Fig 5. Glycine negatively influences THN migration.

(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 S9 Video. (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 S10 Video. (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 Fig 4C. 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 Fig 4D. (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 Fig 2G, and are statistically not significant. (F) FFTs contain frequencies of a similar spectrum compared to controls from Fig 2I. (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 Fig 4C and 4D. 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 [45,46], 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 S5 Fig. Data depicted in the graphs can be accessed in S1 and S3 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.