Saccomanno V et al. (2021), The early development and physiology of Xenopus...

XB-ART-58578

J Neurophysiol 2021 Nov 01;1265:1814-1830. doi: 10.1152/jn.00618.2020.

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The early development and physiology of Xenopus laevis tadpole lateral line system.

Saccomanno V , Love H , Sylvester A , Li WC .

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Xenopus laevis has a lateral line mechanosensory system throughout its full life cycle, and a previous study on prefeeding stage tadpoles revealed that it may play a role in motor responses to both water suction and water jets. Here, we investigated the physiology of the anterior lateral line system in newly hatched tadpoles and the motor outputs induced by its activation in response to brief suction stimuli. High-speed videoing showed tadpoles tended to turn and swim away when strong suction was applied close to the head. The lateral line neuromasts were revealed by using DASPEI staining, and their inactivation with neomycin eliminated tadpole motor responses to suction. In immobilized preparations, suction or electrically stimulating the anterior lateral line nerve reliably initiated swimming but the motor nerve discharges implicating turning was observed only occasionally. The same stimulation applied during ongoing fictive swimming produced a halting response. The anterior lateral line nerve showed spontaneous afferent discharges at rest and increased activity during stimulation. Efferent activities were only recorded during tadpole fictive swimming and were largely synchronous with the ipsilateral motor nerve discharges. Finally, calcium imaging identified neurons with fluorescence increase time-locked with suction stimulation in the hindbrain and midbrain. A cluster of neurons at the entry point of the anterior lateral line nerve in the dorsolateral hindbrain had the shortest latency in their responses, supporting their potential sensory interneuron identity. Future studies need to reveal how the lateral line sensory information is processed by the central circuit to determine tadpole motor behavior.NEW & NOTEWORTHY We studied Xenopus tadpole motor responses to anterior lateral line stimulation using high-speed videos, electrophysiology and calcium imaging. Activating the lateral line reliably started swimming. At high stimulation intensities, turning was observed behaviorally but suitable motor nerve discharges were seen only occasionally in immobilized tadpoles. Suction applied during swimming produced a halting response. We analyzed afferent and efferent activities of the tadpole anterior lateral line nerve and located sensory interneurons using calcium imaging.

???displayArticle.pubmedLink??? 34705593
???displayArticle.pmcLink??? PMC8794055
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Species referenced: Xenopus laevis
GO keywords: swimming behavior [+]

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	Figure 1. High-speed video analyses of tadpole responses to suction at different levels. A: single frames from a 500 fps video showing tadpole turning behavior in the head-toward-nozzle configuration at 10 ms intervals after water level starts to rise inside the suction pipette (0 ms). The maximal bend used to estimate latencies. B: tadpole turning and swimming incidences increase with suction strengths in the head-toward-nozzle configuration while suction applied to tail mainly evokes swimming (tail-toward-nozzle, Pearson’s chi-squared test, P < 0.01, n = 8 tadpoles). C: the likelihood to evoke initial turning increases at high suction levels in the head-toward-nozzle orientation (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.001, n = 8) but in the tail-toward-nozzle orientation turning incidence is lower (independent-samples Mann–Whitney U test for −4 kPa suction trials, P < 0.001, n = 7). D: the success rate for tadpoles swimming out of the suction nozzle increases with suction strength (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.01, n = 8). E: tadpoles produce the first bend to suction consistently toward the exposed side in the head-toward-nozzle (P < 0.05, one-sample Wilcoxon signed rank tests to median of 100%, n = 7) but not in the tail-toward-nozzle orientation. F: the latency from the beginning of suction to the maximum swimming bend decreases with suction strength (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.01, n = 8). In C, E, and F, circles stand for outliers and asterisks are for extremes. G: the latency to the maximum turning bend decreases with suction strength (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.01, n = 8). H: turning bends have longer duration than swimming bends (paired t test, n = 13, *P < 0.001).
	Figure 2. Staining tadpole lateral line neuromasts using 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide (DASPEI) in control and after neomycin treatment. A: converting a color photo of a tadpole after DASPEI staining to gray scale after enhancing the red and yellow channels. B: clustering of neuromasts in a stage 41 tadpole (square). C: DASPEI staining of tadpoles at various stages (only eye region shown). D: neuromast counts for each side of tadpoles at stages 32, 35, 39, and 41 (n = 10 tadpoles each, P < 0.05 and *P < 0.01, one-way ANOVA). E: the appearance of posterior lateral line (LL) neuromasts. Photo shows the red rectangle area in the drawing on the top. F: DASPEI staining of stage 40 tadpoles in control, treatment with 3 mg/mL neomycin for 30 min, and 24 h after neomycin treatment. Orientation of the tadpoles is the same in B, C, E, and F. White arrowheads point at some example neuromasts labeled with DASPEI. Scale bars: 500 µm in A and E; 100 µm in B, C, and F.
	Figure 3. Fictive motor responses in immobilized tadpoles elicited by local suction close to the left eyecup. A: experimental setup diagram showing the tadpole anatomy, the position of the suction pipette, and the recording electrodes. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: examples of initial motor responses elicited by a 500 ms suction pulse at three different strengths (trials 1–4, gray shading) and the processing of trial 4 recording for the calculation of the asymmetry index, which is 0.78. The gray rectangle in the demeaned and rectified trial 4 encircles fictive swimming cycles, whose average motor nerve (m.n.) burst peak amplitude is used for normalizing the integrated m.n. activity. Traces are color-coded to match electrodes. Arrowheads indicate time with simultaneous m.n. bursts on both sides. C: tadpole initial m.n. bursts to suction show preference to the left (from 68 ± 11% in control to 87 ± 10%, P < 0.05) or right side (from 64 ± 19% in control to 17 ± 13%, P < 0.001, both paired t test, n = 8 in each group). D: latencies of first m.n. discharges decrease with suction strengths (linear regression, P < 0.05 in 10 tadpoles. Black circles and dotted line are for the tadpole in B. Gray regression lines are for 9 other tadpoles). E: asymmetry indices are larger in suction-evoked responses than in control (n = 16 tadpoles, related-samples Wilcoxon signed rank test). Significance at P < 0.05 and *P < 0.01 in C and E.
	Figure 4. Suction stops on-going fictive swimming. A: experimental setup showing tadpole anatomy and the position of the LED light, suction pipette, and recording electrodes. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: consecutive fictive swimming episodes initiated by dimming the LED light in control (trials 1–5) and with 500 ms suction applied ∼20 s into swimming (trials 6–10). Only activity from the left motor nerve (m.n.) is shown. C: summary of fictive swimming lengths after suction in 12 individual tadpoles (all P < 0.01 except P = 0.012 in the tadpole indicated by an arrow, independent sample t test, n = 8–12 trials in each tadpole).
	Figure 5. Stimulating the anterior lateral line nerve (stim. aLLN) electrically does not produce clear turning response but stops on-going fictive swimming. A: experimental setup showing tadpole anatomy and the position of the stimulation and recording electrodes. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; m.n., motor nerve; sc, spinal cord. B: examples of stimulation of the aLLN (arrowhead; 0.5 ms pulses, other parameters given above traces) not evoking any prolonged burst before regular fictive swimming. C: single electrical stimulation (arrowheads) applied at various points after swimming initiation stops fictive swimming (gray traces; black traces are controls). D: summary of electrical stimulation of aLLN shortening fictive swimming episodes (significance at P < 0.05, *P < 0.01, paired t tests). The time set for aLLN stimulation from the beginning of fictive swimming episodes in each group is also indicated by the horizontal lines inside each gray bar. Numerals inside black control bars indicate the number of tadpoles tested for each time point.
	Figure 6. Anterior lateral line nerve (aLLN) afferent activities. A: experimental setup for recording aLLN afferent activity. The recording electrode directly sucks onto the cut end of aLLN. B: two successive examples of the aLLN activity around the time of suction (−4 kPa and −5 kPa, shaded regions). The inset (top, boxed area) shows the events triggered by setting the threshold at ±5SD in the aLLN recording trace. C: average binned events in 14 tadpoles showing increased activity during a 7-s suction period (bin width: 0.5 s). Dotted fitting curve is for activity during suction: y = 47.33 X−0.61 (R2 = 0.98).
	Figure 7. Anterior lateral line nerve (aLLN) efferent activities. A: setup for recording the aLLN efferent activity where fictive swimming is started by dimming an LED light. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: an example of aLLN efferent recording during a fictive swimming episode. The box region is expanded below to show the timing of the efferent activity relative to motor nerve (m.n.) bursts on both sides. *Examples of multiple unitary discharges within a single swimming cycle. C: raster plot of unitary aLLN efferent discharge phases in 10 swimming episodes from one tadpole. Top and bottom borders of color bands indicate phase from 0 to 1 as marked in episode 1. Color band length indicates episode duration. D: pooled phase plot of all 525 unitary discharges in C. E: number of unitary discharges per swimming cycle in the first 200 cycles after fictive swimming is started (averaged from 3 episodes from each of 10 tadpoles). Gray bars are SD. F: normalized distribution of the phases of aLLN unitary discharges calculated relative to their immediate fictive swimming cycles, defined by the m.n. bursts on the ipsilateral side at the 5th muscle cleft (100 spikes from each of 10 tadpoles, longitudinal time delays not calibrated). Peak phase is 0.11 (Z of Rayleigh statistic is 22.6, P < 0.001).
	Figure 8. Locating anterior lateral line (aLL) nerve sensory interneurons using calcium imaging. A: experimental setup for calcium imaging using a ×10 water immersion objective. The brain is open and the preparation is tilted so that the left side of the hindbrain stays roughly flat to facilitate the imaging of many neurons in a single focal plane. Images have been acquired at 10 Hz. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: three frames captured at the indicated time points in D (1, 2, and 3). Lines in frame 1 show the dorsal and ventral boundaries of the hindbrain (dotted rectangle region in A). Circles in frame 1 indicate regions of interest (ROIs) where calcium activities are given in C. C: calcium activity of 11 neurons outlined by color-coded circles in B. Different types of responses are grouped as labeled. D: simultaneous motor nerve (m.n.) and suction recordings with inset showing initial m.n. bursts. E: summary of locations of neurons showing increased calcium activity time-locked with suction stimulation. F: examples of neurons in the lateral line (LL) nucleus region, rostral hindbrain, and midbrain to show differences in their calcium activity latencies and peak time. Summary of fluorescence peak value (G), number of frames to reach 20% peak fluorescence (H), and number of frames to reach peak fluorescence (I, all independent-samples Kruskal–Wallis test in G–I) in ROIs in the three brain areas following suction that has evoked swimming. J: comparing fluorescence increase at the beginning of swimming evoked by suction and at the beginning of spontaneous swimming (related-samples Wilcoxon signed rank test). Numbers of neurons used in statistics are given in brackets below the charts and significance levels in G–J: P < 0.05; *P < 0.01. The same color-coding applies to E–J.

References [+] :

Bin, Imaging Myelination In Vivo Using Transparent Animal Models. 2016, Pubmed, Xenbase


	Figure 1. High-speed video analyses of tadpole responses to suction at different levels. A: single frames from a 500 fps video showing tadpole turning behavior in the head-toward-nozzle configuration at 10 ms intervals after water level starts to rise inside the suction pipette (0 ms). The maximal bend used to estimate latencies. B: tadpole turning and swimming incidences increase with suction strengths in the head-toward-nozzle configuration while suction applied to tail mainly evokes swimming (tail-toward-nozzle, Pearson’s chi-squared test, P < 0.01, n = 8 tadpoles). C: the likelihood to evoke initial turning increases at high suction levels in the head-toward-nozzle orientation (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.001, n = 8) but in the tail-toward-nozzle orientation turning incidence is lower (independent-samples Mann–Whitney U test for −4 kPa suction trials, P < 0.001, n = 7). D: the success rate for tadpoles swimming out of the suction nozzle increases with suction strength (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.01, n = 8). E: tadpoles produce the first bend to suction consistently toward the exposed side in the head-toward-nozzle (P < 0.05, one-sample Wilcoxon signed rank tests to median of 100%, n = 7) but not in the tail-toward-nozzle orientation. F: the latency from the beginning of suction to the maximum swimming bend decreases with suction strength (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.01, n = 8). In C, E, and F, circles stand for outliers and asterisks are for extremes. G: the latency to the maximum turning bend decreases with suction strength (related-samples Friedman’s two-way analysis of variance by ranks, P < 0.01, n = 8). H: turning bends have longer duration than swimming bends (paired t test, n = 13, *P < 0.001).
	Figure 2. Staining tadpole lateral line neuromasts using 2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide (DASPEI) in control and after neomycin treatment. A: converting a color photo of a tadpole after DASPEI staining to gray scale after enhancing the red and yellow channels. B: clustering of neuromasts in a stage 41 tadpole (square). C: DASPEI staining of tadpoles at various stages (only eye region shown). D: neuromast counts for each side of tadpoles at stages 32, 35, 39, and 41 (n = 10 tadpoles each, P < 0.05 and *P < 0.01, one-way ANOVA). E: the appearance of posterior lateral line (LL) neuromasts. Photo shows the red rectangle area in the drawing on the top. F: DASPEI staining of stage 40 tadpoles in control, treatment with 3 mg/mL neomycin for 30 min, and 24 h after neomycin treatment. Orientation of the tadpoles is the same in B, C, E, and F. White arrowheads point at some example neuromasts labeled with DASPEI. Scale bars: 500 µm in A and E; 100 µm in B, C, and F.
	Figure 3. Fictive motor responses in immobilized tadpoles elicited by local suction close to the left eyecup. A: experimental setup diagram showing the tadpole anatomy, the position of the suction pipette, and the recording electrodes. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: examples of initial motor responses elicited by a 500 ms suction pulse at three different strengths (trials 1–4, gray shading) and the processing of trial 4 recording for the calculation of the asymmetry index, which is 0.78. The gray rectangle in the demeaned and rectified trial 4 encircles fictive swimming cycles, whose average motor nerve (m.n.) burst peak amplitude is used for normalizing the integrated m.n. activity. Traces are color-coded to match electrodes. Arrowheads indicate time with simultaneous m.n. bursts on both sides. C: tadpole initial m.n. bursts to suction show preference to the left (from 68 ± 11% in control to 87 ± 10%, P < 0.05) or right side (from 64 ± 19% in control to 17 ± 13%, P < 0.001, both paired t test, n = 8 in each group). D: latencies of first m.n. discharges decrease with suction strengths (linear regression, P < 0.05 in 10 tadpoles. Black circles and dotted line are for the tadpole in B. Gray regression lines are for 9 other tadpoles). E: asymmetry indices are larger in suction-evoked responses than in control (n = 16 tadpoles, related-samples Wilcoxon signed rank test). Significance at P < 0.05 and *P < 0.01 in C and E.
	Figure 4. Suction stops on-going fictive swimming. A: experimental setup showing tadpole anatomy and the position of the LED light, suction pipette, and recording electrodes. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: consecutive fictive swimming episodes initiated by dimming the LED light in control (trials 1–5) and with 500 ms suction applied ∼20 s into swimming (trials 6–10). Only activity from the left motor nerve (m.n.) is shown. C: summary of fictive swimming lengths after suction in 12 individual tadpoles (all P < 0.01 except P = 0.012 in the tadpole indicated by an arrow, independent sample t test, n = 8–12 trials in each tadpole).
	Figure 5. Stimulating the anterior lateral line nerve (stim. aLLN) electrically does not produce clear turning response but stops on-going fictive swimming. A: experimental setup showing tadpole anatomy and the position of the stimulation and recording electrodes. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; m.n., motor nerve; sc, spinal cord. B: examples of stimulation of the aLLN (arrowhead; 0.5 ms pulses, other parameters given above traces) not evoking any prolonged burst before regular fictive swimming. C: single electrical stimulation (arrowheads) applied at various points after swimming initiation stops fictive swimming (gray traces; black traces are controls). D: summary of electrical stimulation of aLLN shortening fictive swimming episodes (significance at P < 0.05, *P < 0.01, paired t tests). The time set for aLLN stimulation from the beginning of fictive swimming episodes in each group is also indicated by the horizontal lines inside each gray bar. Numerals inside black control bars indicate the number of tadpoles tested for each time point.
	Figure 6. Anterior lateral line nerve (aLLN) afferent activities. A: experimental setup for recording aLLN afferent activity. The recording electrode directly sucks onto the cut end of aLLN. B: two successive examples of the aLLN activity around the time of suction (−4 kPa and −5 kPa, shaded regions). The inset (top, boxed area) shows the events triggered by setting the threshold at ±5SD in the aLLN recording trace. C: average binned events in 14 tadpoles showing increased activity during a 7-s suction period (bin width: 0.5 s). Dotted fitting curve is for activity during suction: y = 47.33 X−0.61 (R2 = 0.98).
	Figure 7. Anterior lateral line nerve (aLLN) efferent activities. A: setup for recording the aLLN efferent activity where fictive swimming is started by dimming an LED light. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: an example of aLLN efferent recording during a fictive swimming episode. The box region is expanded below to show the timing of the efferent activity relative to motor nerve (m.n.) bursts on both sides. *Examples of multiple unitary discharges within a single swimming cycle. C: raster plot of unitary aLLN efferent discharge phases in 10 swimming episodes from one tadpole. Top and bottom borders of color bands indicate phase from 0 to 1 as marked in episode 1. Color band length indicates episode duration. D: pooled phase plot of all 525 unitary discharges in C. E: number of unitary discharges per swimming cycle in the first 200 cycles after fictive swimming is started (averaged from 3 episodes from each of 10 tadpoles). Gray bars are SD. F: normalized distribution of the phases of aLLN unitary discharges calculated relative to their immediate fictive swimming cycles, defined by the m.n. bursts on the ipsilateral side at the 5th muscle cleft (100 spikes from each of 10 tadpoles, longitudinal time delays not calibrated). Peak phase is 0.11 (Z of Rayleigh statistic is 22.6, P < 0.001).
	Figure 8. Locating anterior lateral line (aLL) nerve sensory interneurons using calcium imaging. A: experimental setup for calcium imaging using a ×10 water immersion objective. The brain is open and the preparation is tilted so that the left side of the hindbrain stays roughly flat to facilitate the imaging of many neurons in a single focal plane. Images have been acquired at 10 Hz. fb, forebrain; hb, hindbrain; m, myotome; mb, midbrain; sc, spinal cord. B: three frames captured at the indicated time points in D (1, 2, and 3). Lines in frame 1 show the dorsal and ventral boundaries of the hindbrain (dotted rectangle region in A). Circles in frame 1 indicate regions of interest (ROIs) where calcium activities are given in C. C: calcium activity of 11 neurons outlined by color-coded circles in B. Different types of responses are grouped as labeled. D: simultaneous motor nerve (m.n.) and suction recordings with inset showing initial m.n. bursts. E: summary of locations of neurons showing increased calcium activity time-locked with suction stimulation. F: examples of neurons in the lateral line (LL) nucleus region, rostral hindbrain, and midbrain to show differences in their calcium activity latencies and peak time. Summary of fluorescence peak value (G), number of frames to reach 20% peak fluorescence (H), and number of frames to reach peak fluorescence (I, all independent-samples Kruskal–Wallis test in G–I) in ROIs in the three brain areas following suction that has evoked swimming. J: comparing fluorescence increase at the beginning of swimming evoked by suction and at the beginning of spontaneous swimming (related-samples Wilcoxon signed rank test). Numbers of neurons used in statistics are given in brackets below the charts and significance levels in G–J: P < 0.05; *P < 0.01. The same color-coding applies to E–J.