Figure 4. Effect of NH₄Cl treatment and low vitamin D diet on performance in Open Field Test.

(A–C) Photographs of the Open Field arena with representative tracings of an untreated, male wild-type mouse (WT) (A), a male, klotho-hypomorphic mouse (kl/kl) treated with 280 mM NH₄Cl solution (B) and a male, NH₄Cl treated kl/kl mouse under vitamin D deficient diet (C). (D–K) Arithmetic means ± SEM (n = 12–30) of (D) average speed measured in the whole observation area, (E) total distance travelled during the observation time, (F) time spent in the border area of the Open Field arena, (G), distance travelled in the border area, (H), time spent in the corners of the Open Field arena, (I) number of visits in the center area, (J) distance travelled in the center area, (K) time spent in the center area of wild-type mice (WT, white bars) and kl/kl mice (kl/kl black bars) either untreated (Control, left bars), treated with 280 mM NH₄Cl solution (NH₄Cl, middle bars) or treated with NH₄Cl and a vitamin D deficient diet (LVD, right bars). *(p < 0.05), **(p < 0.01), ***(p < 0.001) indicates statistically significant differences from untreated wild-type mice (Control); ^##(p < 0.01). ^###(p < 0.001) indicates statistically significant differences from NH₄Cl treated kl/kl mice on control diet. (ANOVA).