable aVOR during angular acceleration stimulation. C–H, Compatible with the behavior, the activity of the abducens nerve innervating the left lateral rectus in a stage 49 (F–H) but not in a stage 47 (C–E) Xenopus was modulated in response to the same stimuli. D, G, One cycle of the recordings in C and F, respectively, at an extended time scale; the average ± SD (red area) of five animals over 30 cycles is summarized in E and H, respectively. Pos, Position; Vel, velocity; Lat, lateral.

Figure 4.

Ontogenetic progression of gravitoinertial and angular VOR in larval Xenopus. From very early stages, the utricular system of larval Xenopus responds well to stimulation with gVOR eye velocity gains consistently >0.6. In contrast, angular acceleration of ±400°/s² is unable to reliably trigger an aVOR in larval Xenopus until stage 49, 9 d after free swimming has commenced. Error bars indicate SD.

Figure 5.

Electrical and pressure activation of horizontal canal afferent responses in stage 47 Xenopus larvae. A, B, Electrical stimulation of the horizontal canal nerve triggered spike activity in both the contralateral (Contra) lateral rectus (A) and ipsilateral (Ipsi) medial rectus nerves (B) as observed in six superimposed single sweep responses. Averaged responses (red) demonstrate a 3 ms difference between response onset in the two nerves. C, D, Lateral rectus nerve activity transiently increased (C) after a brief (1 s) pressure pulse into the contralateral horizontal canal and was inhibited (D) after a pressure pulse into the ipsilateral horizontal canal; mean (n = 3) ± SD (red area). E, Lateral rectus nerve activity exhibited a graded inhibitory response to increasingly longer pressure pulses into the ipsilateral horizontal canal. In all cases, neural activity returned to baseline levels suggesting the cupula was not damaged by the stimulus. Other abbreviations are the same as in Figure 1. Data in A–D are from the same animal.

Figure 6.

Semicircular canals increase in size during Xenopus larval development. A–D, Photomicrographs of the left otic capsule, viewed dorsally from developmental stages 46–49, illustrate the increase in dimensions of the horizontal semicircular canal. Black arrows in A–D indicate the location where the canal lumen radii were measured. Superimposed in D is a schematic illustrating the parameters used to determine circuit and lumen radii. E, Plot of horizontal canal lumen and circuit radius (mean ± SD) at different developmental stages before and after aVOR onset. Parameters before (stage 47) and after aVOR onset (stage 48) were significantly different (**p ≤ 0.01, Mann–Whitney U test); numbers of specimens used for the analysis at each stage are indicated in E. The scale bar in D applies to A–C. Ant, Anterior; Amp, ampulla; Lag, lagena; R_a, R_b, large and small axes of canal circuit radius, respectively; r, radius of canal lumen; Sac, saccule. Other abbreviations are the same as in Figure 1.

Figure 7.

Extraocular nerve discharge during vertical axis rotation in larval Xenopus. A–F, Vertical axis rotation designed to minimize utricle activation and maximally activate one vertical canal pair (C, inset) modulates the spike discharge of the superior (Sup) oblique nerve in stage 47 animals (A–C) but does not modulate nerve activity in stage 46 animals (D–F). B and E show one cycle of the recordings in A and D at an extended time scale, respectively; the average ± SD (red area) of five animals over 30 cycles is summarized in C and F, respectively. G, H, A brief pressure pulse of endolymph Ringer's solution into the ipsilateral (Ipsi) posterior (Post) (G) and contralateral (Contra) anterior (Ant) vertical semicircular canal (H) in the same stage 46 specimen shown in D–F modulated the discharge of the superior oblique nerve. The scale bar in H also applies to G.

Figure 8.

Photomicrographs of the left otic capsule from stage 46 and 48 Xenopus larvae. A, B, Lateral view of the anterior (Ant) and posterior (Post) vertical canals in left otic capsule. Black arrows indicate location where canal lumen radii were measured. C, Plot of vertical canal lumen radius (anterior and posterior canals combined) and horizontal lumen radius (mean ± SD) at different developmental stages before and after aVOR onset. Canal lumen radii before and after horizontal aVOR onset (stage 47 and 48) and before and after vertical aVOR onset (stage 46 and 47) were significantly different (**p ≤ 0.01; Mann–Whitney U test); numbers of specimens used for the analysis at each stage are indicated in C. Other abbreviations are the same as in Figure 1.

Figure 9.

Plots of mean canal lumen radius (r) versus mean canal circuit radius (R) of the horizontal canals of larval Xenopus and zebrafish. A, Superimposed are canal dimensions for selected classes of vertebrates adapted from the study by Muller (1999). Oblique lines represent theoretical endolymph displacement in micrometers for a given angular velocity (1 radian/s or 57.3°/s) and the dashed gray line is the regression line of r = 38.9 × r^1.60 all as calculated by Muller (1999). B, The solid blue line is the regression line of r = 42.2 × r^1.59 for stage 47–52 larvae. Measurements for the horizontal canal lumen and circuit radii for larval zebrafish and mouse are from previous studies [5, 20 dpf (Bever and Fekete, 2002); 35 dpf and behavior (Beck et al., 2004b); mouse (Calabrese and Hullar, 2006)]. The mouse lumen radius is shown at its smallest estimated size. For stage 48 Xenopus, the horizontal aVOR rises above threshold for the first time and becomes fully active at stage 49. Based on the behavioral response of Xenopus and zebrafish larvae, the oblique solid red line is a generalized sensitivity threshold for vertebrate aVOR behavior for this stimulus.