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Figure 2. Effect of pancuronium, benzocaine and MS-222 on the horizontal aVOR in Xenopus tadpoles.A, Schematic of the experimental setting depicting the semi-intact preparation, horizontal sinusoidal turntable positional changes (Tpos; 0.5 Hz, ±10°) and video capture of eye motion. B,C, Single sweeps of co-aligned horizontal positional oscillations of the left (green) and right (red) eye, extracted from video sequences during turntable rotation (B) and average responses (± SEM, shaded areas; n = 8) over a single cycle (C); note that table and eye motion are oppositely directed in agreement with aVOR behavior. D-I, Effect of bath-applied pancuronium (0.12 mg/ml, D,G), benzocaine (0.05%, E,H) and MS-222 (0.05%, F,I) on single sweeps of horizontal sinusoidal turntable motion (Tpos)-evoked eye movements (D-F) and average response (± SEM, shaded areas; n = 8, respectively) over a single cycle (dashed line; G-I); recordings (same color code in G-I) were obtained before (Control, black traces), 5 min (orange traces) and 15 min (red traces) after onset of drug application as well as 1 hour (blue traces, D-F) and 3 hours after beginning of the wash-out (brown trace, F). J, Progression of pharmacological influence on eye motion gain (ratio of eye/table motion magnitude) after application/wash-out of pancuronium, benzocaine and MS-222. K, Dose-response curve of MS-222 impact on the gain of rotation-induced eye motion 5 and 15 min after drug application; the significance of difference in J,K is indicated: *, p≤0.01; **, p≤0.001; ***, p≤0.0001, with respect to control values. Calibration bars in D,G also apply to E,F and H,I, respectively.
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Figure 3. Effect of pancuronium, benzocaine and MS-222 on extraocular motoneuronal discharge in Xenopus tadpoles.A, Schematic of the experimental setting depicting the semi-intact preparation, roll-axis turntable positional changes (Tpos, 0.5 Hz, ±10°) and extracellular recordings of spontaneous and motion-evoked multiple-unit discharge of the superior oblique (SO) eye muscle-innervating trochlear nerve. B-D, Single sweeps of sinusoidal roll motion (Tpos)-evoked discharge modulation (B1,C1,D2), spontaneous firing of the trochlear nerve (D1) and average firing rate modulation (± SEM, shaded areas) over a single cycle (dashed line; B2,C2,D3) before (Control, gray traces and plots), 5 min (orange traces and plots), 15 min (red traces and plots) and after wash-out (blue traces and plots) of bath-applied pancuronium (0.12 mg/ml, B), benzocaine (0.05%, C) and MS-222 (0.05%, D); inset in B2 shows relative peak firing rates before (gray) and after pancuronium application (red); insets in D1,3 show relative resting rates (D1) and peak firing rates during roll motion stimulation (D3) before and during drug application and after wash-out; number of experiments in parentheses in insets; while no significant change (n.s., p = 0.975) of the peak firing rate occurred after adding pancuronium to the bath (B), benzocaine (C) and MS-222 (D) blocked the motor responses entirely (***, p≤0.0001). E, Dose-response curve of benzocaine and MS-222 (n = 20, respectively) on spontaneous and motion-triggered peak modulation values of multiple-unit trochlear motor spike discharge.
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Figure 4. Effect of benzocaine on extraocular motoneuronal discharge in Xenopus tadpoles.A-B, Single sweeps of spontaneous (A), roll motion (Tpos)-evoked (B1) multiple-unit discharge of the superior oblique (SO) eye muscle-innervating trochlear nerve and average firing rate modulation (± SEM, shaded areas; B2) over a single cycle (dashed line; B2) before (Control, gray traces and plot), 10 min after application of dimethylsulfoxide (Control w/DMSO, green traces and plot), 5 min (orange traces) and 15 min after application of benzocaine (0.05%; red traces) and four hours after wash-out (blue traces and plot); inset in A show relative resting rates before (gray), in the presence of DMSO (green), benzocaine (orange and red, complete block) and after wash-out (blue); number of experiments in parentheses; while no significant change (n.s., p = 0.880) of the peak firing rate occurred after adding DMSO to the bath, benzocaine (0.05%) blocked the motor responses entirely (***, p≤0.0001).
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Figure 5. Effect of MS-222 on the spike discharge of mechanoreceptor sensory afferent fibers in Xenopus tadpoles.A, Schematic of the experimental setting depicting the semi-intact preparation, vibrational stimulation of lateral line neuromasts (10 Hz, ±150 µm) and extracellular recordings of the multiple-unit discharge of the peripheral portion of the posterior lateral line nerve (PLLN). B,C, Single sweeps of spontaneous (B1) and vibration-evoked discharge modulation (C1) in the PLLN before (Control, gray traces and plots), 5 min (orange traces and plots) and 15 min after bath-application (red traces and plots) and after wash-out (blue traces and plots) of MS-222 (0.05%); relative resting (B2) and peak firing rates (C2) before, during and after wash-out show the reversible drug effect; number of experiments in parentheses. D, Dose-response curve of MS-222 on multiple-unit spontaneous and sensory stimulus-evoked spike discharge modulation in mechanosensory (lateral line and vestibular) nerve fibers. E, Schematic of the experimental setting depicting the semi-intact preparation, roll-axis turntable positional changes (Tpos, 0.5 Hz, ±10°) and extracellular recordings of the multiple-unit discharge of the peripheral portion of N.VIII (vestibular). F,G, Single sweeps of spontaneous (F1) and roll motion-evoked discharge modulation (G1) of the N.VIII and average firing rate modulation (± SEM, shaded areas; n = 8, respectively) over a single cycle (dashed line; G2) before (Control, gray traces and plots), 5 min (orange traces and plots) and 15 min after bath-application (red traces and plots) and after wash-out (blue traces and plots) of MS-222 (0.05%); #,## in G1,2 indicate activated responses in different nerve fibers by either direction of the sinusoidal roll motion; relative resting (F2) and peak firing rates (G3) before, during and after wash-out show a complete block and partially reversed drug effect; number of experiments in parentheses; ***, p≤0.0001; n.s., not significant.
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Figure 1. Semi-intact Xenopus laevis preparation with functional sensory and motor elements for in vitro pharmacological experimentation.
A-C, Photomicrographs, depicting an isolated head of a Xenopus laevis tadpole before (A1) and after opening of the dorsal cranium (A2) and removal of the skin to disconnect the forebrain (FB, white dotted line in A2) and to make the brainstem, otic capsule (OtC in A1,
B1,2) and extraocular neuromuscular system (i.e. N.IV motor innervation of the SO eye muscle in C) accessible for extracellular single- and multiple-unit recordings and bath-application of drugs; further removal of the entire hindbrain (HB, white dashed line in B1) yielded access to the peripheral part of the N.VIII for electrophysiological recordings of afferent fibers innervating the different vestibular sensory endorgans in the otic capsule (B2). D,E, A computer-controlled turntable (D) allowed spatially specific application of sinusoidal rotational stimuli of semi-intact preparations in the horizontal (red arrows) and vertical planes (e.g. left AC - right PC, blue arrows in E). F,G, Typical multiple-unit discharge modulation, recorded from N.IV (trochlear nerve, F) and the anterior branch of N.VIII (vestibular nerve, G) during 5 cycles of rotation (0.5 Hz) in the AC - PC plane, illustrated in E. H, In vitro optical motion recording of the left eye (E) during 5 cycles of rotation in the horizontal plane (0.5 Hz). Scale bar is 1 mm in A1,2 and 0.2 mm in B1,C; calibration bar and positional excursion amplitude of the rotational stimulus in F also apply to G,H; AC, PC, anterior, posterior vertical semicircular canal; LR, MR, SR, lateral, medial, superior rectus eye muscle; SO, superior oblique eye muscle; OT, optic tectum, Tpos, table position.
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