Adult male C57BL/6J mice (8–12 weeks old) were supplied by Charles River Laboratories (Yokohama, Japan) and CLEA Japan (Tokyo, Japan). All mice were housed at 25 $\pm$ 2 °C on a 12-h light (08:00 to 20:00)/12-h dark (20:00 to 08:00) cycle and were allowed ad libitum access to food (0.3% sodium containing diet for rodents, #CE-2, CLEA Japan, Inc., Tokyo, Japan) and water. Mice were housed in groups of four to five per cage. This study was approved by the Animal Care Committee of Ohu University (Nos. 2016-29, 2017-34, and 2018-29). All animal procedures were performed in accordance with the guidelines of the Animal Care Committee of Ohu University. Principles of laboratory animal care were followed, with special care taken to minimize animal distress and to use the minimum number of animals needed for all experiments. This study was carried out in compliance with the ARRIVE guidelines. All behavioral tests were performed between 13:00 and 16:00 and were conducted and analyzed by investigators who were blinded to the group assignments. Data acquisition and analysis of all animal behavioral tests results were performed using ANY-maze software (version 6.35, Muromachi Kikai Co. Ltd., Tokyo, Japan).

A total of 47 male C57BL/6J mice (8–12 weeks old) were used for the TMT-induced inescapable innate fear test (26 mice), with mineral oil (MO) used as the control (21 mice). All mouse were habituated to the arena of the TMT test box for 15 min one day prior to the test. Each mouse was placed in the center of an acrylic plastic open field box arena (W: 294 mm $\times$ D: 294 mm $\times$ H: 297 mm). The bottom and four inner walls of the box were covered by non-reflective paper and the top of the box was made of clear plexiglass to allow video recording with a web camera. A circular filter paper (D: 2 cm, Cat#; 1001-025; Whatman GE Healthcare Life Science, Little Chalfont, UK) infused with 20 $\mu$L of TMT was placed directly in the corner of the odor exposure box. The TMT was introduced 5 min after the mice were placed in the test box, and fear-related behaviors were observed throughout the test (15 min). To measure the avoidance response to TMT odor, the floor was divided into two areas by a diagonal line. Five test boxes were prepared and 3 to 5 mice per day were tested for TMT odor exposure. The TMT odor in each box was removed between experiments by treatment with 5% bleaching agent overnight and then carefully washing with detergent and water, and wiping clean with 70% ethanol.

To evaluate the avoidance response to TMT, the mean time an individual mouse spent in the non-TMT diagonal side of the arena was compared with the time it spent in the TMT side [39]. The TMT stress-elicited freezing behavior was used as a measure of fear and was defined as the absence of any movement, except for those necessary for breathing [40]. The percentage of time spent in freezing was calculated for each mouse during the 10 min period after TMT was added to the filter paper in the corner of the box. Freezing time was analyzed every min (1 min/bin) or in two 5-min blocks (the first at 5–10 min and the second at 10–15 min).

All TSTs were performed during the light phase (12:00–16:00) of the light–dark cycle. Each mouse was individually suspended by its tail using adhesive tape placed approximately 2 cm from the tip of the tail and via two hanging hooks connected to the ceiling of the test box, which was located 42 cm above the bench top. Each mouse was suspended for 10 min and recorded with a digital video camera in the absence of an investigator. Another investigator, who did not perform the experiment measured the immobility time during the TST. This was performed after completion of the experiments and using ANY-maze software. The investigators were thus blinded to the allocated groupings. Mobility was defined as movement of the hind legs and/or other escape-oriented behaviors, including twisting of the body, but not breathing Mouse immobility was considered to represent the immobility time in this paradigm. Mice with a 2% NaCl intake that spent a shorter time immobile compared to those with water intake without escape-oriented behaviors were considered to demonstrate active coping behavior. The duration of immobility was calculated as the sum of the time periods during which the mouse was motionless for at least 2 s. Immobile behavior sensitivity was set to 70%, and the mouse needed to be immobile for 1 s to initiate scoring of immobility. Mice were returned to their home cages at the completion of the TST [41, 42]. To provide an index of learned despair, 10 min of the test session was divided into two periods, the initial 5 min (0–5 min) and the final 5 min (5–10 min) [41, 43], in addition, the immobility time for 5 min from 1 to 6 min during TST were also analyzed for evaluating the level of the behavioral despair [44].

A total of 21 male C57BL/6J mice (8–12 weeks old) were used for elevated plus maze (EPM). EPM is a reliable measurement instrument for anxiety-like behavior in animals and is used to evaluate emotionality in rodents [45]. The apparatus is comprised of two open arms (300 $\times$ 50 mm) without walls, two enclosed arms (300 $\times$ 50 mm) with walls that are 250 mm in height, and a central square (50 $\times$ 50 mm) that connects the arms. The four grey crossed arms form a plus sign with the central platform (Brain Science idea. Co., Ltd., Osaka, Japan). The apparatus was placed 400 mm above the ground, with the mice able to move freely in all directions. The EPM test was conducted in a quiet room, and the illumination level of the central area was 100 lux [46]. Each tested mouse faced the open arm and was positioned in the center of the apparatus. Their behavior was recorded for 5 min. The apparatus was cleaned with 70% ethanol to prevent the mice from being affected by other odors. The distance traveled, the number of entries into the open arms, and the time spent in the open arms were recorded by a video camera.

The Student’s t-test was used for comparisons between two groups. Two-way repeated measures analysis of variance (ANOVA) was followed by one- or two-way ANOVA to analyze each time block. This was followed by the Bonferroni post-hoc test for comparison of the groups. The correlation coefficient was analyzed by Pearson’s correlation test, and Spearman’s rank correlation coefficient analysis was performed to determine whether two variables were significantly correlated. Fisher z-transformation was performed to test the difference between two Pearson’s correlation coefficients. Statistical analyses were performed using EZR (Easy R) software [47] (version 1.38; Saitama Medical Center, Jichi Medical University, Saitama, Japan) and BellCurve software (version 3.20, Social Survey Research Information Co., Ltd., Tokyo, Japan) [48]. All data in the bars indicate the mean $\pm$ standard error of the mean (SEM). Statistical significance was set at *p 0.05 or **p 0.01. NS signifies no significant difference.

To investigate whether the 2% NaCl intake for 5 days affected the mice’s innate fear sensitivity against TMT, we measured the percentage of freezing time before and during TMT exposure for 5 and 10 min, respectively. Two-way repeated measures ANOVA (NaCl $\times$ TMT) revealed that the TMT affected the time course of freezing behaviors (F(3, 749) = 360.59, p = 5.94 $\times$ 10${}^{-32}$, Fig. 1E). Bonferroni post hoc test indicated that both the water- and sodium-intake mice exhibited freezing behavior upon TMT exposure compared to for MO (TMT–H${}_2$O: p = 4.14 $\times$ 10${}^{-27}$ vs. MO–H${}_2$O; TMT–NaCl: p = 3.03 $\times$ 10${}^{-25}$ vs. MO–NaCl, Fig. 1E). However, sodium-intake did not affect the freezing behavior (MO–NaCl: p = 1.000 vs. MO–H${}_2$O; TMT–NaCl: p = 1.000 vs. TMT– H${}_2$O; Fig. 1E). Two-way repeated measures ANOVA revealed that freezing time for 5 min bin before and after the TMT exposure were affected (F(3, 146) = 344.96, p = 4.71 $\times$ 10${}^{-31}$, Fig. 1F). Although One-way ANOVA indicated that there was no significant difference during 5 min before and after the MO or TMT (before: F(3, 18) = 1.566, p = 0.2937, Fig. 1F), TMT affected the freezing time during both of first and last 5 min of TMT exposure (1st 5 min; F(3, 48) = 79.251, p = 5.50 $\times$ 10${}^{-18}$; last 5 min: F(3, 48) = 712.27, p = 6.40 $\times$ 10${}^{-38}$, Fig. 1F). Bonferroni post hoc test indicated that the TMT significantly induced the freezing behaviors in both water and sodium-intake mice during first and last 5 min (first 5 min: TMT-H${}_2$O: p = 1.11 $\times$ 10${}^{-13}$ vs. MO-H${}_2$O; TMT-NaCl: p = 8.87 $\times$ 10${}^{-13}$ vs. MO-NaCl; last 5 min: TMT-H${}_2$O: p = 7.35 $\times$ 10${}^{-32}$ vs. MO-H${}_2$O; TMT-NaCl: p = 3.23 $\times$ 10${}^{-32}$ vs. MO-NaCl; Fig. 1F). Two-way repeated measures ANOVA revealed that the total distance traveled in the whole area of the test box for the 5-min bin before exposure to TMT and for 10 min during exposure was also affected (F(3, 140) = 22.81, p = 5.45 $\times$ 10${}^{-9}$, Fig. 1G). One-way ANOVA for each 5-min bin indicated that the last 5 min of TMT exposure affected the total distance traveled in whole area of the test box (before TMT for 5 min: F(3, 121) = 0.642, p = 0.589; TMT exposure for the first 5 min: F(3, 121) = 1.493, p = 0.2198; TMT exposure for the last 5 min: F(3, 121) = 63.689, p = 8.88 $\times$ 10${}^{-25}$; Fig. 1G). Bonferroni post-hoc test indicated that distance traveled was significantly different in the last 5 min of TMT exposure for MO and TMT exposed-mice, but not water- or sodium-intake mice (TMT–H${}_2$O: p = 0.985 vs. TMT–NaCl; MO–H${}_2$O: p = 1.000 vs. MO–NaCl; p = 1.000 vs. non-TMT–H${}_2$O; TMT–NaCl: p = 1.000 vs. TMT–H${}_2$O, Fig. 1G). In the next, we investigated the correlation between the freezing level and the distance traveled in water- and sodium-intake mice. Spearman’s rank correlation coefficient analysis indicated that only the percent of freezing time during the first 5 min and the distance traveled during the first 5 min of TMT exposure exhibited significant correlations in only water-intake mice (H${}_2$O: R${}^2$ = 0.6803, p = 0.000907; NaCl: R${}^2$ = 0.358, p = 0.1025, Fig. 1L). This indicates that the water-intake mice exhibited a correlation between freezing level and the distance traveled during the first 5 min of exposure to TMT, but sodium-intake mice did not.

Fig. 1.

Effect of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) on the freezing behavior and the distance traveled in the test box. (A–D) Traces of mice moving in the box during the test under the mineral oil (MO) as a control. Traces of mice moving in the box for 5 min before (a: –5–0 min), the first 5 min (b: 0–5 min), and the second 5 min (c: 5–10 min) during MO exposure for H${}_2$O-intake mice (A), MO exposure for NaCl-intake mice (B), TMT exposure for H${}_2$O-intake mice (C), and TMT exposure for NaCl-intake mice (D). (E) Time course of the freezing behaviors before and during TMT exposure. The graph shows the percent of freezing time per min for 15 min (H${}_2$O-intake mice, n = 11; NaCl-intake mice, n = 15). (F) The percent of freezing time for 5 min (–5–0 min) before MO and TMT exposure and the first (0–5 min) and second 5 min (5–10 min) under the presence of MO or TMT (H${}_2$O–intake mice, n = 11; NaCl–intake mice, n = 15). (G) Distance traveled in the test box for 5 min (–5–0 min) before MO and TMT exposure and the first (0–5 min) and second 5 min (5–10 min) under the presence of MO or TMT (H${}_2$O-intake mice, n = 11; NaCl-intake mice, n = 15). (H–M) Correlation between freezing behavior and the distance traveled in the test box before MO (H), during the first 5 min of MO exposure (I), during the second 5 min of MO exposure (J), before TMT exposure (K), during the first 5 min of TMT exposure (L), and during the second 5 min of TMT exposure (M) (H${}_2$O-intake mice, n = 11; NaCl-intake mice, n = 15). All the data are mean $\pm$ SEM. R${}^2$ is Pearson’s correlation coefficient.

During the TMT exposure, sodium-intake mice exhibited preference for the central zone in the test box compared to water-intake mice, although the preference of the central zone in sodium-intake mice were similar level with water-intake mice. Two-way repeated measures ANOVA revealed that the distance traveled, the time spent, and the number of entries in the central zone were affected by TMT exposure (distance traveled in the central zone: F(3, 140) = 31.19, p = 7.20 $\times$ 10${}^{-11}$, Fig. 2C; number of entries in central zone: F(3, 140) = 30.78, p = 8.72 $\times$ 10${}^{-11}$, Fig. 2D; time spent in the central zone: F(3, 140) = 14.53, p = 1.12 $\times$ 10${}^{-6}$; Fig. 2E). One-way ANOVA was followed by Bonferroni comparison test, which indicated that the distance traveled, the time spent, and the number of entries in the central zone were affected by sodium intake during the first 5 min of TMT exposure (one-way ANOVA: distance traveled in the central zone: F(3, 46) = 34.93, p = 1.34 $\times$ 10${}^{-11}$, Fig. 2C; number of entries in the central zone: F(3, 46) = 36.83, p = 6.00 $\times$ 10${}^{-12}$, Fig. 2D; time spent in the central zone: F(3, 46) = 5.10, p = 0.0042, Fig. 2E; Bonferroni post hoc test: distance traveled: NaCl-intake mice, p = 0.0444 vs. H${}_2$O-intake mice, Fig. 2C; number of entries in the central zone: NaCl-intake mice, p = 0.0411 vs. H${}_2$O-intake mice, Fig. 2D; time spent: NaCl-intake mice, p = 0.0384 vs. H${}_2$O-intake mice; Fig. 2E). Spearman’s rank correlation coefficient revealed that the distance traveled in the central zone in both the water- and sodium-intake mice was correlated with freezing time during the first 5 min of exposure to TMT (Pearson’s correlation coefficient: H${}_2$O-intake mice: R${}^2$ = 0.5625, NaCl-intake mice: R${}^2$ = 0.4927; Spearman’s rank correlation: H${}_2$O-intake mice: p = 0.000153; NaCl-intake mice: p = 0.0146, Fig. 2I). In addition, the number of entries in the central zone during the first 5 min of exposure to TMT was also correlated with freezing time in both water- and sodium-intake mice (number of entries: Pearson’s correlation coefficient: H${}_2$O-intake mice: R${}^2$ = 0.6392, NaCl-intake mice: R${}^2$ = 0.4889; Spearman’s rank correlation: H${}_2$O-intake mice: p = 5.74 $\times$ 10${}^{-6}$; NaCl-intake mice: p = 0.0099; Fig. 2J). However, the time that water-intake mice spent in the central zone was not correlated with freezing time during the first 5 min of exposure to TMT, and the freezing time in sodium-intake mice during the first 5 min of exposure to TMT was not correlated with the time spent in the central zone during the first 5 min of exposure to TMT (Pearson’s correlation coefficient: H${}_2$O-intake mice: R${}^2$ = 0.0213, NaCl-intake mice: R${}^2$ = 0.5716; Spearman’s rank correlation: H${}_2$O-intake mice: p = 0.2443; NaCl–intake mice: p = 0.0028; Fig. 2K). These results indicated that mice with a higher preference of the central zone during the first 5 min of exposure to TMT exhibited shorter freezing times.

Fig. 2.

The effect of 2% NaCl intake on the central preference and the correlation between freezing time and the central preference during inescapable innate fear caused by 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) exposure. (A) Schedule of the time course before and during the first and second 5 min of TMT exposure. (B) The location of the central zone and mineral oil (MO) or TMT in the test box. (C–E) Distance traveled (C), number of entries (D), and time spent (E) in the central zone in the test box before and during the first and second 5 min of MO or TMT exposure. (F–H) Correlation between the percent of freezing time during the first 5 min of MO exposure and the distance traveled (F), number of entries (G), and time spent (H) in the central zone in the test box for both water- and sodium-intake mice. (I–K) Correlation between the percent of freezing time during the first 5 min of TMT exposure and the distance traveled (I), number of entries (J), and time spent (K) in the central zone in the test box for both water- and sodium-intake mice. All the data are mean $\pm$ SEM. Statistical significance (*p 0.05, **p 0.01) is noted. R${}^2$ is Pearson’s correlation coefficient.

We next investigated whether the NaCl intake affected mice’s active coping behavior during the TST as an aversive situation for the mice. The immobility time during TST in each group gradually increased, and the two-way repeated measures ANOVA revealed that the TMT and NaCl affected the immobility time during TST (F(3, 469) = 10.65, p = 2.31 $\times$ 10${}^{-5}$, Fig. 3B). One-way ANOVA suggested that the immobility time for 1-min bins from 1–2 to 9–10 min were also affected, and the immobility time in mice with sodium intake and exposure to TMT were longer than those of mice with water intake and TMT exposure (0–1 min: F(3, 243) = 0.220, p = 0.8827; 1–2 min: F(3, 243) = 7.022, p = 0.000151; 2–3 min: F(3, 243) = 5.446, p = 0.00122; 3–4 min: F(3, 243) = 5.603, p = 0.000990; 4–5 min: F(3, 243) = 5.000, p = 0.00220; 5–6 min: F(3, 243) = 4.708, p = 0.00326; 6–7 min: F(3, 243) = 6.636, p = 0.000252; 7–8 min: F(3, 243) = 5.895, p = 0.000672; 8–9 min: F(3, 243) = 4.536, p = 0.00409; 9–10 min: F(3, 243) = 2.769, p = 0.0423; Bonferroni post hoc test: 0–1 min: p = 0.572; 1–2 min: p = 4.32 $\times$ 10${}^{-32}$; 2–3 min: p = 8.86 $\times$ 10${}^{-26}$; 3–4 min: p = 3.39 $\times$ 10${}^{-16}$; 4–5 min: p = 3.81 $\times$ 10${}^{-23}$; 5–6 min: p = 7.88 $\times$ 10${}^{-19}$; 6–7 min: p = 1.69 $\times$ 10${}^{-32}$; 7–8 min: p = 3.50 $\times$ 10${}^{-29}$; 8–9 min: p = 1.84 $\times$ 10${}^{-22}$; 9–10 min: p = 2.57 $\times$ 10${}^{-16}$; Fig. 3B). Many papers have suggested that a higher immobility time during the first 6 min, except the first 1 min, reflects despair behavior, which is used for the assessment of antidepressants. Therefore, we investigated whether the innate fear induced by TMT exposure affected the immobility time during TST 1 h after the TMT. Multiple comparisons among four groups were performed by one-way ANOVA, which was followed by Bonferroni post hoc test and indicated that the TMT exposure increased the immobility time in water-intake mice compared to MO exposure (F(3, 46) = 9.857, p = 4.54 $\times$ 10${}^{-5}$; Bonferroni post hoc test: H${}_2$O–TMT mice: p = 0.0031 vs. H${}_2$O–MO mice, Fig. 3C). However, NaCl intake suppressed the increase in the immobility time after the TMT exposure (NaCl–TMT mice: p = 0.344 vs. H${}_2$O–TMT mice, Fig. 3C), indicating that the NaCl intake prevented the TMT-induced despair behavior. In our previous work, we demonstrated that the learned despair behavior could be observed in the last 5 min of 10-min TST in subsequent of the first 5 min [41, 43]. Therefore, we investigated whether the NaCl intake affected the induction of learned despair behavior. Two-way repeated measures ANOVA revealed that NaCl intake and TMT exposure affected the immobility time during TST (3, 93) = 10.33, p = 3.03 $\times$ 10${}^{-5}$, Fig. 3D). Bonferroni comparison test indicated that the immobility time during the last 5 min in every group was increased compared to that in the first 5 min of TST (H${}_2$O–MO mice in the first 5 min: p = 1.42 $\times$ 10${}^{-32}$ vs. H${}_2$O–MO mice in the last 5 min; H${}_2$O–TMT mice in the first 5 min: p = 1.49 $\times$ 10${}^{-13}$ vs. H${}_2$O-TMT mice in the last 5 min; NaCl–MO mice in the first 5 min: p = 1.91 $\times$ 10${}^{-13}$ vs. NaCl–MO mice in the last 5 min; NaCl–TMT mice in the first 5 min: p = 1.28 $\times$ 10${}^{-10}$ vs. NaCl–TMT mice in the last 5 min; Fig. 3D). These results indicated that the NaCl intake enhanced the active coping behavior and induced fear-based despair-like behavior, while the learned despair-like behavior was not affected by NaCl intake. There was no correlation between the immobility time during the first or last 5 min and the freezing time during the first 5 min in each group when the mice were exposed to MO (Pearson’s R: H${}_2$O–MO in TST (0–5 min): R${}^2$ = 0.034; NaCl–MO in TST (0–5 min): R${}^2$ = 0.2898; H${}_2$O–MO in TST (5–10 min): R${}^2$ = 0.034; NaCl–MO in TST (5–10 min): R${}^2$ = 0.2898; Fig. 3E,F). However, NaCl intake showed a correlation between the immobility time during the first or last 5 min and the freezing time during the first 5 min in each group when the mice were exposed to the TMT (Pearson’s R: H${}_2$O–TMT in TST (0–5 min): R${}^2$ = 0.0027; NaCl–TMT in TST (0–5 min): R${}^2$ = 0.3438; H${}_2$O–TMT in TST (5–10 min): R${}^2$ = 0.063; NaCl–TMT in TST (5–10 min): R${}^2$ = 0.6394; Spearman’s rank correlation: H${}_2$O–TMT in TST (0–5 min): p = 0.4277; NaCl–TMT in TST (0–5 min): p = 0.0426; H${}_2$O–TMT in TST (5–10 min): p = 0.4431; NaCl–TMT in TST (5–10 min): p = 0.0045; Fig. 3G,H). This indicates that mice with lower freezing levels exhibited shorter immobility times. Fisher’s r-to-z transformation revealed that during the second 5 min of TMT exposure, Pearson’s correlation coefficient of 2% NaCl intake was significantly different from that of water-intake mice (z = 2.967, p = 0.00301, Fig. 3H). These results suggest that the sodium-intake mice exhibited lower freezing levels during inescapable innate fear of TMT exposure and shorter immobility times during the first 5 min of exposure to TST.

Fig. 3.

The effect of 2% NaCl intake on the immobility time during tail suspension test (TST) and the correlation between the immobility time during TST and the freezing time during the first 5 min of mineral oil (MO) or 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) exposure. (A) Schematic drawings indicate the location of the central zone and the MO or TMT and the schedule of TST. (B) The time course of immobility time for 10 min of the tail suspension test (TST) in H${}_2$O–MO (n = 9), H${}_2$O–TMT (n = 15), NaCl–MO (n = 11), and NaCl–TMT mice (n = 11). (C) Immobility time during the first 6 min, except the first 1 min, of TST exposure in H${}_2$O–MO (n = 9), H${}_2$O–TMT (n = 15), NaCl–MO (n = 11), and NaCl–TMT mice (n = 11). (D) Summary of cumulative immobility time during the first (0–5 min) and last 5 min (5–10 min) 1 h after TMT exposure in H${}_2$O–MO (n = 9), H${}_2$O–TMT (n = 15), NaCl–MO (n = 11), and NaCl–TMT mice (n = 11). (E–D) Correlation between percent of freezing time during the first 5 min and the immobility time during the first 5 min of TMT under MO exposure (E), during the second 5 min of TST under MO exposure (F), during the first 5 min of TST under TMT exposure (G), and during the second 5 min of TST under TMT exposure (H) for both water- and sodium-intake mice. All the data are mean $\pm$ SEM. Statistical significance (**p 0.01, *p 0.05) is noted. Statistically significant differences between two Pearson’s correlation coefficients by Fisher’s r-to-z transformation (${}^{\mathrm{\#}\mathrm{\#}}$p 0.01) are indicated. NS means no significant. R${}^2$ is Pearson’s correlation coefficient.

Because the freezing time during the first 5 min of exposure to TMT was correlated with the central preference, such as distance traveled, the number of entries, and the time spent in the central zone (Fig. 2I–K), we investigated whether the immobility time during TST was correlated with the preference of the central zone during the first 5 min of exposure to TMT to induce innate fear. The immobility time during both the first and last 5 min of TST in all groups was not correlated with any preference for the central zone during MO exposure (distance traveled: H${}_2$O–MO in TST (0–5 min): R${}^2$ = 0.0018, p = 0.855; NaCl–MO in TST (0–5 min): R${}^2$ = 0.0216, p = 0.519, Fig. 4A; H${}_2$O–MO in TST (5–10 min): R${}^2$ = 0.00718, p = 0.855; NaCl–TMT in TST (5–10 min): R${}^2$ = 0.0568, p = 0.259, Fig. 4B; number of entries: H${}_2$O–MO in TST (0–5 min): R${}^2$ = 0.0015, p = 0.894; NaCl–MO in TST (0–5 min): R${}^2$ = 0.1158, p = 0.250, Fig. 4E; H${}_2$O–MO in TST (5–10 min): R${}^2$ = 0.00263, p = 0.947; NaCl-TMT in TST (5–10 min): R${}^2$ = 0.021, p = 0.535, Fig. 4F; time spent: H${}_2$O–MO in TST (0–5 min): R${}^2$ = 0.0003, p = 0.603; NaCl–MO in TST (0–5 min): R${}^2$ = 0.0023, p = 0.709, Fig. 4I; H${}_2$O–MO in TST (5–10 min): R${}^2$ = 0.0040, p = 0.777; NaCl–TMT in TST (5–10 min): R${}^2$ = 0.0359, p = 0.433, Fig. 4J). However, although the immobility time during TST in both the first and last 5 min in water-intake mice were not correlated with any central preference during first 5 min of TMT exposure, NaCl intake showed a correlation between the immobility time during TST in both the first and last 5 min and central preference during first 5 min of TMT exposure, excepting the time spent in the central zone and the immobility time during last 5 min of TST in sodium-intake mice (distance traveled: H${}_2$O–TMT in TST (0–5 min): R${}^2$ = 0.0525, p = 0.334; NaCl–TMT in TST (0–5 min): R${}^2$ = 0.526, p = 0.0165, Fig. 4C; H${}_2$O–TMT in TST (5–10 min): R${}^2$ = 0.003, p = 0.781; NaCl–TMT in TST (5–10 min): R${}^2$ = 0.5589, p = 0.0233, Fig. 4D; number of entries: H${}_2$O–TMT in TST (0–5 min): R${}^2$ = 0.0368, p = 0.504; NaCl–TMT in TST (0–5 min): R${}^2$ = 0.452, p = 0.0251, Fig. 4G; H${}_2$O–TMT in TST (5–10 min): R${}^2$ = 0.0171, p = 0.725; NaCl–TMT in TST (5–10 min): R${}^2$ = 0.637, p = 0.0139, Fig. 4H; time spent: H${}_2$O–TMT in TST (0–5 min): R${}^2$ = 0.1009, p = 0.713; NaCl–TMT in TST (0–5 min): R${}^2$ = 0.494, p = 0.000807, Fig. 4K; H${}_2$O–TMT in TST (5–10 min): R${}^2$ = 0.0055, p = 0.8894; NaCl–TMT in TST (5–10 min): R${}^2$ = 0.517, p = 0.0956; Fig. 4L). Fisher’s r-to-z transformation revealed that during the first 5 min of TMT exposure and the immobility time, Pearson’s correlation coefficient of sodium-intake mice was significantly different from that of water-intake mice (distance traveled: TST (0–5 min): z = 2.5248, p = 0.0116, Fig. 4C; TST (5–10 min): z =2.2393, p = 0.0251, Fig. 4D; number of entries: TST (0–5 min): z = 2.21184, p = 0.0270, Fig. 4G; time spent: TST (0–5 min): z = 2.6847, p = 0.00726, Fig. 4H). These results indicate that the NaCl intake caused mice to adapt to the fear and aversive environment, and the preference of the central zone during TMT exposure may be a coping behavior to prevent fear stress–induced despair during TST.

Fig. 4.

Correlation between immobility time and preference of the central zone during mineral oil (MO) and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) exposure. (A–D) Correlation between the distance traveled in the central zone during the first 5 min of MO and the immobility time during the first 5 min of tail suspension test (TST) (A), during the first 5 min of MO and the immobility time during the second 5 min of TST (B), during the first 5 min of TMT and the immobility time during the first 5 min of TST (C), and during the first 5 min of TMT and the immobility time during the second 5 min of TST (D). (E–H) Correlation between the number of entries in the central zone during the first 5 min of MO and the immobility time during the first 5 min of TST (E), during the first 5 min of MO and the immobility time during the second 5 min of TST (F), during the first 5 min of TMT and the immobility time during the first 5 min of TST (G), and during the first 5 min of TMT and the immobility time during the second 5 min of TST (H). (I–L) Correlation between the time spent in the central zone during the first 5 min of MO and the immobility time during the first 5 min of TST (I), during the first 5 min of MO and the immobility time during the second 5 min of TST (J), during the first 5 min of TMT and the immobility time during the first 5 min of TST (K), and during the first 5 min of TMT and the immobility time during the second 5 min of TST (L) for H${}_2$O–MO (n = 9), H${}_2$O–TMT (n = 15), NaCl–MO (n = 11), and NaCl–TMT mice (n = 11). Statistical significance (**p 0.01, *p 0.05) are noted. NS means not significant. R${}^2$ is Pearson’s correlation coefficient.

The sodium-intake mice exhibited a central preference in the test box during the TMT exposure. It has been suggested that decreased levels of anxiety increase exploratory behavior in the open field box, while increased anxiety levels result in less locomotion and staying close to the walls of the open field box for longer [49, 50], although this is controversial because higher preference of the central zone does not correlate with anxiety levels in mice. Therefore, we examined whether NaCl intake affected the anxiety level using the EPM [45, 51] and whether the NaCl intake produced a correlation between anxiety level and immobility time during TST. The distance traveled in the open arm in sodium-intake mice was similar to that of water-intake mice (NaCl-intake mice; t(17) = 1.4026, p = 0.1787 vs. H${}_2$O-intake mice; Fig. 5C). However, both the number of entries and the time spent in the open arm were significantly increased in the sodium-intake mice compared to the water-intake mice (number of entries: NaCl-intake mice: t(17) = 4.4462, p = 0.000354 vs. H${}_2$O-intake mice, Fig. 5D; time spent: NaCl-intake mice: t(17) = 1.8758, p = 0.0380, Fig. 5E). In addition, the sodium-intake mice spent more time in the central zone than the water-intake mice, although both groups had a similar number of entries in the central zone (number of entries: NaCl-intake mice: t(17) = 1.6436, p = 0.1186 vs. H${}_2$O-intake mice, Fig. 5F; time spent: NaCl-intake mice: t(17) = 2.1429, p = 0.0453; Fig. 5G). Two-way repeated measures ANOVA revealed that the time course of the immobility time during TST in sodium-intake mice was similar to that of water-intake mice (F(1, 197) = 0.8694, p = 0.3622, Fig. 5H). The immobility time during the first 5 min, except the first 1 min (1–6 min), in sodium-intake mice was also similar to that of water-intake mice (NaCl-intake mice: t(17) = 0.4795, p = 0.6377, Fig. 5I). In addition, learned despair during the second 5 min compared to the first 5 min in sodium-intake mice was at a similar level to water-intake mice (F(1, 41) = 0.6168, p = 0.4419, Fig. 5J). In the next experiment, we investigated whether the distance traveled, number of entries, and the time spent in the open arm were correlated with the immobility time during the first and second 5 min of TST. However, Spearman’s signed rank test revealed that no behavior was correlated with the immobility time during the first and second 5 min of TST (distance traveled and first 5 min of TST: H${}_2$O-intake mice: p = 0.6362, NaCl-intake mice: p = 0.9787, Fig. 5K; number of entries and first 5 min of TST: H${}_2$O-intake mice: p = 0.7514, NaCl-intake mice: p = 0.8944, Fig. 5L; time spent and first 5 min of TST: H${}_2$O-intake mice: p = 0.8028, NaCl-intake mice: p = 0.1509, Fig. 5M; distance traveled and second 5 min of TST: H${}_2$O-intake mice: p = 0.5526, NaCl-intake mice: p = 0.0872, Fig. 5N; number of entries and second 5 min of TST: H${}_2$O-intake mice: p = 0.9867, NaCl-intake mice: p = 0.7495, Fig. 5O; time spent and second 5 min of TST: H${}_2$O-intake mice: p = 0.5334, NaCl-intake mice: p = 0.7495, Fig. 5P). These results indicate that the anxiety level in sodium-intake mice was not correlated with their immobility time during TST. Therefore, central preference in sodium-intake mice might not reflect their anxiety level.

Fig. 5.

The effect of 2% NaCl intake on mouse anxiety levels while using the elevated plus maze (EPM) and the correlation between immobility time during tail suspension test (TST) and the anxiety level. (A) Schematic drawing shows the EPM and the schedule of TST. (B) Traces of water- and sodium-intake mice moving on the EPM. (C–E) Comparison between water- and sodium-intake mice in terms of distance traveled (C), number of entries (D), and time spent (E) in the open arm of the EPM. (F, G) Comparison between the number of entries (F) and time spent (G) in the central zone of the EPM for water-intake mice (n = 10) and sodium-intake mice (n = 11). (H) The time course of immobility time during 10 min of TST for water-intake mice (n = 10) and sodium-intake mice (n = 11). (I) Immobility time during the first 6 min, except the first 1 min, of TST for water-intake mice (n = 10) and sodium-intake mice (n = 11). (J) Summary of cumulative immobility time during the first (0–5 min) and last 5 min (5–10 min) 1 h after exposure to inescapable innate fear for water-intake mice (n = 10) and sodium-intake mice (n = 11). (K–M) Correlation between immobility time during the first 5 min of TST and the distance traveled (K), number of entries (L), and time spent in the open arm of the EPM (M) for water-intake mice (n = 10) and sodium-intake mice (n = 11). (N–P) Correlation between immobility time during the second 5 min of TST and the distance traveled (N), number of entries (O), and time spent in the open arm of the EPM (P) for water-intake mice (n = 10) and sodium-intake mice (n = 11). Statistically significant differences between two Pearson’s correlation coefficients by Fisher’s r-to-z transformation (${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}}$p 0.05) are indicated. NS means not significant. R${}^2$ is Pearson’s correlation coefficient.