Figure 4: Mixture enhances proliferation in brain, modifies neural cell populations and behaviour.

(a) Dorsal views of brains of mixture exposed wild type NF45 X. laevis tadpoles. Immunohistochemistry used the anti-PH3 antibody (red, mitosis) and DAPI (blue, nucleus). Scale bar, 200 μm. (b) Numbers of proliferating cells in (PH3 + cells) in tadpole brains following mixture exposure. n = 13 brains per condition, 5 independent experiments pooled (representative number of positive cells in each brain). Statistics used 2-way ANOVA and Dunnett’s post-test (Medians ± SDs, *p < 0.05,****p < 0.0001) (c) CLARITY imaging illustrating the region of interest delimited for analysis in (d–g) on hind brain. Examples of control (left) and mix 1x exposed (right) double transgenic tadpoles Tg(nβt:DSRED) (neurons, red) and Tg(Mmu.mbp:NTR-eGFP) (oligodendrocyte, green). Scale bar, 200 μm. (d–g) Quantification of CLARITY signals obtained for each fluorescent signal in hindbrain. Neuron (d) and oligodendrocyte (e) numbers and cell volumes (f,g), n = 3 to 5 brains, Statistics used non parametric Kruskal Wallis ANOVA and Dunn’s post-test (Means ± SDs, *p < 0.05, **p < 0.01) (h) Wild type NF45 X. laevis tadpoles were exposed to DMSO (CTRL), or mixture (0.1x, 1x, 10x), T₃ 5 × 10⁻⁹ M for 72 h for mobility analysis. Example of total distance covered in 10 mins under 30 secs/30 secs light (blue lines)/dark (grey lines) cycles by one tadpole per condition (i) Mean distance covered during 10 mins under different conditions. Distance is normalized versus controls for 4 independent experiments with n = 12 per experiment. Representation uses scattered dot plots Mean +/− SD. Statistics used meta-analysis with Kruskal-Wallis. Note that stars directly over a group indicates significant difference with CTRL group (Error bars indicate s.e.m, **p < 0.01, ****p < 0.0001).