Evidence for dynamic rearrangements but lack of fate or position restrictions in premigratory avian trunk neural crest

Neural crest (NC) cells emerge from the dorsal trunk neural tube (NT) and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the NT have similar or differential ability to contribute to NC cells and their derivatives. To this end, we precisely labeled NT precursors at specific dorsoventral levels of the chick NT using fluorescent dyes and a photoconvertible fluorescent protein. NT and NC cell dynamics were then examined in vivo and in slice culture using two-photon and confocal time-lapse imaging. The results show that NC precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the NT, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the NT to contribute to NC derivatives, with the exception of sympathetic ganglia, which appeared to be ‘filled’ by the first population to emigrate. Rather than restricted developmental potential, however, this is probably due to a matter of timing

Evidence for dynamic rearrangements but lack of fate or position

Neural crest cells emerge from the dorsal trunk neural tube and migrate ventrally to colonize neuronal derivatives, as well as dorsolaterally to form melanocytes. Here, we test whether different dorsoventral levels in the neural tube have similar or differential ability to contribute to neural crest cells and their derivatives. To this end, we precisely labeled neural tube precursors at specific dorsoventral levels of the chick neural tube using fluorescent dyes and a photoconvertible fluorescent protein. Neural tube and neural crest cell dynamics were then examined in vivo and in slice culture using 2-photon and confocal time-lapse imaging. The results show that neural crest precursors undergo dynamic rearrangements within the neuroepithelium, yielding an overall ventral to dorsal movement toward the midline of the neural tube, where they exit in a stochastic manner to populate multiple derivatives. No differences were noted in the ability of precursors from different dorsoventral levels of the neural tube to contribute to neural crest derivatives, with the exception of sympathetic ganglia, which appeared to be "filled" by the first population to emigrate. Rather than restricted developmental potential, however, this is likely due to a matter of timing

Gephyrin-Independent GABA_AR Mobility and Clustering during Plasticity

<div><p>The activity-dependent modulation of GABA-A receptor (GABA_AR) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABA_AR clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABA_AR declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABA_AR declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABA_ARs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or <em>N</em>-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABA_ARs revealed that excitation-induced enhancement of GABA_AR lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABA_AR lateral diffusion on the cell surface and inhibition of a Ca²⁺ dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABA_AR-dependent regulatory mechanism and a GABA_AR-independent one. Our results also indicate that GABA_AR mobility and clustering after sustained excitatory activity is independent of gephyrin.</p> </div

Suppression of 4AP-induced reduction in gephyrin immunofluorescence by GABA_AR immobilization under increased levels of cytosolic Ca²⁺.

<p><b>A</b>, <b>B</b>: Top: Representative pseudocolor images of hippocampal neurons loaded with fluo-4 AM without (<b>A</b>) or with (<b>B</b>) surface GABA_AR XL, before (Con) and 10 s after 4AP application (4AP). Scale bars: 20 µm. Bottom: Time-course plots of F/F0 ratio changes (averages ± SEM) measured on proximal dendrites with the addition of 4AP, in the absence (<b>A</b>) or presence (<b>B</b>) of GABA_AR XL. 4AP was applied at time = 0 as indicated by the gray horizontal bar in the traces. <b>C</b>, <b>D</b>: Peak amplitudes (<b>C</b>) and areas under the curve (<b>D</b>) for the F/F0-time plot during 90 s after addition of 4AP. Values indicate averages ± SEM. NS: <i>p</i>>0.05, Welch's <i>t</i>-test. −XL: n = 30 cells, +XL: n = 28, from 3 cultures. Note that normal increase in Ca²⁺ was induced by 4AP even under XL conditions. <b>E</b>: Examples of gephyrin-immunoreactive clusters in dendrites with (+XL) or without (−XL) surface GABA_AR XL in the presence (4AP) and absence (Con) of 4AP treatment for 10 min. Scale bar, 5 µm. <b>F</b>, <b>G</b>: Effects of GABA_AR XL and 4AP treatment on the normalized number of clusters (<b>F</b>) and normalized fluorescence intensities (<b>G</b>) of gephyrin clusters (averages ± SEM). NS: <i>p</i>>0.05; ***: <i>p</i><0.005, Welch's <i>t</i>-test, n = 30 cells/condition (3 cultures). 4AP-induced reduction in gephyrin cluster size was completely suppressed by GABA_AR XL.</p

GABA_AR-independent gephyrin declustering during sustained activity induced by NMDA stimulation.

<p><b>A</b>–<b>D</b>: Effect of CysA treatment on NMDA-driven dispersal of GABA_AR and gephyrin clusters. Left: Examples of GABA_AR (<b>A</b>, <b>B</b>) and gephyrin (<b>C</b>, <b>D</b>) immunoreactivity in neurons incubated with NMDA for 30 min, with (<b>B</b>, <b>D</b>) and without CysA (<b>A</b>, <b>C</b>). Scale bars, 5 µm. Right: Normalized fluorescence intensities (averages ± SEM) of synaptic GABA_AR (<b>A</b>, <b>B</b>) and gephyrin (<b>C</b>, <b>D</b>) clusters. *: <i>p</i><0.05, ***: <i>p</i><0.005, Welch's <i>t</i>-test. Con, CysA, CysA+NMDA: n = 30 cells/condition, NMDA: n = 25 cells/condition (3 cultures). CysA suppressed NMDA-induced dispersal of GABA_AR clusters, but not that of gephyrin clusters. <b>E</b>: NMDA-induced gephyrin dispersal under GABA_AR XL. Top: Gephyrin immunoreactive clusters in neurons with (+XL) and without (−XL) surface GABA_AR XL after NMDA stimulation. Bottom: Effects of GABA_AR XL and NMDA treatment on the normalized fluorescence intensity of gephyrin clusters (average ± SEM). ***: <i>p</i><0.005, Welch's <i>t</i>-test, n = 30 cells/condition (3 cultures). <b>F</b>–<b>H</b>: Comparison of the Ca²⁺ influx level induced by 4AP and NMDA. Increase in Ca²⁺ after the addition of 4AP (black) or NMDA (red) (<b>F</b>). Gray horizontal bars in the traces indicate the presence of 4AP or NMDA. Peak amplitudes (<b>G</b>) and areas under the curve (<b>H</b>) for F/F0-time plots during 90 s after the onset of stimulation. Values indicate averages ± SEM. ***: <i>p</i><0.005, Welch's <i>t</i>-test. 4AP: n = 21 cells, NMDA: n = 23 cells (3 cultures).</p

Time-course analysis of 4AP-induced decrease in GABA_AR- and gephyrin-associated immunofluorescence.

<p><b>A</b>–<b>C</b>: Representative examples of immunoreactivity associated with synapsin (<b>A</b>), GABA_AR (<b>B</b>), and gephyrin (<b>C</b>) on the dendrites of hippocampal neurons (21–27 DIV) treated with 50 µM 4AP for 0–10 min. Scale bars: 5 µm. <b>D</b>–<b>F</b>: Time-course plots of changes in normalized fluorescence intensities (averages ± SEM) of total clusters of synapsin (<b>D</b>), GABA_AR (<b>E</b>), and gephyrin (<b>F</b>) following 4AP treatment. <b>G</b>, <b>H</b>: Time-course plots of 4AP-induced reduction in the normalized fluorescence intensities of synaptic GABA_AR (<b>G</b>) and gephyrin (<b>H</b>) clusters. NS: <i>p</i>>0.05, *: <i>p</i><0.05, ***: <i>p</i><0.005, Tukey's range test in ANOVA, n = 40 cells/condition (4 cultures).</p

Activity-dependent decrease in synaptic clusters of GABA_AR preceding that of gephyrin.

<p>Left: Representative examples of immunoreactivity associated with GABA_AR (<b>A</b>, <b>C</b>) and gephyrin (<b>B</b>, <b>D</b>, <b>E</b>) in the presence (<b>A</b>, <b>B</b>: 4AP, <b>C</b>–<b>E</b>: NMDA) or absence (Con) of stimulation for the indicated times. Right: Normalized fluorescence intensities (averages ± SEM) of synaptic GABA_AR (<b>A</b>, <b>C</b>) and gephyrin (<b>B</b>, <b>D</b>, <b>E</b>) clusters following stimulation. Note that fluorescence intensity of gephyrin was unchanged 60 s after the onset of stimulation (4AP: <b>B</b>, NMDA: <b>D</b>), while that of GABA_AR significantly decreased at 60 s (4AP: <b>A</b>, NMDA: <b>C</b>). Scale bars: 5 µm. NS: <i>p</i>>0.05, ***: <i>p</i><0.005, Welch's <i>t</i>-test, n = 30 cells/condition (3 cultures).</p

Inhibition of GABA_AR lateral diffusion by cross-linking (XL).

<p><b>A</b>: Examples of cross-linked GABA_AR clusters on the dendrites of hippocampal neurons with (XL+4AP) or without (XL) 4AP stimulation. Scale bar: 5 µm. <b>B</b>: Normalized average fluorescent intensities of cross-linked GABA_AR clusters with (XL+4AP) or without (XL) 4AP stimulation (averages ± SEM). XL: n = 39 cells; XL+4AP: n = 40 cells, from 4 cultures. <b>C</b>: Representative trajectories of GABA_AR-QDs recorded for 38.9 s with (+XL) or without (−XL) XL in the presence or absence of 4AP, inside (red) and outside (blue) the synapse. Scale bar: 1 µm. <b>D</b>–<b>G</b>: Effects of XL and 4AP treatment on diffusion coefficients (<b>D</b>), percentage of immobile receptors (<b>E</b>), synaptic confinement sizes (<b>F</b>), and synaptic dwell times (<b>G</b>) of GABA_AR-QDs. <b>D</b>: The diffusion coefficients of GABA_AR-QDs (median ± IQR). The number of GABA_AR-QDs analyzed: Con: n = 556; 4AP: n = 418; XL: n = 321; XL+4AP: n = 321. <b>E</b>: The percentage of immobile GABA_AR-QDs (average ± SEM). Con: n = 15 cells; 4AP: n = 14; XL: n = 14; XL+4AP: n = 19. <b>F</b>: The confinement size (average ± SEM) of synaptic GABA_AR-QDs. Con: n = 366 QDs; 4AP: n = 203; XL: n = 200; XL+4AP: n = 196. <b>G</b>: The average dwell times (±SEM) of GABA_AR-QDs in synapses. Con: n = 2039 events; 4AP: n = 1576; XL: n = 473; XL+4AP: n = 493. Data in <b>D</b>–<b>G</b> were obtained from 3 cultures. NS: <i>p</i>>0.05, **: <i>p</i><0.01, ***: <i>p</i><0.005, Welch's <i>t</i>-test for <b>B</b>, <b>E</b>–<b>G</b>, and Mann–Whitney <i>U</i> test for <b>D</b>.</p