Journal: PLoS Biology

Article Title: Neurotransmitter-mediated activity spatially controls neuronal migration in the zebrafish cerebellum

doi: 10.1371/journal.pbio.2002226

Figure Lengend Snippet: (A) Overlay of fluorescent and transmitted-light images of a 30 hpf embryo co-expressing H2B-GCaMP6s and ChR2-YFP. At this developmental stage, only nuclei at the ventral end of the MHB express H2B-GCaMP6s at high enough levels to detect fluorescence signal increases over the background fluorescence from ChR2-YFP (nuclei 1 and 2, compare to nucleus 3 in the central region of the MHB). Nuclei at the URL, however, express only ChR2-YFP and serve as negative control (nucleus 4). Scale bar: 25 μm. (B) Nuclei co-expressing both markers show highly regular calcium transients (nuclei 1 and 2), while negative control THNs do not (nuclei 3 and 4). (C) Fourier transformation reveals that the frequency of calcium transients in nuclei 1 and 2 strongly coincides with the expected frequency of one signal per min, or 16.7 mHz (arrows). Nucleus 3 shows a weak peak at this frequency. (D) Tracking of THNs expressing either control (YFP-CAAX), ChR2-YFP, or SwiChR-YFP along the MHB (colored dots) demonstrates that hyperpolarization slows THNs down, while depolarization increases speed. Transmitted light images on the left provide positional information on THNs within the tissue; boxes indicate the region shown at greater magnification in the fluorescence images. Migration progress over time is schematically indicated on the right, in which arrows indicate the start and end points for the shown frames. Elapsed time is indicated at the top. Scale bar: 25 μm. See also . (E) Individual THNs (orange) were tracked in SIMI°BioCell software for quantification. An example is marked in dark blue. The tracks were corrected for tissue shifts using reference points (cyan). See also . (F) THNs reduce their migration speed as they progress along the MHB from approximately 25% to −35% of the distance to the ventral end. Zero percent is the dorsal and 100% is the ventral end of the MHB. Track distribution is given at the top. (G) Migration speeds are affected by de- and hyperpolarization irrespective of starting point. Means ± SEM are plotted per 10% MHB bin. (H) Tracks were grouped as either dorsal (≤ −27.5% MHB) or ventral (≥27.5% MHB) to test the significance of de- and hyperpolarization compared to control level. Only significant differences are indicated. Data depicted in the graphs can be accessed in . CAAX, PM-targeting signal derived from K-Ras; ChR2, channelrhodopsin; F/F0, fluorescence over background; GCaMP6, circular permutated green florescent protein-Calmodulin-M13 peptide 6s; H2B, Histone 2B; hpf, hours post fertilization; MHB, midbrain-hindbrain boundary; n.s., not significant; PM, plasma membrane; SwiChR, mutated channelrhodopsin; THN, tegmental hindbrain nuclei neuron; URL, upper rhombic lip; YFP, yellow fluorescent protein.

Article Snippet: Tracks were measured in SIMI°BioCell (SIMI reality motion systems) and analysed with “Phainothea” ( https://github.com/IaniiNN/CMC ).

Techniques: Expressing, Fluorescence, Negative Control, Transformation Assay, Migration, Software, Derivative Assay

Journal: PLoS Biology

Article Title: Neurotransmitter-mediated activity spatially controls neuronal migration in the zebrafish cerebellum

doi: 10.1371/journal.pbio.2002226

Figure Lengend Snippet: (A) Single cell tracking along the MHB shows that glycine as well as overexpression of a glycine receptor subunit slows THNs down. For control, a point mutation was introduced in the GlyRa1 subunit that abolishes ligand binding (Y226F). Their progress is schematically indicated on the right. Elapsed time is indicated at the top. Scale bar: 25 μm. See also . (B) Glycine-treated THNs, as well as GlyRa1 wt or Y226F overexpressing THNs (orange, dark blue), were tracked in SIMI°BioCell software. References for correction are shown in cyan. See also . (C) Glycine effects are strongest in the central region of the MHB. For control, embryos were treated with glycine together with MK801 or strychnine. Control values from experiment shown in . Means ± SEM are plotted per 10% MHB bin. (D) Statistic analysis confirms that the effects on THN migration speeds induced by glycine occur in the central region of the MHB and depend on the glycine receptor; control values from experiment shown in . (E) Calcium transient rates decrease in THNs upon exposure to glycine, but their strength does not differ from controls. Maximal F/F0 values from each transient are compared to controls from , and are statistically not significant. (F) FFTs contain frequencies of a similar spectrum compared to controls from . (G) Overexpression of the wt GlyRa1 subunit mimics the effects of excess glycine without adding extracellular glycine. When substrate binding is abolished (Y226F), THNs migrate similar to untreated control cells. Control values from experiment shown in . Means ± SEM are plotted per 10% MHB bin. (H) The hyperpolarizing effect of glycine depends on the co-expression of the channel KCC2. This gene is very weakly expressed in developing neurons as reported previously [ , ], but could be observed in the spinal cord and the hindbrain of a 30 hpf wt embryo (top panel, bottom panel: sense probe for control). Boxes indicate regions shown in (G). Scale bar: 200 μm. (I) Magnification of region indicated in (F) to show that KCC2 may be present at the MHB and the ventral region of the rostral hindbrain at 30 hpf, although its expression is weak. Scale bar: 50 μm. See also . Data depicted in the graphs can be accessed in and Data. F/F0, fluorescence over background; FFT, fast Fourier transform; GFP, green fluorescent protein; Gly, glycine; GlyRa1, glycine receptor alpha1 subunit; hpf, hours post fertilization; KCC2, solute carrier family 12 (potassium/chloride transporter), member 5b; MHB, midbrain-hindbrain boundary; MK801, dizocilpine; n.s., not significant; PM, plasma membrane; Stry, strychnine, THN, tegmental hindbrain nuclei neuron; URL, upper rhombic lip; wt, wild-type; Y226F, glycine receptor alpha1 subunit Y226 mutated to F.

Article Snippet: Tracks were measured in SIMI°BioCell (SIMI reality motion systems) and analysed with “Phainothea” ( https://github.com/IaniiNN/CMC ).

Techniques: Single Cell Tracking, Over Expression, Mutagenesis, Ligand Binding Assay, Software, Migration, Binding Assay, Expressing, Fluorescence

Journal: PLoS Biology

Article Title: Neurotransmitter-mediated activity spatially controls neuronal migration in the zebrafish cerebellum

doi: 10.1371/journal.pbio.2002226

Figure Lengend Snippet: (A) Following THNs along the MHB suggested a cell speed increase upon AMPA receptor block, whereas NMDA causes a speed decrease. CNQX and hexamethonium applied simultaneously produced no speed change in ventral tracks. Progress over time is indicated. Elapsed time is given at the top. Scale bar: 25 μm. See also and Videos. (B) Individual tracks (orange, dark blue) are represented after tracking and correction (cyan markers) in SIMI°BioCell. See also and Videos. (C) Migration speed analysis shows that blocking AMPA receptors increases speeds strongly in the ventral part of the MHB. NMDA produces the opposite effect again in the ventral region. Blocking AMPA and nicotinic ACh receptors simultaneously leads to an increase in THN speed in dorsal tracks, whereas in the ventral part, the effects outbalance each other. Control values from experiment shown in Figs and . Means ± SEM are plotted per 10% MHB bin. (D) Statistic analysis confirms that the effects on THN speeds by the glutamate system are confined mostly to the ventral part of the MHB; control values from experiment shown in . Bars are means ± SEM. Data depicted in the graphs can be accessed in . ACh, acetylcholine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; Hexam, hexamethonium; MHB, midbrain-hindbrain boundary; MK801, dizocilpine; NMDA, N-methyl-D-aspartate; THN, tegmental hindbrain nuclei neuron.

Article Snippet: Tracks were measured in SIMI°BioCell (SIMI reality motion systems) and analysed with “Phainothea” ( https://github.com/IaniiNN/CMC ).

Techniques: Blocking Assay, Produced, Migration